Identification of critical kinase(s) required to trigger CDC-25.1
degradation during embryogenesis in C. elegans
Christopher St-François
Department of Biology, McGill University
Montreal, Quebec, Canada
April 2015
A thesis submitted to McGill University in fulfilment of the
Requirements of the degree of Master of Science
© Christopher St-François 2015
ABSTRACT
In eukaryotic cells, CDC25 phosphatases play precise roles in positively regulating the cell cycle
during embryonic growth and development. Proper control of CDC25 expression levels is
pivotal for the correct formation of tissues and organs. In mammalian cells, CDC25A removes
inhibitory phosphates on CDK2 and is a critical G1/S phase target that is regulated downstream
of DNA damage signals. This is controlled through its timely degradation which is initiated by a
highly conserved F-box protein called β-TrCP, upon recognition of phosphorylated serine
residues within the CDC25A phosphodegron motif. In a genetic screen for tissue-specific cell
cycle regulators in C. elegans, we isolated a cdc-25.1 gf mutation that disrupts this domain,
presumably affecting its appropriate recognition, allowing it to resist degradation by βTrCP/LIN-23. As a result, this critical cell cycle regulator persists and causes a supernumerary
round of cell divisions, exclusively in the intestine. Since phosphorylation of the CDC-25.1
phosphodegron likely precedes this event, we are trying to identify the kinase(s) responsible for
this/these critical modifications. We performed a reverse genetic screen by eliminating all the
predicted C. elegans serine/threonine kinases using RNA interference. This screen was carried
out in a elt-2::gfp background and we hoped to observe intestinal hyperplasia to identify the
kinases that are involved in triggering this event. We successfully discovered many kinases that
caused embryonic lethality that are required for embryogenesis and require further investigation.
Finally, we narrowed our search down to a specific a C. elegans kinase family called NEver in
mitosis Kinase-Like (NEKL) in which are closely related to the fungi NIMA kinase family. We
further characterized this kinase family by exposing the worms with various RNAi treatments
and studying NEKL mutants in a elt-2::gfp background.
Further analysis of nekl-3 RNAi
resulted in embryonic lethality and lead to the strongest hyperplasia phenotype of the NEKL
gene family.
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RÉSUMÉ
Dans les cellules Eucaryote, la phosphatase CDC25 jouent un rôle dans la régulation positive du
cycle cellulaire durant l’embryogenèse et le développement.
Le contrôle des niveaux
d’expression de CDC25 est essentiel pour la bonne formation des tissues et organes. Dans les
cellules mammifère, CDC25A enlève les phosphorylations inhibiteurs sur CDK2 et est essentiel
pour la phase G1/S parce qu’il est régularisé par les signaux émient par le dommage d’ADN. La
dégradation du CDC25A (CDC-25.1 dans les C. elegans) dépend de la phosphorylation du site
conservé DSG(X)4S reconnu par la protéine F-box appelé β-TrCP/LIN-23. On a déjà isolé une
mutation cdc-25.1(gf) qui affecte ce domaine phosphodegron, qu’on présume l’attribution de la
résistance à la dégradation par β-TrCP/LIN-23. Cette mutation résulte à la persistance du
régulateur CDC-25.1 et cause un surplus de division exclusivement dans l’intestin. Comme la
phosphorylization du phosphodegron de CDC-25.1 précède sa dégradation, on essaye d’identifier
la kinase qui est responsable pour cette modification essentielle. On a performé un crible
génétique d’interférence d’ARN qui élimine les kinases sérine/thréonine. Ce crible a été fait
avec des vers elt-2::gfp et on voulait observer hyperplasie intestinale pour identifier les kinase
responsable pour la création de cette événement. La découverte de plusieurs kinases qui étaient
responsable pour la cause de la létalité embryogénèse a réussi mais il faut beaucoup plus
d’investigation. Finalement, on a raccourci notre recherche sur une famille de kinase spécifique
au C. elegans, appelé NEver in mitosis Kinase-Like (NEKL), qui est relié à la famille NIMA des
champignons. On a caractérisée cette famille en plus de détaille en exposant les vers à de
différent type de interférence d’ARN et en étudiant les mutants NEKL dans une souche elt2::gfp.
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ACKNOWLEDGEMENTS
To begin with, I would like to thank my supervisor, Richard Roy, for his continuous guidance
and support during my two years in the laboratory. Rick has allowed me the freedom to come up
with my own ideas and let my skills development as an independent scientist. I also appreciated
the way he was always available for discussion when I dropped by his office. Moreover, I
admire the way that he brings the lab together and brought us all out for numerous dinners,
birthday celebrations, retreats, and conferences and always made us feel like we were family.
I am also grateful to all Roy lab members for all the help, encouragement, advice, friendship and
many laughs. Thanks to Michaël Hebeisen and Shaolin li who taught me everything when I first
arrived from picking worms to various scientific protocols. A special thanks to Yu Lu for caring
enough to take the time to explain and help me understand various protocols and I appreciate all
our great conversations that led to lots of laughs, especially for our “special” connection. I
would also like to thank Julie Mantovani, Émilie Demoinet, Meng Xie and Emily Wendland for
all the advice, help and good conversation, you guys are great. I am thankful to Monique Zetka
and all her laboratory members for all the constructive comments and advice during our
numerous combined lab meetings.
I am grateful to my Supervisory committee members
Christian Rocheleau and François Fagotto for their help, advice and encouragement.
I am grateful to my family especially my mom for her everlasting love and support. Thank you
for always believing, constantly being there for me and always trying to help me in any possible
way. I would also like to thank Kelly for her constant positive support and for the reward of
seeing her beautiful smile at the end of every day. Last but not least, is a family member who I
would like to thank the most of all and I am infinitely in debt to, my brother and best friend,
Abbas Ghazi. I am deeply grateful for your constant support, motivation and help. Thank you
for all the vast biochemistry expertise you taught me, bringing original scientific ideas to my
attention and for guiding me through some of the protocols. I appreciated all the amazing talks
we had on life and the last two years have been an absolute blast, I have never laughed so hard in
my life. I could not have asked for a better friend to spend my time with. Thank you.
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PREFACE
This is a non-manuscript-based thesis presented in accordance with the “Guidelines for thesis
preparation” from the faculty of Graduate Studies and Research.
(http://www.mcgill.ca/gps/programs/thesis/guidelines/preparation/).
This thesis is composed of three chapters; an Introduction (Chapter I), which encompasses a
literature review, as well as, the rationale of the study, two research chapter (Chapter II and III).
This thesis is prepared and written by the candidate in collaboration of his thesis supervisor
Richard Roy.
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TABLE OF CONTENTS
ABSTRACT.................................................................................................................................................. 2
RÉSUMÉ ...................................................................................................................................................... 3
ACKNOWLEDGEMENTS .......................................................................................................................... 4
PREFACE ..................................................................................................................................................... 5
TABLE OF CONTENTS .............................................................................................................................. 6
LIST OF FIGURES ...................................................................................................................................... 8
LIST OF TABLES ........................................................................................................................................ 9
CHAPTER 1: .............................................................................................................................................. 10
LITERATURE REVIEW ........................................................................................................................... 10
THE CELL CYCLE................................................................................................................................ 11
The cell cycle phases: roles of CDK/cyclin complexes ...................................................................... 11
Regulation of the activation of CDK/cyclin complexes ..................................................................... 14
THE DEGRADATION VIA THE PROTEASOME .............................................................................. 16
Ubiquitination ..................................................................................................................................... 16
Proteolysis ........................................................................................................................................... 16
The ubiquitin proteasome degradation pathways control over the cell cycle ..................................... 17
The APC/C (anaphase-promoting complex/cyclosome) pathway ...................................................... 17
The SCF (Skp1/Cullin/F-box complex) pathway ............................................................................... 18
Regulation of the CDC25 phosphatase and the DNA damage checkpoint pathway........................... 19
THE CELL CYCLE UNDER DEVELOPMENTAL REGULATION................................................... 21
Caenorhabditis elegans a model organism to study the cell cycle during development .................... 21
Reverse genetics.................................................................................................................................. 21
Regulation of the embryonic cell cycle in Caenorhabditis elegans.................................................... 22
The CDK inhibitors............................................................................................................................. 23
The ubiquitin-mediated degradation directs cell cycle regulation ...................................................... 23
Post-translational alteration required for proper cell cycle control ..................................................... 24
RATIONAL OF STUDY OF MY MASTER OF SCIENCE WORK .................................................... 27
CHAPTER 2: .............................................................................................................................................. 29
ABSTRACT............................................................................................................................................ 30
INTRODUCTION .................................................................................................................................. 31
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MATERIALS AND METHODS ............................................................................................................ 34
Strains and culture............................................................................................................................... 34
RNA interference ................................................................................................................................ 34
Microscopy and image processing ...................................................................................................... 34
RESULTS ............................................................................................................................................... 35
Genetic screen for intestinal hyperplasia ............................................................................................ 35
Functional classification of kinases that cause embryonic lethality. .................................................. 36
Hypomorphic RNA-interference......................................................................................................... 36
Tissue-specific RNA-interference....................................................................................................... 37
Classification of embryonic lethal kinases .......................................................................................... 38
DISCUSSION ......................................................................................................................................... 42
CHAPTER 3: .............................................................................................................................................. 53
INTRODUCTION .............................................................................................................................. 54
MATERIAL AND METHODS .......................................................................................................... 56
RESULTS ........................................................................................................................................... 58
CONCLUSION ................................................................................................................................... 62
GENERAL DISCUSSION ......................................................................................................................... 73
NEKL-3 involvement in CDC25-1 regulation .................................................................................... 73
REFERENCES ........................................................................................................................................... 77
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LIST OF FIGURES
Figure 1: C. elegans - A Model Organism……………………………………………...…...…p.26
Figure 2: CDC-25.1 And Its Critical Destruction Motif……………………………………....p.44
Figure 3: Classification of Genes Resulting in Embryonic Lethality………………….………p.48
Figure 4: Known Interactions In The Innate Immune Response In C. elegans…………….....p.50
Figure 5: Kinases Impinging On Proper Morphogenesis During Embryogenesis……………p.51
Figure 6: L4440 Expression Vector And 3’UTR Cloning……………………………………p.67
Figure 7: Microscopy Of nekl-3 3’UTR Injected Worms….…………………………………p.69
Figure 8: Various MR1619 Embryonic Lethal Egg Phenotypes…………………………..….p.71
Figure 9: NEKL-3 Amino Acid Sequences Alignment……………………………………..…p.72
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LIST OF TABLES
Table 1: Kinase screen for intestinal hyperplasia phenotype in elt-2::gfp background.......….p.45
Table 2: Individual Classification of Genes Resulting in Embryonic Lethality……….……...p.49
Table 4: L1 Intestinal cell number of bacteria feed nek-like 3’UTR RNAi clones…………...p.65
Table 3: L1 Intestinal cell number of bacteria feed nek-like RNAi clones…………………....p.66
Table 5: Intestinal cell number of young adult injected nek-like 3’UTR dsRNA…………….p.68
Table 6: Intestinal count of nekl mutants strain with integrated elt-2::gfp………………........p.70
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CHAPTER 1:
LITERATURE REVIEW
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THE CELL CYCLE
Eukaryotic cell cycle progression is controlled by a fundamental evolutionarily conserved
regulatory network that has been elucidated by numerous studies performed in two yeast species;
Schizosaccharomyces pombe and Schizosaccharomyces cerevisiae [1]. Cell cycle regulators
described in work performed with these yeasts have evolutionarily conserved orthologs that have
been generally identified in every higher organism studied thus far, whereas the mechanisms that
underlined this cell cycle control have been much conserved among eukaryotes [2]. Taken as a
whole, this signifies that findings resulting from research performed in lower organisms can be
biologically relevant when studying the functions of the cell cycle machinery.
The cell cycle phases: roles of CDK/cyclin complexes
The cell cycle includes several phases, that lead up to two main events; S-phase (DNA synthesis
phase) and M-phase (mitotic phase). During these events, genetic material is correctly replicated
in S phase and then packaged into the sister chromatids, which are faithfully separated into two
identical daughter cells in M phase. There are two intermediary Gap phases, G1 and G2, which
govern the entry into S and M correspondingly [1]. The START point in yeast or the restriction
point in mammals is the instant where the cell becomes insensitive to any cellular signal late in
G1 and passing through this point commits the cell to the mitotic cycle [3-4]. Signals such as
growth factors and accessibility to nutrients will allow the cell to decide whether it is favorable
to begin this cycle and enter S phase. In G2, once the cell has verified the successful replication
of its genome, it can make a decision whether or not to divide. These decisions are all based on
internal and external signals, that once present, allow the dividing cell to bypass various blocks
to cell cycle progression [5]. Achieving a complete mitotic cycle is dependent on the cells’
ability to assure that each phase occurs successfully. After a cell divides into two identical
daughter cells, each daughter cell can choose to either exit the cell cycle permanently or
temporarily and enter a state (G0) where the cell is dormant or quiescent [6].
Various cell cycle regulators systematically govern the shift from one cell cycle phase to the
next. Genetic and biochemical studies demonstrate that cell division in eukaryotes is mediated
by cyclin proteins and the cyclin-dependent kinases (CDKs) a family of serine/threonine protein
kinases [2, 7-8]. There are thirteen known CDKs identified in diverse organisms and most of
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these kinases require an association with a cyclin partner to be active [9-11]. Many of these
CDKs are required for transcription or post-transcriptionally, but five of them play very
important roles in cell cycle progression [11]. Although, it was initially assumed that the
different stages of cell division are regulated by cell-cycle-stage-dependent accumulation and
proteolytic degradation of different cyclin subunits that associate with their respective CDKs.
For example, the association of cyclin D/CKD4-6 primarily governs the exit from G1, cyclin
B/CDK1 regulates the G2/M transition and accumulation of cyclin A/CDK2 and cyclin E/CDK2
elicits the G1/S transition and entry into S phase, correspondingly, there is most likely
considerable redundancies between the kinases since disruptions of many of these kinases have
only mild phenotypes in higher organisms [12]. CDKs promote cell cycle progression by
phosphorylating a recognizable CDK motif (S/TPXK/R) on critical downstream substrates to
alter their activity [13]. The cyclin/CDK substrates seem to constitute an intricate network of
various cellular events that control mitosis, chromatin condensation, cytoskeletal reorganization,
centrosome duplication, transcription, DNA replication, and translation.
Role of CDKs in G1/S transition
Studies in mammalian cells have shown that toward the end of G1, the transcriptional repressor
pRB (the tumor suppressor retinoblastoma protein) is shown to be hyperphosphorylated by the
cyclin D/CDK4-6 complex [14]. This pRB phosphorylation allows for the dissociation of the
pRB/E2F complex and enables the initiation of transcription of target genes involved in S phase
succession, such as CDK2, cyclins E and A and factors of the DNA pre-replication complex [12,
15]. In late G1 and during the onset of S phase, the phosphorylation of E2F by cyclin A/CDK2
inhibits its own transcription which allows for systematic progression through S phase [16].
A mitotically dividing cell must maintain genomic integrity by faithfully duplicating its genome
once per cell cycle through stringent controls during S phase, to ensure the generation of two
identical sets of daughter chromosomes for the completion of M phase. This means that each
distinct origin of replication must fire only once per cell cycle, and this is accomplished by
ensuring that the replicative DNA helicase can be loaded only at origins during G1 phase, but can
only be activated at the same time during the subsequent S phase. CDC6 and CDT1 are loaded
on to the chromatin at the origins of replication and help load the origin recognition complex
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(ORC1-6) and the MCM2–7 helicase [17-19].
Studies in budding yeast show that the
Sld3/Cdc45 complex is recruited to the origins of replication, possibly by direct association with
the MCM2–7 complex. Once the origin recognition complex (Sld3/Cdc45) and the MCM2–7
helicase are in place, they remain idle until the initiation of S phase and activation by Cdc7 and
CDKs [20]. The MCM2-7 complex gets phosphorylated by the Cdc7 kinase which induces a
structural change, in addition, the Cdk kinases phosphorylate and recruits Sld2 and Sld3 which in
turn form a complex with Dpb11 allowing the recruitment of the DNA polymerase to origins
[21]. Activation of the MCM2–7 helicase unwinds the origin and allows the priming of leading
and lagging strands by various DNA polymerase α [21].
As the DNA helix unwinds and the genome is replicated the origins of replication are no longer
licensed due to the same CDKs that induce its chromatin binding affinities. The licensing factors
become phosphorylated by cyclin A/CDK2 which inhibits re-licensing and/or targeting them for
nuclear export or degradation via the ubiquitin-mediated pathway [17, 22-23]. During S and M
phase DNA licensing is prevented by geminin in higher eukaryotes [24-25]. Once M phase has
been completed, the licensing factors binding affinity for chromatin is re-established due to the
downregulation of CDK activity and degradation of geminin via the APC/C (anaphasepromoting complex/cyclosome). This allows pre-RCs to bind to replication origins during early
G1 and prime the DNA for the next round of the cell cycle. Therefore, the origin of replication
can only become licensed, if M phase is completed, ensuring that the chromosomes are only
replicated once per cell cycle.
Role of CDKs in G2/M transition
The entry into M phase is initiated by the cyclin A/CDK1 complex at the end of G 2 whereas M
phase is driven by cyclin B/CDK1 [22]. Many proteins that are required in mitosis have been
shown to be important substrates of cyclin B/CDK1 such as nuclear lamins (breakdown of
nuclear envelop), microtubules (mitotic spindle formation), histones and condensins
(chromosomes condensation) [7, 26-28].
These proteins must be reset by dephosphorylation
performed by various phosphatase in order to exit mitosis and to restore the nuclear envelop at
the initiation of cytokinesis, decondensate the chromosomes, and remodel the cytoskeleton [2930].
The crucial onset of M phase by cyclin B/CDK1 can only happen after successful
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termination of S phase and this switch is controlled by post-translational modifications of
numerous cyclin/CDK subunits such as dephosphorylation, phosphorylation and degradation
[31]. Therefore, the synchronization and systematic progression of the cell cycle is dependent
upon successive completion of DNA replication followed by mitosis.
Regulation of the activation of CDK/cyclin complexes
Cell cycle dynamics are regulated by several pathways that utilize different cyclin/CDK
complexes as their downstream effectors [32]. As the cell cycle advances through each phase,
the various protein levels of nearly all CDKs remain invariable. Meanwhile, their activity is
constantly controlled by post-transcriptional regulation, such as, forming complexes with
different cyclins, phosphorylation (by inhibitors), dephosphorylation (by activators) and by
subcellular relocalization.
Cyclins CDK complexes
The substrate specificity of the CDKs (can) depend on its association with individual cyclin
subunits, such as cyclins A, B, D, and E. Differential transcriptional activation and ubiquitinmediated degradation of these cyclin subunits in a phase-dependant manner assure the proper
expression of subunits during specific cell cycle phases [33]. This regulation of cyclin levels
guarantees that each cell cycle phase progresses in a proper and efficient order, where the
beginning of one phase requires the completion of the other. In mammals, the CDK/cyclin
specificity is further increased by alternative splicing creating multiple functional isoforms [33].
Cyclin/CDK regulation
CDK activity is also dependent on its phosphorylation state and this allows for the proper
progression of the cell cycle. CDK1 and CDK2 must be activated by phosphorylation of other
CDK/cyclin complexes on specific CDK residues [34]. Such post-translational modifications are
carried out by CDK-activating kinases, such as CAK (composed of CDK7/cyclinH), which
induce conformation changes by phosphorylating conserved threonine residues on CDK1 and
CDK2 [34]. On the other hand, phosphorylation of two conserved residues within the active
sites of CDKs by negative regulators such as WEE1 and MYT kinases can inhibit its function.
Whereas, removal of this inhibition by dual specificity phosphatase family members such as the
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highly conserved cell division cycle 25 (CDC25) allows for positive progression of the cell cycle
by activating CDK/cyclin complexes [35-40].
The CDC25 family of dual specificity
phosphatases include paralogs that engage in distinct and specific yet redundant phase-and/or
tissue-specific roles. For example, G1/S and G2/M transitional control is governed by three
separate CDC25 phosphatases (CDC25A/B/C) in mammals [41]. Cell cycle regulation is mostly
under the control of CDC25A, which participates in G1/S and G2/M, while CDC25B and
CDC25C play important roles in mitosis [42-43]. CDC25s are also under strict phase dependent
post-transcriptional regulation by various
modifications of their N-termini.
Such
phosphorylation-dependent modifications of CDC25 activity and/or degradation are performed
by numerous kinases implicated in the cell cycle such as PIM1, Aurora A, polo-like kinases, in
addition to checkpoint kinases CHK1, CHK2, Nek11 and p38 [42, 44-46]. Therefore, CDK
post-translational modification influences the progression through each cell cycle phase.
CDC25 regulation
During periods of growth and proliferation it is imperative that each cell cycle phase is
completed successfully and in a timely manner.
This involves the precisely controlled
expression and degradation through of critical positive cell cycle regulators. CDC25 paralog
levels oscillate in a similar fashion to the cyclin subunits in each specific cell cycle phase. The
levels of CDC25A and B oscillate in a phase dependent manner, whereas levels of CDC25C stay
invariable during each cell cycle phase [47]. The expression of CDC25A increases from mid to
late G1 [48-49] and once S phase is initiated, CDC25A is targeted for degradation in a
CHK1/SCF-dependent manner, while at the end of M phase, CDC25A is targeted in a APC/Cdependent manner [43, 50-53]. CDC25B levels begin to accumulate at S phase until the end of
G2, where the levels begin to drop due to ubiquitin-mediated degradation [54]. As a result, the
timely progression of the cell cycle requires the precise regulation of CDC25 levels during each
phase. Interestingly, the same mechanisms under normal conditions that are used to control the
protein levels of CDC25 are also involved in the cellular damage response pathways to evoke a
prompt and effective cell cycle arrest to allow time to restore the cellular integrity.
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DEGRADATION VIA THE PROTEASOME
The regulation of numerous cellular processes is under the control of the ubiquitin-mediated
degradation pathway.
Such processes include, but are not limited to, cellular response to
extracellular stresses and effectors, differentiation and development, programmed cell death, cell
cycle regulation and DNA repair [55-57]. Uncoupling of this important ubiquitin-proteasome
pathway is related to the onset of many human diseases as well as malignant transformation [58].
In order for degradation via the ubiquitin-proteasome pathway, substrates must first be polyubiquitinated to allow for recognition by the 26S proteasome machinery, which results in its
degradation.
Ubiquitination
Three classes of enzymes, E1 through E3, are required to covalently poly-ubiquitinate their
substrate proteins. Ubiquitin is first activated by an ubiquitin activating enzyme (E1) and then
covalently bound to E1 via by an ATP-dependent reaction. The ubiquitin molecule is then
transferred to an ubiquitin-conjugating enzyme (E2), which is, in turn, catalyzed by an ubiquitinprotein enzyme (E3) and ubiquitin is then bound by a lysine residue to the substrate protein.
Sequential addition to the of activated ubiquitin molecule to each other occurs at a specific
lysine48 residue that cause the substrate protein to become poly-ubiquitinated [55]. The targeting
of protein substrates depends on the recognition of specific destruction signal by the E3 ligase.
In higher organisms, there are numerous E3 ligase families that can recognize specific protein
targets [55, 59-60]. It has been reported that there are over 1000 different E3 ligases present in
the human genome [61].
Proteolysis
Recognition and subsequent degradation performed by the 26S proteasome requires the polyubiquitination of the target protein. Two identical subcomplexes make up the 26S proteasome: a
20S barrel catalytic core and a 19S base cap and lid that closes the 20S core on both sides. The
19S base cap and lid recognizes poly-ubiquitinated substrates and inserts them into the 20S
barrel.
Once the target substrates enter the 20S catalytic core, they are degraded into
16
oligopeptides and the poly-ubiquitin chain is broken back down into single ubiquitin units so that
they can be reused by the E1 enzymes [62-63].
The ubiquitin proteasome degradation pathways control over the cell cycle
There are four E3 ligase families that vary according to the constitution of their motifs; RINGfinger-type, PHD finger-type, HECT-type and U-box-type [64]. The biggest family of E3 ligases
is the RING-finger-type and this family can be further divided into four subfamilies, where some
E3 ligases can act alone and other are part of a multi-protein complex. The E3 ligases that form
multi-protein complexes contain a RING subunit and Cullin or Cullin homology domain [64-65].
The anaphase-promoting complex/cyclosome (APC/C) and the Skp1/Cullin/F-box (SCF) are the
two major Cullin-RING-based E3 ligases which are accountable for the cyclic degradation of
essential cell cycle regulators such as the cyclins and CDC25 phosphatases [64, 66]. These two
key ubiquitin E3 ligases facilitate the accurate and timely progression through each of the cell
cycle phases by controlling the degradation of important cell cycle regulators. Intriguingly, after
genotoxic stress, the checkpoint pathways rely on the same E3 ligases to stall or arrest the cell
cycle’s forward progression and permit the cell machinery to repair damage or undergo the
initiation of apoptosis. This ability to halt the cell cycle prevents the incorporation of detrimental
mutations and genomic instability by allowing the cell the appropriate time required to restore its
integrity [44, 67].
The APC/C (anaphase-promoting complex/cyclosome) pathway
The APC/C complex has two very well studied activators, CDC20 and CDH1, with important
roles in cell cycle regulation. APC/CCDC20 and APC/CCDH1 are functional during two distinct
times in the cell cycle; APC/CCDC20 begins to be active during anaphase until late mitosis while
APC/CCDH1 is active from late mitosis to late G1 [64]. The APC/C complex substrate specificity
is completely dependent on the activator (CDC20 or CDH1) it is associated with during meiotic
or mitotic phases [68]. Different substrate specificity lies in the activator’s ability to recognize
and interact with different KEN- or D-box destruction motif present on various cell cycle
regulators [68].
17
APC/CCDC20 contributes to the initiation of chromosome segregation and anaphase by degrading
inhibitors that link sister chromatids together at their kinetochores [68-69].
Early Mitotic
Inhibitor (EMI1) ensures that APC/CCDC20 remains inactive in early mitotic events so that the
APC/CCDC20 does not initiate chromosome segregation.
Furthermore, EMI1 allows for the
initiation of S phase by inhibiting APC/CCDH1 in late G1 which allows for the stabilization of
cyclin A levels [70]. APC/CCDH1 remains inactive in early mitosis due to CDK1/cyclin B
phosphorylation of CDH1. After metaphase, this inhibition is relieved due to the degradation of
cyclin B by APC/CCDC20 [71]. Once APC/CCDH1 is activated it becomes the most abundant
complex because it degrades CDC20, CDC25A and most mitotic cyclins [43, 69]. This
degradation triggers mitotic exit and ensures low cyclin levels in G1 so that the cell can stay
dormant until signaled otherwise [72].
Moreover, in G1, CDK activity is inhibited by accumulation of negative cell cycle regulators
such as p21, p27 and p57, and WEE1 kinases because of APC/CCDH1 ability to target Tome1 and
SKP2 which are responsible to target these CDK inhibitors [73-74]. In addition, APC/CCDH1
targets DNA licensing factors ORC1 and CDC6 during G1 as well as many mitotic protein such
as polo-like kinase PLK1 and Aurora A kinase [72, 75-76]. In conclusion, the APC/C allows for
the mediated coordination and irreversible progression between M and late G1 phases.
The SCF (Skp1/Cullin/F-box complex) pathway
From late G1 to early M phase, the major coordinator of cell cycle protein levels is the
Skp1/Cullin/F-box (SCF) E3 ligase and its activity complements the APC/C in a temporal
regulatory loop to dictate cell cycle progression [77]. The SCF complex is made up of three
proteins; the adaptor protein SKP1, a Cullin CUL1, a RING-finger protein RBX1/ROC1 and an
exchangeable F-box protein that interacts with SKP1 and is required for substrate specificity
[64]. The F-box proteins are classified according to three specific domains WD40 repeats
(FBXW), leucine-rich repeats (FBXL) or other domains (FBOX). The most studied F-box
proteins include SPK2, FBW7 and β-TrCP which are all involved in cell cycle regulation.
SCFSKP2 recognizes many of the CDK inhibitors; p27, p21 and p57, numerous cyclins; cyclin A,
cyclin D1 and cyclin E, in addition to E2F, ORC1, CDT1, CDK9 and MYC and is involved in
18
their proteolysis [64]. Since SKP2 targets many CKIs, its overexpression has been observed in
aggressive cancers, while its loss-of-function causes slow growth along with centrosome over
duplication and polyploidy [78]. SCFFWB7 was first discovered as a negative regulator of LIN12/Notch in a genetic screen done in C. elegans [79]. SCFFWB7 is believed to be a tumor
suppressor and its substrates seem to be many oncoproteins such as cyclin E, MYC, JUN, and
Notch [64, 77].
β-TrCP identifies substrates that contain a conserved F-box DSG(X)4S destruction motif, called
phosphodegron, and recognition of this destruction box is dependent upon phosphorylation of
both serine residues by particular kinases [80]. SCFβ-TrCP substrates include β-catenin, subunits
of IĸB, circadian regulator along with the prolactin and interferon receptors [80]. Central cell
cycle regulators are also subject to ubiquitin dependent degradation via SCFβ-TrCP, such as the
CDK inhibitor WEE1, the APC/C inhibitor EMI1 and the CDK activators CDC25A and
CDC25B [81]. The post-transcriptional modifications of the phosphodegron allows for the
timely expression of different SCFβ-TrCP substrates during a given cell cycle phase. For example,
the phosphorylation of the F-box recognition motif and subsequent degradation of WEE1 during
the initiation of M phase allows for the activation of CDK1 by CDC25A and proper mitotic
progression [82].
The timely expression and degradation the APC/C and SCF and their associated proteins enables
for the critical cross regulation between them. This allows the APC/C and SCF to coordinate
progression of different cell cycle phases in an irreversible fashion.
Regulation of the CDC25 phosphatase and the DNA damage checkpoint pathway
The dual specificity phosphatase CDC25 is a conserved positive cell cycle regulator that drives
entry into S and M phases [83-84]. Therefore, controlling the levels of such a vital regulator is
crucial in maintaining proper timely completion of each phase under normal conditions. When
cells encounter various genotoxic stresses, there are numerous checkpoint pathways that impinge
on CDC25 to halt the cell cycle and allow for subsequent DNA damage repair [50, 85].
19
These checkpoint pathways act during key cell cycle phases such as G1/S, intra-S, G2/M to
ensure proper DNA and cellular integrity. DNA damage caused by ionizing radiation (IR) or
ultra-violet light (UV) and incomplete DNA replication activates the DNA damage checkpoint
pathway. The ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases
activate the checkpoint kinases, CHK1 and CHK2, through phosphorylation [66-67]. These two
important checkpoint kinases phosphorylate numerous cell cycle regulators including CDC25
phosphatases.
CHK1 and CHK2 prime CDC25A for degradation via SCFβ-TrCP mediated
proteolysis causing cell cycle arrest due to inhibition of CDK/cylin activity. They also target
CDC25C which causes its nuclear export via the binding of 14-3-3 which inhibits CDC25C
interaction with CDK1/cyclin B in the nucleus [43-44, 66]. As mentioned previously, in order
for recognition by β-TrCP substrates, their conserved phosphodegron motif (DSG(X)4S) must be
phosphorylated.
Interestingly, even though CHK1 and CHK2 phosphorylate CDC25A
downstream of ATM and ATR pathways, they do not phosphorylate the specific serine residues
in the conserved motif [44]. Therefore, CDC25A phosphorylation by the CHK1 kinase during
interphase seems to inactivate its activity while keeping protein levels fairly constant. This
inactivation is rapidly increased downstream of the DNA damage pathway by the recruitment of
CHK2 to aid CHK1 with hyper phosphorylation of CDC25A [44]. CHK1 kinases are also
responsible for activating CDK inhibitors such as WEE1 family members to further support cell
cycle arrest [86]. In has also been reported that in response to osmotic stress, microtubule
depolarization, disruption of the cytoskeleton, heat stress and UV light a p38 (MAPK)-dependent
checkpoint pathway is shown to inhibit CDC25B and CDC25C activity on the same
phosphorylation site as CHK1 [87-88]. This phosphorylation causes nuclear export of CDC25B
and C, similar to CHK1, and is performed by p38 downstream effector MAPK-associated protein
kinase-2 in a 14-3-3-dependent fashion [89].
The G1/S, intra-S, G2/S transition phases are initiated by CDC25 phosphatases and thus proper
CDC25 control guarantees faithful completion of each phase before the initiation of the
transition into the next phase [66-67]. These checkpoint pathways impinge on positive cell cycle
regulators, such as CDC25 phosphatase, to enable the cell to repair damage and allow for proper
reproduction and cellular survival. There is a strong correlation found in many human cancers
20
between defects found in SCFβ-TrCP E3 ligase machinery and the overexpression and stabilization
of the CDC25 phosphatase [81].
THE CELL CYCLE UNDER DEVELOPMENTAL REGULATION
Caenorhabditis elegans a model organism to study the cell cycle during development
Caenorhabditis elegans is a small round worm that belongs to the nematode family.
As
compared to unicellular organisms such as yeast, multicellular organisms can give better insight
on the coordination of cell division during developmental processes such as embryogenesis,
larval development and germline differentiation. Furthermore, many of these regulators control
cell cycle progression in mammals have orthologs that function in a similar manner in C. elegans
[90]. In addition, because C. elegans is transparent every single cell lineage is completely
mapped out in detail for both embryogenesis and post-embryonic development [91-92]. The
mapped lineage and the transparency of the cuticle have facilitated the identification of
mutations that effect cell cycle control by using reporter genes that are exclusively expressed in
certain cells (figure 1). With the ever-increasing genomic, genetic and molecular resources that
are available, that make C. elegans has become an ideal system to study developmental
regulation of the cell cycle.
Reverse genetics
The real strength of C. elegans, as a model organism, lies in the ability to characterize the
function of various genes using multiple genetic techniques. Forward genetic methods involve
mutagenesis and cloning the mutant gene based on the phenotype of interest in developmental
events. This approach has been indispensable in the field and has led to the isolation of many
gain-of-function, loss-of-function and temperature sensitive alleles. However, this technique is
time consuming and this may take many months to isolate and characterize these mutant genes.
In 1998, Andrew Fire and Craig Mello uncovered a new pathway of RNA mediated interference
(RNAi) in C. elegans that changed the face of genome wide studies of gene function [93]. The
RNA mediated interference (RNAi) response in the complete organism can be triggered by
21
systematic inactivation of a gene product initiated in any area of the body [94]. This allows for
the high-throughput genome wide RNAi investigations through RNAi induction by means of
injection, soaking and feeding [95]. In addition, 80% of human cancer genes that cause
germline mutations have C. elegans orthologs and that 53% of these orthologs show a visible
RNAi phenotype [96].
A genome wide RNAi library of IPTG inducible bacterial clones
expresses dsRNA capable of targeting most of the C. elegans genome [97]. Many of the
pathways involved in cancer are conserved in C. elegans and combining this information with
the ability to conduct such genomic screens in C. elegans is a powerful tool that can reveal many
novel genes involved in development pathways and cellular functioning. Also, the use of C.
elegans as a model organism for biomedical research is further aiding in the comprehension of
various molecular pathways involved in the aetiology of several human diseases [98].
Regulation of the embryonic cell cycle in Caenorhabditis elegans.
C. elegans embryos acquire maternal gene products in the adult germline to drive early
embryonic cell division and function to set the positional expression of zygotic patterning genes
[92, 99]. During the early embryonic cell divisions, there are no gap phases; therefore the
embryos undergo quick cleavage-like cell division alternating between DNA replication and
mitosis, where the cells lose size and volume as they divide. At the 24-cell stage, the first gap
phase, is introduced in the descendants of the intestinal precursor cell “E” [100]. The onset of
gastrulation highlights the timely introduction of gap phases to specific lineages, in addition to
specific maternal to zygotic gene transition (MZT) [100-102]. When embryogenesis is nearly
complete most of the cells have ceased dividing except for fifty five blast cells that continue to
divide to form the reproductive system and develop a more intricate nervous system of the adult
worm [91].
There are many specific spatiotemporal controls that impinge on many different components of
each individual cell and their respective lineage that allows for the proper development of an
entire organism. CDK complexes and their regulators are the core of cell cycle progression and
their control is a key factor in proper development and a summary of C. elegans cell cycle
regulators will follow.
22
The CDK inhibitors
In C. elegans as in yeast, CDK inhibitors (CKI) bind to CDK/cyclin complexes to finely tune cell
cycle phase succession. The Cip/Kip family of CKIs are important for cell cycle arrest and cell
cycle phase exit because of their ability to bind cyclin E, D, B, A/CDK complexes during normal
development, differentiation and in the presence of cellular stressors [103]. During C. elegans
embryogenesis, cki-1, the homolog of mammalian p27/KIP1, is only expressed in post-mitotic
differentiating cells, while in L1 larvae it is expressed in a timely manner in various lineages to
control cell cycle progression [104-105]. Thus, overexpression of cki-1 or cki-2 causes G1 arrest
in embryonic cells and specific ectopic expression of cki-1 in larval cells causes arrest in G1.
Alternatively, cki-1(lf) mutants induce differentiation and morphogenesis defects by allowing
uncontrolled over proliferation of embryonic and postembryonic lineage cell cycle progression.
The hyperplasia produced by the loss of cki-1 enables changes in cell fate specification during
the management of cell cycle progression [104-105].
Ubiquitin-mediated degradation directs cell cycle regulation
Cell cycle control, in C. elegans, is under the strict regulation by the ubiquitin-proteasomal
pathway. The APC/C ubiquitin E3 ligase has a well characterized role in mitotic exit; therefore
mutations in the APC/C members trigger metaphase-to-anaphase transition defects in processes
such as spindle and chromosomal rearrangements [106-107]. Embryonic arrest occurs at the
one-cell stage in APC/C null mutants, where the cells arrest in metaphase because the sister
chromatids remain paired [108].
A C. elegans Cullin component of the SCF E3 ubiquitin ligase, cul-1, was the first Cullin gene
discovered to be required for cell cycle exit. Severe over proliferation occurs in nearly all
embryonic and post embryonic lineages due to the loss of cul-1. [109]. Moreover the loss of
other SCF components, such as lin-23 (ortholog of the F-box protein β-TrCP), skr-1 and skr-2
(SKP-related proteins) phenocopy the same hyperplastic embryonic and post embryonic
proliferation observed in the cul-1(lf) [109-111]. Since the SCF E3 ligase complex (CUL-1,
SKR1/2 and LIN-23) is responsible for the G1/S transition, loss of function of anyone of these
23
components allows cell to break their terminal differentiation by enabling increased levels of cell
cycle regulators [77, 110]. On the other hand, G1 arrest is observed in cul-2 null mutant germ
cells because CKI-1 escapes CUL-2-dependent degradation and cul-2(lf) causes chromosome
condensation, in addition to segregation defects during early embryogenesis [112-114]. CUL-4 is
required to degrade CDT-1, which is an important pre-replication factor, as well as to inhibit
DNA replication during S phase [115-116]. Following to the loss of cul-4, cells can acquire a
DNA content up to 100C due to an immense amount of DNA synthesis [117]. The cullin gene
family seems to be highly conserved throughout evolution, whereas the other SCF components
families, F-box and Skr seemed to have expanded greatly in C. elegans lineage [111, 118].
Furthermore, the severe overproliferation caused by lin-23(RNAi) has been shown to trigger E
lineage hyperplasia by two different mechanisms; generation of additional E cell fate
specification and stabilization of CDC25.1 [119]. GFP::CDC25.1 expression seems to persist
during development in lin-23(RNAi) treated wild-type worms beyond its wild-type control
expression. This was further confirmed in GFP::CDC-25.1[G47D] gain of function variant that
increased stabilization of this positive cell cycle regulation because it could not be properly
recognized by LIN-23 for Ubiquitin-mediated degradation [119]
Post-translational alteration required for proper cell cycle control
In C. elegans, there is very stringent post-transcriptional control over the activity of CDK/cyclin
complexes due to the presence of four different CDC25 phosphatases paralogs (cdc-25.1, cdc25.2, cdc-25.3 and cdc-25.4) and three distinct members of the Wee/Myt family (wee-1.1, wee1.2 and wee-1-3) [120-121]. WEE-1.1 is believed to regulate early cellular division events in
various cells due to wee-1.1 mRNA expression in the AB and E blastomeres along with temporal
wee-1.1 expression during the 12-16 cell stage during embryogenesis [121]. Intriguingly, the
onset of gastrulation creates the first gap phase (G2) in the “E” intestinal daughter cells which
follows the initiation of wee-1.1 mRNA expression in the E precursor [100]. Moreover, wee1.3(lf) mutations in the C-terminus cause CDK1 activation in oocytes and, in contrast, wee1.3(gf) results in a G2/M arrest in tissue-specific spermatogenesis [122-123].
24
The key positive cell cycle regulator CDC-25.1 has the ability to remove inhibitory phosphates
from CDK/cyclin complexes to promote cell cycle progression [124-125].
This critical
phosphatase has been revealed to incorporate spatiotemporal signals to the coordination of the
timely cell cycle progression in development. Loss of cdc-25.1 causes mitotic arrest in the
germline and early embryos [126-127]. Interestingly, the discovery of two gain-of-function
CDC-25.1 mutation, caused by a point mutation in the phosphodegron motif recognized by the
SCF E3 ligase component β-TrCP/LIN-23, resulted in hyperplasia exclusively in the intestinal
cells which initiated during a specific window during intestinal development [119, 128-129].
Hence, the proper regulation of the E lineage requires stringent control over CDC-25.1 because
its stabilization creates intestinal-specific hyperplasia. This may also reflect other possible roles
that CDC-25.1 expression might impose on other lineages during embryogenesis.
25
Figure 1: C. elegans - A Model Organism
A
B
A: C.elegans lineage map form from a one cell embryo to a fully developed multicellular organism
B: transparent cuticle allows for studies of internal organs
modified from Kipreos et.al, 2005
26
RATIONAL OF STUDY OF MY MASTER OF SCIENCE WORK
Regulation of the cell cycle, in multicellular organisms, is indispensable to control cell numbers
in tissues and organs during development. For several years, C. elegans have been exploited as a
model for the analysis for cell cycle control during developmental processes, and many groups
are currently researching various aspects of cell cycle regulation. While significant progress has
been made in identifying molecular pathways that affect CDC-25.1 regulation during
development, there is still much to be discovered about the upstream effectors that impinge on its
function.
In addition, this key positive cell cycle regulator has been well studied in the
mammalian system because of many checkpoint pathways that inactivate this phosphatase
downstream of DNA damage or cellular instability. In C. elegans, embryonic cell cycle
progression, specifically intestinal development, is largely under the control of the critical
positive cell cycle regulator CDC-25.1.
The intestinal-specific hyperplasia observed in C.
elegans cdc-25.1(gf) is indicative of multiple types of human cancers where such positive cell
cycle regulators are stabilized and creates tumorigenesis in analogous tissues or organs.
It is evident that accurate CDC-25.1 regulation through its timely degradation is important for
establishing faithful mitotic divisions and formation of many tissues and organs during
embryogenesis. Stabilization of CDC-25.1 causes intestinal specific hyperplasia at the E8-cell
stage of embryogenesis, as seen in cdc-25.1(gf) mutants [119]. This mutation found in cdc25.1(gf) is believed to disrupt the phosphorylation of the β-TrCP/LIN-23-like motif, by an
unknown kinase, and allows for its stabilization and persistence, through the loss of recognition
by the F-box protein LIN-23 of the phosphorylated motif, during the early cell cycles in
embryogenesis [119, 128, 130]. Therefore, using a reverse genetic approach, we will knock
down the kinase(s) required to phosphorylate this β-TrCP/LIN-23-like motif which will most
likely stabilize CDC-25.1 and cause intestinal hyperplasia or embryonic lethality depending on
the plethora of its function and targets.
Uncovering, the kinase(s) involved in CDC-25.1
regulation would be useful in understanding how misregulation of this kinase(s) leads to human
cancer predisposition syndromes and its timely and temporal control of early embryonic cell
cycle during development.
27
28
CHAPTER 2:
IDENTIFICATION OF SERINE/THREONINE KINASES THAT PHOSPHORYLATE
CDC-25.1 DURING EMBRYOGENESIS DURING INTESTINAL DEVELOPMENT IN
C. elegans.
29
ABSTRACT
In eukaryotic cells, CDC25 phosphatases play precise roles in positively regulating the cell cycle
during embryonic growth and development. Proper control of CDC25 expression levels is
pivotal for the correct formation of tissues and organs. In mammalian cells, CDC25A removes
inhibitory phosphates on CDK2 and is a critical G1/S phase target that is regulated downstream
of DNA damage signals. This is controlled through its timely degradation which is initiated by a
highly conserved F-box protein called β-TrCP/LIN-23, upon recognition of phosphorylated
serine residues within the CDC25A (CDC-25.1 in C. elegans) phosphodegron motif. We have
isolated a cdc-25.1 gf mutation that disrupts this domain, presumably affecting its appropriate
recognition, allowing it to resist degradation by β-TrCP/LIN-23. As a result, this critical cell
cycle regulator persists and causes a supernumerary round of cell divisions, although exclusively
in the intestine. Since phosphorylation of the CDC-25.1 phosphodegron likely precedes this
event, we are trying to identify the kinase(s) responsible for this/these critical modifications. We
have made use of bioinformatics to identify candidate C. elegans serine/threonine kinases, the
activities of which, we have eliminated using RNA interference. By screening for intestinal
hyperplasia in a elt-2::gfp background, we hope to identify all the kinases that are involved in
triggering this event. Because the cdc-25.1 gf mutant causes hyperplasia exclusively in the gut,
all candidate kinases that cause a supernumerary round of intestinal cell division will be
subjected to a secondary screen in transgenic worms expressing GFP in an alternative lineage
(MS), where the cdc-25.1 gf mutant has no obvious effect on proliferation. This screen will
allow us to narrow down candidates by eliminating kinases involved in other developmental
processes (i.e. cell fate specification). Our screen has identified numerous kinases that caused
embryonic lethality and abnormalities. Further characterization of their developmental roles
and/or ability to directly phosphorylate CDC25.1 will allow us to have better insight on C.
elegans embryonic development.
30
INTRODUCTION
Developmental pathways and cell cycle machinery have to be under very strict control to allow
the proper growth and differentiation of cells and organs. This control occurs at two specific
time points, the G1/S and G2/M phase transition. Crucial cell cycle regulators alter these
transitions by modifying the activity of cyclin/CDK complexes [8, 32, 131]. The modulation of
these cyclin/CDK complexes take place on various levels such as subcellular relocalization,
ubiquitin-mediated degradation, activating or inhibitory phosphorylations and/or controlling
expression of cyclins, CDKs and cyclin-dependent kinase inhibitors (CKIs). All of these factors
cooperate to guarantee that each cell cycle occurs in a faithful and reproducible manner [132136].
Two major ubiquitin E3 ligases, the anaphase promoting complex/cyclosome (APC/C) and the
Skp1/cullin/F-box (SCF) E3 ligase manage the degradation of the major cell cycle regulators to
elicit the irreversible succession into the each cell cycle phase under normal conditions [64, 72,
137]. The same E3 ubiquitin ligases are involved in checkpoint pathways when DNA damage or
incomplete replication happens and avoids genomic instability by arresting the cell cycle phase
transition to allow the cell to repair the problem [44, 67]. The positive cell cycle regulator
CDC25 dual specificity phosphatase has been shown to activate both S and M phase CDKs by
removing phosphates from cyclin/CDK complexes [124-125]. Many higher organisms have
multiple CDC25 paralogs with redundant, but different phase and tissue specific roles. In
humans, CDC25A is required for the activation of cyclinE(A)/CDK2 for the initiation of S
phase, whereas in the transition into M phase requires assistance from CDC25B and CDC25C
[42]. At the end of M phase the degradation of the CDC25 paralogs occurs via the ubiquitinmediated pathway and is performed by the APC/CCDH1 (activator = Cdc20 homologue) via
recognition of a KEN box motif. During the S/G2 transition, CDC25A degradation is performed
by the SCFβ-TrCP (F-box SCF β-transducin repeat-containing protein) E3 ubiquitin ligase via
recognition of a phosphorylated β-TrCP-like destruction box motif (DSGX4S) [50-51, 85].
After DNA damage induced during S and G2 phase, the degradation of CDC25A by SCFβ-TrCP
appears to be increased by the ATM/CHK2-ATR/CHK1-dependent pathway [53, 138].
31
There are four CDC-25 paralogs (CDC-25.1/2/3/4) found in C. elegans of which cdc-25.1 has
the highest homology with the cdc25A gene of human [120]. Germline proliferation is also
maintained by CDC-25.1 in the adult hermaphrodite gonad [127]. Oocytes and early embryos
have maternal cdc-25.1 gene products, that are present up to the 28-cell stage, to ensure that
meiosis and early mitotic division are completed [127]. CDC25A is deemed to act as a protooncogene in human because of its function as a positive cell cycle regulator and overexpression
of CDC-25.1 in C. elegans results in premature S phase initiation [128]. Embryonic hyperplasia
of the intestinal (E) lineage has been described by our laboratory and others due to a reported
gain-of-function (gf) mutation [128-129]. This mutation stabilizes CDC-25.1 and allows it to
persist in all blastomeres past the 100-cell stage.
The hyperplasia caused by the gf cdc-
25.1(rr31) creates an unscheduled supernumerary division in the intestinal cells after the E8stage.
The resulting cdc-25.1 (gf) mutation was found to affect β-TrCP-like DSGX4S
phosphodegron motif in the N-terminus of the phosphatase that was key for the recognition of
the SCF E3 ligase F-box protein LIN-23/β-TrCP for the degradation of CDC-25.1 during a
timely window in embryogenesis (figure 2) [119]. Mammalian CDC25A studies have also
shown that the SCFβ-TrCP E3 ligase is responsible for ubiquitin-mediated degradation of CDC25A
during each phase of the cell cycle after DNA damage [50, 85]. Nevertheless, there have been
numerous kinases, such as CHK1, CHK2, GSK3, PLK3, that are known to phosphorylate
residues of CDC25A, therefore, allowing the destabilization of CDC25A by the SCFβ-TrCP E3
ligase.
What remains to be discovered is the identity of the kinase responsible for the
phosphorylation of the serines within the phosphodegron motif that permits the F-box β-TrCP to
bind and subsequently polyubiquitinate CDC25A for degradation [43, 139].
In addition, mutations in specific cell cycle regulators bring about analogous tissue- or organspecific tumourigenesis as seen in the intestinal-specific hyperplasia in C. elegans and are
observed in multiple types of human cancers. The identity of the candidate kinase, that regulates
the levels of CDC-25.1 phosphatase during embryonic cell cycle progression and allows for the
possible mediation of cell cycle arrest after DNA damage-induced checkpoint activation, is still
not known in C. elegans, or in any other organism.
To this end we will use a reverse genetic approach that will allow us to identify the kinase that
carries out the priming phosphorylation that targets CDC-25.1 for degradation. Like the cdc32
25.1(gf) strain, its compromise should also give rise to supernumerary cell division in the
intestine due to inappropriate elimination of CDC-25.1.
33
MATERIALS AND METHODS
Strains and culture
All C. elegans strains were maintained at 15°C and grown according to standard procedures
(Brenner, 1974), unless otherwise stated. N2 Bristol was used as the wild type strain. The
following alleles and transgenes were used. cdc-25.1(rr31)I; rrIs01[elt-2::gfp; unc-119+]X;
rrEx128/129/130[end-3::gfp::cdc-25.1(wt);
25.1(rr31);unc-119+];
unc-119+];
rrEx131/132/133[end-3::gfp::cdc-
rrEx138/139/140[end-3::gfp::cdc-25.1(rr31);
elt-2::gfp]
MR156
rrIs01[elt-2::gfp; unc-119(+)]X. MR142 cdc-25.1(rr31)I; rrIs01(X). MR931 rde-1(ne219)V;
rrIs01(X); rrEx235[end-3::rde-1; elt-2::rde-1; inx-6::gfp].
DNA constructs
pMR208 3’UTR-nekl-3, pMR209 3’UTR-nekl-1, pMR210 3’UTR-nekl-2, and pMR211 3’UTRnekl-4 were generated by inserting the 163bp, 103bp, 73bp or 194bp XbaI/XhoI fragments into
L4440 respectively. pMR207 2xFLAG::nekl-3 was generated by cloning the 909bp BamHI/XbaI
NEKL-3 cDNA generated by RT-PCR between the BamHI/XbaI site of pcDNA3::2xFLAG
(donated by Arnim Pause’s Lab). pMR212 gst::cdc-25.1(wt) and pMR213 gst::cdc-25.1(rr31)
was generated by cloning the first 180 bp of cdc-25.1(wt) and cdc-25.1(rr31) XhoI/NotI into
pGEX-6P-3, respectively.
RNA interference
nekl-3(RNAi) was performed by injecting 1µg/µl nekl-3 dsRNA into young adults (Fire et al.,
1998). Embryos were dissected from 36 and 48h post-injection hermaphrodites and mounted for
microscopic observation.
Feeding RNA interference experiments were done according to
standard procedures (Kamath et al., 2003). Hypomorphic RNAi was achieved by shortening the
induction time during which animals were fed on dsRNA producing bacteria.
Microscopy and image processing
Light microscopy, image analysis and processing were performed essentially as previously
described (Kostic and Roy, 2002).
34
RESULTS
In order to study the developmental regulation of the cell cycle, the C. elegans intestine
exemplifies an excellent model for many purposes. The intestine arises from a single progenitor
“E” blastomere at the 7 cell stage of embryogenesis. The cells of the intestinal E lineage will
then divide mitotically until the intestine processes 20 cells at the end of embryogenesis where
the L1 larvae hatch with a functional digestive tract [92]. The observation and lineaging of the E
daughters, as well as any alteration of the patterning of the intestinal cell divisions can be readily
observed with the use of many embryonic intestinal specific GFP markers. Intestinal specific
markers such as, the elt-2::gfp nuclear marker, can mark intestinal cells from the E16 cells stage
to adulthood [140]. We and many others have performed genetic screens and characterized
mutations using these elt-2::gfp animals to study the role of developmental cell cycle regulators
in many embryonic and postembryonic intestinal cell division patterns [104, 128-129, 141-143].
Genetic screen for intestinal hyperplasia
To determine which kinase(s) is/are responsible for the regulation of cellular division in the
intestine during embryogenesis, more specifically the kinases that target CDC-25.1 for
degradation, we performed a complete genomic screen utilizing only serine/threonine and
tyrosine kinases found in the Ahringer feeding RNAi library, which contains 16 757 bacterial
clones that are designed to produce dsRNA to target specific C. elegans genes[97]. C. elegans
kinases have close homologs to 81% (419/516) of mammalian kinases and share 153 subfamilies
[144]. The kinase library (kinome) was generated bioinformatically, with the use of wormMart
(www.wormbase.com), by identifying all motifs resembling serine/threonine and tyrosine
kinases in the C. elegans genome. A 96-well plate kinome was then created to facilitate
manipulation for the reverse genetic kinases screen. This kinome array, which comprises 349
individual clone, was performed by feeding dsRNA to L4 larvae, which express the elt-2::gfp
marker, in order to observe an RNAi effect in their offspring. Any bacterial clone that shows
signs of hyperplasia in the L1 hatchings have been rescreened along with all clones that caused
sterility and embryonic lethality to validate their effect. From this initial screen of 349 predicted
serine/threonine kinases, we observed that the RNAi-mediated downregulation of many kinases
produced sterility, embryonic lethality and intestinal cell cycle defects. These defects are
consistent with the candidate kinases partaking in vital roles during early developmental cues
35
such as embryogenesis. These observations also indicated that the alleged kinase, which
phosphorylates CDC-25.1 β-TrCP-like DSG phosphodegron, that plays a role in causing
intestinal specific hyperplasia could be missed using standard RNAi techniques due to their
overall loss-of-function-associated embryonic lethality.
Functional classification of kinases that cause embryonic lethality.
The kinomic screen, resulted in 57/349 kinases that causes embryonic lethal eggs. These kinases
were rescreened in the elt-2::gfp and myo-2::gfp background in to assure reproducibility of
results of the observed phenotype. The lethality observed, due to specific RNAi clones, were
classified as embryos arrested with less than 20 intestinal cells; embryos arrested with wild-type
number of cells and embryos arrested with a random disorganized intestinal cell pattern (table 1).
The kinases were identified and classified according to their GO terms found through WormBase
(www.wormbase.org). The 57 kinases can be classified into 6 groups that include cell cycle,
neural development, embryogenesis/viability, apoptosis, H. Sapiens cancer related and a large
group of candidate kinase did not have a description or fall into any of the classes (figure 2, table
2).
Hypomorphic RNA-interference
A very useful approach to rescue developmental defects is altering the depletion levels of the
gene products using hypomorphic RNAi [108]. This method can allow the embryos laid by the
transferred adult, to eventually bypass the threshold of embryonic defects by permitting basal
levels of the targeted gene product. Since, these 57 candidates caused embryonic lethality or
developmental arrest, the genes were rescreened where the induction time of the young
hermaphrodites’ exposure to the RNAi was reduced to allow the developmental progression
through to the L1 stage so that hyperplasia of the intestine could be scored. The induction time
spend feeding on the RNAi bacteria was changed to 24, 48, and 72 hours. This approach was
successfully used to rescue multiple lethal RNAi effects of target kinases in a dosage-sensitive
manner during development. However, varying the induction time allowed for the rescue of the
severity of the RNAi effect on the number of arrested embryos but this approach did not permit
us to monitor any hyperplasia in the L1 intestine. The longer the adult hermaphrodites were
exposed to the RNAi bacteria the stronger the effect of the dsRNA induced lethality was
36
observed. Even though this method allowed for manipulation of the levels of maternally loaded
genes it was unsuccessful in creating a hypomorph that broke the threshold level required for
proper embryogenesis and resulted in hyperplasia.
Tissue-specific RNA-interference
Previous studies in our laboratory have showed that the cdc-25.1(rr31) gain of function allele,
with the mutated phosphodegron, only caused specific over proliferation of the “E” intestinal
lineage. This was due to the abnormally high levels of CDC-25.1(rr31) protein in the E lineage
during embryogenesis because this mutant protein was able to “escape” ubiquitin mediated
degradation [119]. In addition, the putative kinase responsible for phosphorylating this motif
seems to be required for additional mandatory role during embryogenesis due the resulting
embryonic lethality observed by RNAi. Since, we could not get L1s to hatch with hyperplasia
from a regular or hypomorphic RNAi approach; we adopted a new strategy that would enable us
to bypass this lethality. This strategy allowed us to detect putative intestinal-specific hyperplasia
caused by RNAi of the individual kinase, with emphasis on the regulation of CDC-25.1 during
embryonic development. This tactic involves using a modified rde-1 mutant strain, created by a
past lab member, which rescued rde-1 in the intestine so that the resulting worms would have
intestinal specific RNAi sensitivity. The argonaut-like protein RDE-1 recognizes siRNA and is
involved in the RNA-induced silencing complex (RISC) mediated degradation of specific
mRNA [94, 145]. Therefore, a mutation in rde-1 disrupts the RNAi pathway and makes the
worms insensitive to dsRNA exposure [146]. Worms carrying this mutation along with the elt2::gfp marker were transformed with rde-1 driven by intestinal specific promoters to restore the
RNAi pathway in the intestine.
This resulted in the creation of the
MR931 strain [rde-
1(ne219)V; rrIs01(X); rrEx235[end-3::rde-1; elt-2::rde-1; inx-6::gfp] where only the RNAi
pathway is exclusively reinstated in the intestine, while the rde-1(ne219) mutation renders the
surrounding tissues resistant to RNAi.
The MR931 strain with intestine-specific RNAi sensitivity was screened by feeding RNAi clones
of the 57 kinases that caused embryonic lethality. This screen was performed following the same
protocol as the original screen for hyperplasia performed with the MR156 strain (mentioned
above). This method was valuable in rescuing all the embryonic arrested eggs caused by specific
37
kinase knockdown from the original screen and allowed proper development and hatching of L1
larvae. This meant that all defects observed caused by RNAi of the kinases would only occur
specifically in the intestine. Interestingly, this approach did not allow for the observation of
hyperplasia in the young L1 hatchling intestine caused by the RNAi of our highly sought after
candidate kinase that is required for the phosphorylation of this destruction box on CDC-25.1
that mediates its degradation. This may possibly indicate that there might be more than one
kinase that is responsible for this critical CDC-25.1 phosphorylation event during intestinal
development. Thus, the loss of function due to RNAi knockdown of one kinase may, in fact,
compensate by the overlapping functional role of another kinase.
However, this genetic screen did reveal many essential developmental roles, where individual
RNAi of specific kinases caused embryonic defects or lethality. Further attention to these genes
will allow us to better understand some uncharacterized functions that these kinases play in
embryogenesis and maintaining proper development.
Classification of embryonic lethal kinases
Kinases with known roles in innate immunity
15 years ago, the Toll pathway (toll, pelle, cactus, and dtrf1) was first discovered to be involved
in Drosophila melanogaster dorsal-ventral patterning during embryogenesis [147]. Toll’s role as
a morphogen in dorsal-ventral patterning has been well established in D. melanogaster but
completely overlooked in C. elegans [148]. In recent years, much of the attention has been
focused on this signaling pathway in adult organisms because of its role in the primitive immune
response.
The C. elegans Toll pathway homologs are tol-1, pik-1, ikb-1, and trf-1 have been
well characterized in the worms defense against foreign pathogens [149]. The tol-1(nr2013)
receptor null mutant has been reported embryonic lethal by other C. elegans groups displaying
that it also has a role in early development that evolved before its role in immunity [149]. tol-1
plays homologous roles to Toll in Drosophila during the immune response, therefore, one could
suggest that TOL-1 function in C. elegans during development could be involved in a
homologous morphogen gradient implicated in dorsal ventral axis patterning by triggering a
kinase cascade. Further confirmation was similarly observed in downstream kinase candidates
such as pik-1(RNAi) where milder degree of embryonic lethality was observed. This could
38
possibly be due to the fact that TOL-1 has multiple parallel kinase effectors and PIK-1 is
involved in one of the many downstream intracellular signaling cascades.
Another key pathway in the C. elegans immune response against pathogens is the NSY-1/SEK1/PMK-1/p38 MAPK cascade (figure 4).
Interestingly, this same pathway is involved in
neuronal cell fate carried out by UNC-43 which is the upstream kinase that activates this cascade
[150-151]. RNAi of unc-43, nsy-1 and have caused a small ratio of embryonic arrest when
individually knockdown.
The C. elegans innate immune pathway is accepted to be homologous to the D. melanogaster
and H. sapiens pathway which utilizes TGF-β-activated kinase (TAK1) which signals through
downstream effectors such as the same p38 MAPKs and IkB kinases [150, 152]. Interestingly,
mom-4 is the C. elegans homologue of D. melanogaster tak1 and the MOM-4 pathway acts in
parallel to the Wnt signalling pathway and is genetically known to downregulate POP-1 activity
in specifying anterior-posterior cell fate partitioning [153]. Since, the worm and human immune
responses are genetically conserved, it could explain why some RNAi of these kinases involved
in the innate immune response cause embryonic lethality possibly in some unknown interaction
with anterior-posterior cell fate specificity during the early cellular divisions.
Kinases required for proper morphogenesis during embryogenesis
Appropriate embryonic development relies heavily on the correct formation and patterning of all
various tissues and organs [154]. During development, cells must undergo different size changes
to build tubular organs, extend to form sheets or organize themselves into several precise
patterns. Moreover, dynamic modification of the cell’s shape and location depends on the
organization of the cytoskeleton, which is modelled by the contractions of the myosin light
chains (MLC). The MLC activity is, in turn, regulated by the RHO family of Ras-like GTPases
and their downstream effectors; the RHO-binding kinases (ROCK) [155]. An example of this in
C. elegans occurs when bean-shaped embryos undergo elongation and become worm shapedlarvae during embryonic morphogenesis. At this stage, LEThal-502 (ROCK), the Rho-binding
kinase is involved in phosphorylating the MLC and causing this contractile elongation of
epidermal cells while phosphatases such as MEL-11 antagonize LET-502 function [156].
Disruption in genes required for morphogenesis such as LET-502 block embryonic elongation
39
and cause embryonic lethality. This lethality was observed in this RNAi screen, where the eggs
would arrest in the comma stage due knockdown of LET-502.
In addition Myosin Light Chain Kinase also has the ability to phosphorylate the smooth muscle
MLC to induce actin-myosin contractions to organize cellular shape [157-158]. MRCK-1 kinase
in C. elegans functions downstream of unc-73 (Rho/Rac GEK, [159]) and pak-1 (p21activated
kinase) act in a parallel pathway to let-502/mel-11 in MLC contraction [160]. In addition, we
observed low penetrance of embryonic lethality due to mrck-1 and pak-1 RNAi phenotype,
consistent with the predicted redundant roles of PAK-1, MRCK-1 and LET-502 in coordinating
the contractility of MLC [161]. Therefore, PAK-1, MRCK-1 and LET-502 could have spaciotemporal regulation of the actin/myosin chains during embryonic elongation. An example of this
is during early cortex contractility during establishment and maintenance of polarization at the
one cell stage. Where RHO-1 acts through LET-502 to establish non-muscle myosin (NMY)
polarization and CDC-42 acts through MRCK-1 for maintaining the NMY polarization and the
cellular cortex during the one cell stage [162].
Interestingly, studies in Xenopus and mammals have shown that Rac and Rho mediate the insulin
receptor pathway during development also impinges on same cellular elongation pathway as let502/mel-11 [163-165]. daf-2, the C. elegans insulin receptor homolog, has been very well
characterized as the mediator of dauer and longevity [166]. Daf-2 mutants strongly enhanced the
mel-11 mutants by increasing larval lethality 50-fold at 25oC and 30-fold at 15oC. To further
confirm that daf-2 was involved in the elongation pathway downstream daf-2 effectors such as
age-1 and daf-16 also show genetic interaction with mel-11 [163]. A summary of key players in
the embryonic elongation pathway can be found in (figure 5) [156].
Our kinomic screen revealed many of these serine/threonine kinases that are involved in
actin/myosin contractility that cause cellular expansion and elongation during embryonic
morphogenesis. Notable mentions are genes described above, such as let-502, where a strong
RNAi lethality can be observed and most of the eggs observed seem to have arrested during or
before the comma stage of development consistent with its role in cellular elongation. The
intestinal cell number remained wild-type and the elt-2::gfp cells stayed grouped together
because development was halted before the intestinal organ could be properly formed. mrck-1
had a lower RNAi penetrance, the phenotype observed ranged from cell arresting early in
40
development where there was not proper differentiation of the intestine which resulted in random
intestinal cell location assortment. This detected early arrest could be due to the loss of tissue
polarity linked to severe downregulation by RNAi of the myotonic dystrophy CDC42-binding
related-kinase, mrck-1 [162]. A small percentage (<1%) of larvae that escaped developmental
arrest had severe motility defects and died shortly after hatching. This could be because MRCK1 is also involved in early elongation events, along with other unknown developmental function
and proper cellular connections could have been distorted during embryogenesis.
Kinases involved in spindle-assembly and checkpoint signaling
Polo-like kinases are critical regulators of both cell cycle progression and to the DNA damage
response and there are three PLK members in C. elegans [167]. PLK-1 is a positive regulator of
mitotic entry involved in numerous roles which consist of chromosome segregation, cytokines,
early embryonic polarity, centrosome maturation, and kinetochore–spindle attachment [168169].
PLK-1 levels are highly regulated to coordinate many mitotic events, therefore as
expected, plk-1 RNAi eggs were non-viable due these crucial roles mentioned above.
Interestingly, PLK-2 is believed to have similar functions to PLK-1 and in also found to be
highly expressed in G1, however, plk-2 RNAi did not cause a large ratio of embryonic arrest
[170].
Aurora kinases are involved in several biological processes and primarily participate in
regulating mitotic events. In C. elegans, AIR-1 (Aurora A kinase) is known to be involved in
regulating centrosome separation and spindle assembly and entry into mitosis. AIR-2 (Aurora B
kinase) is a member of the chromosomal passenger complex and functions in chromosome–
microtubule interactions (chromosome segregation), sister chromatid cohesion, the spindleassembly checkpoint and cytokinesis [171]. RNAi of such important mitotic regulators as the
Aurora kinase caused embryonic lethality at various stages of development due to numerous
mitotic defects.
Another notable group of kinase that caused embryonic lethality are the checkpoint kinases.
Proper control of mitosis requires many checkpoints to assure the cellular integrity of various
events. CHK-1 and CHK-2 are activated by ATM and ATR downstream of DNA damage, and
halt the mitotic progression so that the cell can repair the defects [172]. chk-1 and chk-2 along
41
with Y52D5.2 and Y43H10A.6 (related to chk) RNAi cause significant embryonic lethality as
expected. BUB-1 (budding uninhibited by benzimidazoles 1) is also required in the spindleassembly checkpoint because mitotic exit is inhibited pending the proper connection of all the
kinetochores to the spindles, which explains its lethal phenotype [173].
DISCUSSION
An RNAi kinase screen that included 83% of the C. elegans kinome (349/419) identified 57
kinases that caused embryonic lethality and could potentially target CDC-25.1 upon further
analysis. As expected a large portion of these kinases that caused embryonic arrest were key
players directly involved in cell cycle regulation and/or polarity. Many genes that govern cell
elongation and migration have caused significant developmental arrest. Interestingly, many
kinases involved in the innate immune system also have secondary roles in coordinating proper
development of the early embryo. Several of these genes involved in the different pathways
caused embryonic lethality at various stages with some unknown reasons and/or phenotypes.
Studying these genes using various specific GFP markers will shed some light on the resulting
RNAi lethal phenotype of these kinases to discover further molecular function.
This screen allowed us to identify many kinases that had crucial roles in proper development of
viable L1 hatchling; however we did not find any overwhelming evidence that knockdown of
any of these kinases causes intestinal hyperplasia in the larvae by increasing CDC-25.1 stability
by the inability to phosphorylate its critical DSG motif. Unfortunately, the effectiveness of each
individual clone of the Ahringer RNAi library has not been tested which could account for the
inability to observe supernumerary cell divisions in the intestine. Another possible explanation
could be that our key candidate kinase possesses other secondary and tertiary functions in
addition to phosphorylating the CDC-25.1 for destruction. This would mean that the RNAi of
the candidate kinase could have caused early embryonic arrest eggs due to its essential role in
early cellular division or differentiation.
Various hypomorphic approaches, by varying the
induction time of each hermaphrodite to the each RNAi, rescued many of the non-viable eggs but
did not lead to the discovery of any hyperplasia. Moreover, there is a possibility that there could
be a threshold level required for bypassing early lethality and thereafter cause hyperplasia of the
intestine.
42
The MR142 cdc-25.1(rr31) gain-of-function mutant strain caused hyperplasia by disrupting the
ability of the essential kinase(s) required for proper regulation of CDC-25.1 levels. The feeding
RNAi approach knockdown all maternally loaded genes but the initiation of the transcription of
the zygotic genes could compensate for the lack of the maternally loaded kinase in the intestine
during the critical E8 stage of development.
Finally, we could not rule out that there is a possibility that there are redundantly acting protein
kinases that regulate the activity and levels of CDC-25.1 to properly coordinate cell cycle
progression during various cell cycle events. CDC-25.1 is an important cell cycle regulator and
it would not be surprising if numerous cellular pathways act to regulate its levels by
phosphorylating its destruction motif. If there are in fact multiple kinases involved in targeting
CDC-25.1, we would have missed them in the screen because redundancy would have caused no
observed hyperplasia phenotype.
Another possibility could be that the candidate kinase(s)
involved has secondary functions that cause embryonic arrest. These candidates could be
identified by adjusting the screening strategy to select for potentially essential genes.
Alternatively, we cannot rule out that the RNAi screen only contained 83% of the C. elegans
kinome (349/419) and we would have to further interrogate the remaining kinases for their role
in mediating cell cycle regulation in the embryonic intestine.
43
Figure 2: CDC-25.1 and its critical destruction motif
A


Β-TrCP consensus motif
DSGXXXXS
cdc25.1(wt)
DGSSRDSGVSMTSCSDKS
cdc25.1(rr31)
DGSSRDSDVSMTSCSDKS
cdc25.1(ij 48)
DGSSRDFGVSMTSCSDKS
B
C
Wild type
Cdc25-1(rr31)
A: DSGX4S β-TrCP amino acid recognition motif for proper CDC-25.1 degradation.
B. Diagram showing the phosphorylated recognition motif for subsequent β-TrCP binding.
C. Microscopy of the intestine with elt-2::gfp transgene
modified from Hebeisen, M. et al. 2008
44
Table 1: Kinase Screen For Intestinal Hyperplasia Phenotype In elt-2::gfp and myo-2::gfp
Background
Identification
elt-2::gfp screen
Gene
Name
Cosmid
spe-8
F53G12.6
I-1C01
cdk-2
Riok1
let502
bub-1
Chrom.
HGMP
K03E5.3
I-1D09
ZC123.4c
I-1I09
M01B12.5a
I-M12
C10H11.9
I-2I07
W03G9.5
I-2O17
ZC581.7
I-3A20
H37N21.1
I-3D03
R06C7.8
I-3H11
T21G5.1
I-3I08
F52B5.2
I-4G11
wts-1
T20F10.1
I-5K13
cdk-9
H25P06.2a
Y18D10A.
5
C17F4.6
II-3G05
F59A6.1
II-4M01
F59A6.4
II-4M07
ire-1
C41C4.4
II-6A03
gcy-5
ZK970.6
II-7A18
prk-2
kin19
F45H7.4
III-1P19
c03c10.1
III-2C13
daf-4
C05D2.1a
III-2P04
R151.4a
III-3H20
gsk-3
gcy19
nsy-1
arrested
less than
20cells
arrested
wild type
20cells
myo-2::gfp
randomly
unorganized
cell pattern
early
arrest
randomly
unorganized
pattern
intestinal morphology
messed
+
+
+
+
+
+
arrested eggs are larger
the wt
+
+
+
+
+
+
abnormal L1 motility
horseshoe shaped
arrested eggs
no myo-2::gfp in
arrested eggs
+
+
Comments
+
+
+
+
no elt-2::gfp in arrested
eggs
+
+
+
+
+
gfp scattered all over
I-5N09
+
+
gfp scattered all over
I-6D13
+
+
+
+
F31E3.2a
III-3N09
prk-1
kin18
C06E8.3a
III-3N23
T17E9.1a
III-3O13
plk-1
C14B9.4a
III-4E08
+
+
+
+
arrested eggs are larger
the wt
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
45
+
+
+
+
Identification
elt-2::gfp screen
arrested
randomly
wild
unorganized
type
cell pattern
20cells
myo-2::gfp
Cosmid
D2045.7
III-5J05
lit-1
W06F12.1a
III-6F20
+
+
daf-2
Y55D5A.5a
III-7G12
+
+
tpa-1
B0545.1a
IV-1C03
+
+
K11H12.9
IV-1C04
+
kgb-1
T07A9.3
IV-1I17
+
kgb-2
ZC416.4
IV-1L22
+
F36H12.9
IV-2B14
+
ZK354.2a
IV-2D08
+
Y52D5A.2
IV-2H10
+
R13H9.6
IV-2L05
+
+
+
+
unc82
unc43
kin-4
unc22
pik-1
?chk2
HGMP
early
arrest
randomly
unorganized
pattern
Gene
Name
plk-3
Chrom.
arrested
less
than
20cells
+
Comments
+
arrested eggs are larger
the wt
+
gfp scattered all over
C55C3.4
IV-2P08
W03F8.2
IV-2P09
+
+
F55G1.8
IV-3B20
+
+
half stained gfp eggs
D2024.1
IV-3H21
+
+
morphology
B0496.3
IV-3P11
+
K11E8.1b
IV-5O24
C08F8.6
IV-5P04
C10C6.1
IV-6I17
ZK617.1a
IV-6K06
+
+
gfp spiral pattern
+
arrested after comma
stage
+
+
+
gfp scattered all over
+
gfp scattered all over
arrested eggs are larger
the wt
K09B11.1
IV-7K03
+
+
+
K09B11.5a
IV-7K11
+
+
+
Y38H8A.3
IV-7K21
+
+
Y43D4A.6
IV-8C03
+
+
C49C3.10
IV-8C22
+
+
T08D2.7
V-13E12
+
+
early
+
46
gfp spiral pattern
abnormal pharynx
morphology
arrested eggs are larger
the wt
arrested eggs are larger
the wt
Identification
Gene
Name
Cosmid
Chrom.
HGMP
Arrested
less
than
20cells
elt-2::gfp screen
arrested
randomly
wild
unorganized
type
cell pattern
20cells
myo-2::gfp
early
arrest
randomly
unorganized
pattern
+
+
Comments
Y39H10A.7
a
V-13G06
+
K08B12.5
V-4L05
+
+
air-1
K07C11.2
V-5J24
+
+
gfp scattered all over
pck-1
F57F5.5
V-8G03
+
+
R10D12.10
V-9O13
+
+
morphology
arrested eggs are larger
the wt
K02E10.7
X-1L14
+
+
C36B7.1
X-3P21
+
+
F08F1.1a
X-4E14
+
+
chk-1
mrck1
kin-9
+
+
+
gfp scattered all over
abnormal pharynx
morphology
Cosmids that caused embryogenic lethality in our RNAi screen were identified with a “+” in one or many of the
three column categorized as; arrested with less the 20cells, with 20cells and with a random unorganized display of
intestinal cells.
47
Figure 3: Classification of Genes Resulting in Embryonic Lethality
14%
unknown
22%
cancer related
12%
31%
apoptosis
embryogenesis/viability
19%
cell cycle
2%
neural development
Genes were classified as 31% cell cycle related, 19% embryonic viability related,
14% neural development related, 12% were closely related to oncogenic related
genes, 2% apoptotic related and 22% had unknown developmental functions.
48
Table 2: Individual Classification of Genes Resulting in Embryonic Lethality
Target gene
Locus
K03E5.3
R06C7.8
T17E9.1a
C14B9.4a
K11H12.9
Y52D5A.2
F55G1.8
T08D2.7
cdk-2
bub-1
kin-18
plk-1
K07C11.2
air-1
H25P06.2a
Y18D10A.5
R151.4
c03c10.1
F52B5.2
ZC123.4c
Y39H10A.7a
C10C6.1
Y43D4A.6
cdk-9
gsk-3
T07A9.3
Y55D5A.5a
F59A6.1
C10H11.9
M01B12.5a
K08B12.5
C05D2.1a
B0496.3
K11E8.1b
function
Target gene
Locus
W03G9.5
F59A6.4
F36H12.9
ZK354.2a
R13H9.6
D2024.1
C08F8.6
Y38H8A.3
plk-3
chk-2
neuron development
K09B11.1
pik-1
wts-1
chk-1
kin-4
?chk-2
T20F10.1
T21G5.1
C55C3.4
W03F8.2
C06E8.3a
K09B11.5a
H37N21.1
ZK970.6
W02B12.12a
kgb-1
daf-2
nsy-1
let-502
riok-1
mrck-1
daf-4
unc-82
unc-43
F45H7.4
B0523.1
B0545.1a
ZC416.4
T06C10.3
ZK617.1a
T03D8.5
K02E10.7
F08F1.1a
cell cycle
kin-19
Embryogenesis/
Vitality
49
function
apoptosis
unknown/ cancer
related
prk-1
gcy-5
prk-2
kin-31
tpa-1
kgb-2
unc-22
gcy-22
kin-9
unknown
Figure 4: Known Interactions In The Innate Immune Response In C. elegans.
Modified from Irazoqui et al., 2010
50
Figure 5: Kinases impinging on proper morphogenesis during embryogenesis
Piekny and Mains, 2003
51
52
CHAPTER 3:
NEKL FAMILY REVEALS A POSSIBLE ROLE IN CDC-25.1 REGULATION
53
INTRODUCTION
NIMA kinases are serine/threonine kinases that are found to control many processes such as
spindle organization, chromatin organization, mitotic entry, cytokinesis, and they are involved in
the DNA damage pathway [46, 174-175]. The identification of NIMA (Never-In-Mitosis kinase
A) emerged from a genetic screen performed in Aspergilus nidulans where the temperature
sensitive mutant never entered mitosis [176-177]. Since its initial discovery in filamentous fungi,
most of the characterization has been performed in unicellular organisms. Moreover, the most
important finding in Aspergilus nidulans was that NIMA is believed to be essential for mitotic
entry because of its role in localizing cdc2/cyclinB to the nucleus for initiation of mitosis [178].
Recent investigations have been pursued in the mammalian NIMA-related family composed of
eleven kinases, termed NEK1-NEK11, that share a high identity with the N-terminal catalytic
domain of NIMA.
This suggests that the non-conserved C-terminal of each individual
mammalian NEK kinase member has its own distinct role in the cell cycle [179].
Aside from NIMA-related kinases playing a predominant role in the progression of M phase,
there is compelling evidence that these mammalian NIMA-related kinases play a crucial role in
the G1/S transition [175, 180-181]. The most studied role of the NEK kinases during this
specific cell cycle period has been its function in response to genotoxic stress.
NEK1 is involved in all DNA damage response (DDR) pathways when exposed to various
genotoxic agents such as hydrogen peroxide (H2O2), ionizing radiation (IR), methylmethanesulphufonate (MMS), and cisplatin (CPT). Moreover, NEK1 is involved in the DNA
repair pathway such as double stranded breaks (DSB), non-homologous end joining (NHEJ),
homologous recombination (HR) [181]. In addition, NEK1 depletion has shown to block CHK1
and CHK2 phosphorylation and this results in the prevention of the checkpoint pathway that
causes both G1/S and G2/M arrest. It has also been suggested that NEK1 functions downstream
of ATM/ATR in between the DDR and DSB pathway [182]. Another NIMA-related kinase,
NEK11, is shown to be activated by the induction of DNA-damaging agents and contributes to
S-phase arrest. NEK11 kinase-dead (KD) U2OS cell lines reduced the sensitivity to this S-phase
arrest and these cells where more prone to lethality caused by DNA-damaging agents [175].
54
In addition, NEK cascades have been previously reported such as the NEK9 activation of
NEK6/NEK7 that is required for mitotic progression [183]. Interestingly, NEK2 protein levels
have been found to peak under normal conditions during the S and G2 phase of the cell cycle
where elevated NEK2 levels could be explained by its requirement for initiation of mitosis [184185]. This lead to the discovery of a novel NEK2A activation of NEK11 in G1/S arrested cells
resulting in NEK2A/NEK11 forming a complex that is specifically localized to the nucleus but
has no discovered in cellular relevance [180]. Aside from increased NEK2 levels functioning
during genotoxic stress, there has been no light shed explaining the reason for the accumulation
of NEK2 during unperturbed progression of S-phase.
Lastly, NEK11 has been recently reported to be responsible for the direct phosphorylation of
CDC25A during IR-induced G2/M checkpoint in mammals [46]. This study demonstrates that
CHK1 is required to activate NEK11, subsequently, once activated NEK11 can directly
phosphorylate the CDC25A β-TrCP DSGX4S recognition motif which is required for the timely
degradation of CDC25A. Also, the removal of NEK11 by siRNA inhibits the cells ability to
degrade CDC25A and the cell cycle can bypass the G2/M checkpoint following IR-induced
damage.
NIMA cell cycle functions have been studied in all model organisms except in C. elegans.
Interestingly, there are four NIMA-like kinases identified in C. elegans named NEver-in-mitosis
Kinase-Like (NEKL1-4) and have high homology in the NIMA catalytic N-terminal domain.
The conservation of this kinase family among organisms is compelling evidence of the
possibility that this same NIMA-like family in C. elegans may play a role in the cellular
regulation of CDC-25.1 during embryonic development. We expect that further investigation in
this specific C. elegans NIMA-like related kinase family will lead to discovery of the critical
kinase required to negatively regulate CDC-25.1 during embryogenesis.
55
MATERIAL AND METHODS
Strains and culture
All C. elegans strains were maintained at 15°C and grown according to standard procedures
(Brenner, 1974), unless otherwise stated. N2 Bristol was used as the wild-type strain. The
following alleles and transgenes were used. cdc-25.1(rr31)I; rrIs01[elt-2::gfp; unc-119+]X;
rrEx128/129/130[end-3::gfp::cdc-25.1(wt);
25.1(rr31);unc-119+];
unc-119+];
rrEx131/132/133[end-3::gfp::cdc-
rrEx138/139/140[end-3::gfp::cdc-25.1(rr31);
elt-2::gfp]
MR156
rrIs01[elt-2::gfp; unc-119(+)]X. MR142 cdc-25.1(rr31)I; rrIs01(X). MR931 rde-1(ne219)V;
rrIs01(X); rrEx235[end-3::rde-1; elt-2::rde-1; inx-6::gfp]. RB1455 Y39G10AR.3(ok1662)(I).
VC1733 nekl-2(gk839) I/hT2[bli-4(e937) let-?(q782) qIs48](I;III).
VC1755 +/szT1[lon-
2(e678)](I); F19H6.1(gk506)/szT1(X.) MR1617 Y39G10AR.3(ok1662)(I); rrIs01(X). MR1618
nekl-2(gk839)(I); rrIs01(X). MR1619 nekl-3(vc1155)(X); rrIs01(X).
DNA constructs
pMR208 3’UTR-nekl-3, pMR209 3’UTR-nekl-1, pMR210 3’UTR-nekl-2, and pMR211 3’UTRnekl-4 were generated by inserting the 163bp, 103bp, 73bp or 194bp XbaI/XhoI fragments into
L4440 respectively. pMR207 2xFLAG::nekl-3 was generated by cloning the 909bp BamHI/XbaI
NEKL-3 cDNA generated by RT-PCR between the BamHI/XbaI site of pcDNA3::2xFLAG
(donated by Arnim Pause’s Lab). pMR212 gst::cdc-25.1(wt) and pMR213 gst::cdc-25.1(rr31)
was generated by cloning the first 180 bp of cdc-25.1(wt) and cdc-25.1(rr31) XhoI/NotI into
pGEX-6P-3, respectively.
RNA interference
C. elegans strains were transformed as previously described [186]. nekl-3(RNAi) was performed
by injecting 1µg/µl nekl-3 dsRNA into young adults (Fire et al., 1998). Embryos were dissected
from 36 and 48h post-injection hermaphrodites and mounted for microscopic observation.
Feeding RNA interference experiments were done according to standard procedures (Kamath et
al., 2003).
Microscopy and image processing
Light microscopy, image analysis and processing were performed essentially as previously
described (Kostic and Roy, 2002).
56
Cell Culture, Transfections and Immunoprecipitation
For immunoprecipitation after transient transfection, HEK293T cells were transfected with
expression plasmid (total 2 μg of plasmid DNA/0.5 × 106 cells/60-mm dish) using FuGENE™ 6
reagents (Roche Applied Science) according to the instruction manual. After 15-44 h, cells were
harvested and suspended in Nek11 lysis buffer (20 mM Hepes-NaOH pH 7.5, 420 mM NaCl, 2
mM MgCl2, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2
μg/ml pepstatin A, 2 μg/ml heparin, 1 μM okadaic acid, 1 μM cyclosporin A) on ice for 10 min.
Soluble cell extracts were obtained by centrifugation (12,000 × g) at 4 °C for 10 min. HeLaS3
and U2OS cell extracts were also prepared as above. To prepare cytosolic and nuclear extracts,
HeLaS3 cells were primarily extracted with Nek2 lysis buffer (50 mM Hepes-NaOH pH 7.5, 100
mM NaCl, 5 mM KCl, 10 mM MgCl2, 5 mM MnCl2, 0.1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 2 μg/ml heparin, 1 μM
okadaic acid), and centrifuged (10,000 × g, at 4 °C for 10 min) to obtain the cytosolic extract.
The resulting cell pellets were re-extracted by Nek11 lysis buffer. FLAG-tagged proteins were
immunoprecipitated by anti-FLAG M2 agarose (Sigma), for 2-3 h at 4 °C on a rotating wheel.
57
RESULTS
The accumulated evidence from many mammalian studies has directed us to pursue further
investigation of the NEK-like family in C. elegans. NEK has been shown to play a crucial role
downstream of DNA damage and contributes to various cell cycle checkpoints.
Most
importantly, mammalian NEK11 has been to shown to phosphorylate the CDC25A β–TrCP
destruction motif during the G2/M checkpoint pathway induced by genotoxic stress. This
prompted us to study the developmental role of this NEKL family to verify if these kinases also
functioned to target CDC-25.1 in C. elegans. An RNAi approach was selected to determine if
downregulation of nekl expression levels would give rise to the expected intestinal hyperplasia in
L1 hatchlings.
nekl family specific RNAi rescreening for intestinal hyperplasia
To our discontent, the initial first screen did not identify any intestinal hyperplasia from the
RNAi of the C. elegans nekl genes. In order validate whether the NEKL family of kinases is
responsible for phosphorylating the CDC-25.1 phosphodegron, we first decided to pay more
attention to this family by rescreening the individual clones found in the Ahringer library. The
library contains three of the four nekl members (Y39G10AR.3; nekl-1, ZC581.1; nekl-2, and
F19H6.1; nekl-3) which L4 larvae, that express the elt-2::gfp marker, were feed separate IPTGinducible nekl bacterial clones on agar plates (as described in materials and methods). This time
around, the RNAi experiments (scored in an identical fashion as in Chapter 1) were performed
on a larger scale in the hopes to detect the RNAi induced hyperplasia. When exposing MR156
L4 larvae with the intestinal specific GFP marker with nekl-1(RNAi), nekl-2(RNAi) and nekl3(RNAi), the L1 hatching all appear to have a wild-type number of 20 intestinal cells, but the
nekl-3(RNAi) seem to have a small ratio of embryonic lethality. Thus, there was not enough
evidence to rule hyperplasia as the cause of the small percentage of arrested elt-2::gfp eggs. To
further investigate the grounds of these terminally arrested embryos caused by nekl-3(RNAi), we
fed the MR931 worms, with intestinal specific RNAi sensitivity, to the RNAi induced bacterial
lawn composed of nekl-1(RNAi), nekl-2(RNAi) and nekl-3(RNAi). This approach will allow this
small proportion of embryonic lethal nekl-3(RNAi) eggs to bypass this lethality and hatch as L1s
because only the “E” intestinal lineage is sensitive to RNAi. This strain could better enable us to
58
visibly count the possible hyperplasia seen in these specific MR931 young L1 hatchings. The
bacterial nekl clones showed no robust hyperplasia effect in the intestine of the MR931
(intestinal specific RNAi sensitivity) worms even if there was a small increase in the number of
intestinal cells in the nekl-3 RNAi fed worms (table 3). The feeding results by RNAi of the nekl
family kinases could not determine which specific kinase was the culprit required to cause the
same intestinal hyperplasia phenotype reported in the cdc-25.1(rr31) gain-of-function mutant
[128].
Due to the compelling evidence of the NEK family role in targeting CDC25A
phosphodegron in H. sapiens, a different approach was required to enable us to address the weak
effects of nekl-3(RNAi) in causing intestinal hyperplasia.
Interestingly, the Caenorhabditis
Genetics Center (CGC) reports that the nekl-2(gk839) and nekl-3(gk506) alleles are apparent
homozygous lethal deletions whereas the nekl-1(ok1662) strain is viable with a null nekl-1 copy.
Surprisingly, we were not observing any strong nekl-2 and nekl-3 RNAi lethality in the RNAi
feeding approach.
Moreover, there has been no reported confirmation of the integrity of the each clone in the
Ahringer library, therefore, we set forth a cloning strategy to specifically target each nekl gene’s
3’UTR because precise targeting of the 3’UTR by dsRNA is known to be an effective method to
specifically inhibit gene mRNA translation [187]. This method will give more robust RNAi
phenotypes in the hopes to reproduce observed lethality described by the CGC in the mutant
nekl-2(gk839) and nekl-3(gk506) worms. This approach of constructing our own vectors allowed
us to specifically target each individual gene by their unique 3’UTR sequences.
The
determination of the encoding 3’UTR of each nekl gene was completed using UTRome
(www.UTRome.org) and each individual 3’UTR were inserted into the L4440 vector (Fire Lab)
where the individual nekl 3’UTRs were cloned inside the MCS flanked by two T7 promoters
(figure 6). The 3’UTR nekl family dsRNAi producing clones were individually fed to the
MR156 and MR931 L4 Larvae. The nekl-1 3’UTR (RNAi) and nekl-2 3’UTR (RNAi) fed to the
MR156 adult worms carrying the elt::gfp transgene did not induce intestinal hyperplasia.
Interestingly, there was no embryonic lethality in feeding nekl-2 3’UTR(RNAi) to the L4 larvae
which did not coincide with the reported nekl-2(gk839) mutant lethality where the embryos
where the homozygotes are inviable. Moreover, this 3’UTR RNAi experimental approach did
not cause a hyperplasia phenotype in the eggs of MR156 worms fed with nekl-3 3’UTR(RNAi),
however, this approach did slightly increased the hyperplasia observed in the MR931 worms
59
(table 4). The full effectiveness of the nekl-3 3’UTR(RNAi) penetrance was not 100% and any
potential hyperplasia could not be confirmed in the dead MR156 eggs. Due to the lack of
severity of the expected non-viability resulting from the RNAi feeding experiments, we decided
to inject concentrated 3’UTR dsRNA of nekl gene family members into young adult gonads to
provoke a robust RNAi phenotype. This approach enabled us to further observe a slightly
increased ratio of terminally arrested eggs and a stronger hyperplasia phenotype in the intestinal
cells with the injection of nekl-3 3’UTR(RNAi) (table 5). We further investigated the phenotype
of the MR156 elt-2::gfp eggs and the potentially hyperplastic MR931 L1 hatchlings by high
resolution microscopy. This revealed that the non-viable eggs laid by the MR156 adults injected
with nekl-3 dsRNA 3’UTR arrest early in embryogenesis before the comma stage due to some
unknown early developmental hindrance. Supplementary analysis of these MR156 arrested eggs
disclosed no supernumerary cellular division found in the intestinal cells (figure 7).
The
downregulation of nekl-3 by injecting 3’UTR dsRNA in MR931 adults caused supernumerary
cellular division in the intestine. We quantified the intestinal cellular count up to 30 cells in
some L1 larvae as opposed to the 20 cells scored for the wild-type L1 hatchling. Interestingly,
the phenotype detected in these RNAi injections did not yield the full intestinal hyperplasia as
the 41 cells observed in the cdc-25.1(rr31) gain-of-function mutant (figure 8).
These results reveal that the downregulation of nekl-3 has the ability to cause an intermediate
intestinal hyperplasia in specific RNAi intestinal sensitive MR931 worms when compared to the
CDC-25.1(rr31) mutant. Furthermore, the slight embryonic arrest observed from nekl-3 RNAi
lead us to conclude that NEKL-3 may have additional roles in embryonic development as well as
the possibility of being responsible for the phosphorylation of the CDC-25.1 destruction box in
C. elegans.
Nekl mutants reveal an important development role
The severity of the nekl RNAi phenotypes did not seem to coincide with the 100% lethality of
the nekl-2(gk839) and nekl-3(gk506) null mutant phenotype. However, there is a possibility that
the RNAi approach was not effectively knocking down the full expression of the individual nekl
genes thus resulting in the observed small ratio of embryonic lethality. To properly address
whether the various nekl mutants affected the intestinal cellular divisions during embryogenesis,
we devised a strategy to quantify the number of intestinal cells in mutant strains carrying the nekl
60
deletions; RB1455 (nekl-1(ok1662)), VC1733 (nekl-2(gk839) I/hT2[bli-4(e937) let-?(q782)
qIs48](I;III)), VC1155 +/szT1[lon-2(e678)] I; F19H6.1(gk506)/szT1 X) (the mutant strain for
nekl-4 is not available). The VC1733 (nekl-2) and VC1155 (nekl-3) are balanced heterozygous
deletion alleles where the homozygous null alleles are lethal. In order to visualize and count the
intestinal cells of the mutant strains carrying nelk deletions, we created new strains by
successfully crossing our deletion mutants into the MR156 background which carries the
intestinal GFP marker (rrIs01[elt-2::gfp; unc-119(+)]X). From this genetic cross the following
new strains were successfully created: MR1617 (Y39G10AR.3(ok1662)(I); rrIs01(X)); MR1618
(nekl-2(gk839)(I); rrIs01(X)) and MR1619 (nekl-3(vc1155)(X); rrIs01(X)).
Observation of the early MR1617 (nekl-1(ok1662)) L1 hatchings revealed healthy and viable
larvae that displayed a wild type number of intestinal cells (20 cells). However, the MR1618
(nekl-2(gk839)) and MR1619 (nekl-3(vc1155)) strains were homozygous lethal when carrying
both copies of the deletion allele. The progeny from the heterozygous MR1618 hermaphrodite
that carried both null copies of the gk836 and the MR1619 hermaphrodite eggs that carried both
null copies of the vc1155 were respectively embryonic lethal, as expected from the CGC
description.
Microscopy of the MR1618 and MR1619 embryonic lethal eggs uncovered a
variable phenotype in the intestinal cell number in the arrested eggs or L1 larvae. The eggs
observed from the MR1618 strain seem to arrest early in development before the comma stage.
Moreover, there seemed to be no apparent over proliferation in the number of intestinal cells
found in the null MR1618 eggs. Interestingly, the MR1619 strain’s null eggs had an increased
number of intestinal cells compared to its MR1618 counterpart. We could count up to 31
intestinal cells marked with the elt-2::gfp reporter gene. Closer examination of the MR1619 null
embryos show an abundance of over proliferated cells near the start of the intestine (figure 8),
showing that a loss of nekl-3 causes intestinal hyperplasia but less severe hyperplasia then the
cdc25.1(rr31) mutant.
Due to these intriguing results, there is some genetic evidence that NEKL-3 is the kinase that
could be required to phosphorylate CDC-25.1. We further compared the sequence homology
between C. elegans NEKL-3 and mammalian NEK11 to determine if NEKL-3 could be
orthologous. Both proteins share 26% amino acid sequence identity with high homology in the
conserve NIMA-Like kinase domain (figure 9). It has been reported that suitable docking of E3
61
SCF ligase β‑TrCP which triggers polyubiquitylation and proteolysis of CDC25A requires
NEK11 activation by CHK1 phosphorylation downstream of DNA damage signals [46].
Interestingly, the CHK-1 recognition motif for activation has been previously characterized and
NEKL-3 shares a common CHK-1 activation site with NEK11 which gives us more evidence
that NEKL-3 might be involved in this critical step for CDC-25.1 degradation [188].
Verifying NEKL role in phosphorylating CDC-25.1
CDC25A is shown to be phosphorylated on S76 (CHK1, GSK3-β), S79 (CK1α), T80 (PLK3) and
S82 (NEK11) near the phosphodegron motif which trigger its timely degradation [46, 189].
Moreover, it has been established that NEK11 has been responsible for this critical
phosphorylation of S82 in H. Sapiens but this has not been confirmed in C. elegans even though
strong evidence points to NEKL-3 involvement in targeting CDC-25.1. Investigation into the
NEKL kinase family’s ability to phosphorylate the C. elegans CDC-25.1 phosphodegron will
have to be performed in vitro. Previous studies have shown that due to the conserved expression
mechanisms between C. elegans and H. sapiens it is possible to easily express active worm
kinases in HEK293T cells [190]. Full length nekl-3 cDNA was cloned into the mammalian
expression vector pcDNA3.1 with 3x FLAG tag at the N-terminal. Surprisingly, when the
HEK293T cells were transfected with pcDNA3::nekl-3, we observed that the transfection
significantly halted the growth of the HEK293T cells after 24 hours overexpression of NEKL-3.
This cell growth inhibition was also observed by an independent group when they over expressed
mammalian NEK11 in HEK293T (data not shown)[175]. Moreover, this strongly suggests
evidence that over expression of NEKL-3 can cause cell cycle arrest. It is not clear that indeed
this occurs through its effects on CDC25A. This may also support that NEKL-3 has some shared
homologous function with NEK11. To directly show that NEKL-3 can physically phosphorylate
the CDC-25.1 destruction box in vitro, the N-terminal of expression vector of GST::CDC-25.1
and the peptides containing the CDC25.1 DSG phosphodegron motif were obtained to test
NEKL-3 kinase activity. However, satisfactory data has not been produced at this time.
62
CONCLUSION
Various RNAi methods targeting the nekl family members appeared to help improve the
effectiveness of knocking down our target kinases and creating a phenotype observed in the
affected eggs. Our RNAi feeding method seemed to give the least robust hyperplasia phenotype,
whereas, the 3’UTR injection was a slightly more robust for nekl-3. This study suggests genetic
evidence for many crucial roles for nekl-2 and nekl-3 required for proper development during
embryogenesis. The loss of nekl-3 by RNAi or nekl-3(ok506) worms displayed a phenotype
similar to the intestinal hyperplasia characteristic of cdc-25.1(rr31)gf worms where the stability
of CDC25.1 is increased due to its ability to escape degradation via β-TrCP recognition, which
causes the extra cellular division in the intestine. The most robust hyperplasia phenotype was
observed in the MR1619 strain and the dsRNA injections of the 3’UTR of nekl-3. This approach
still could not reproduce the full 41 cell hyperplasia phenotype of the loss-of-function cdc25.1(rr31). Moreover, it is important to note that the dsRNA injections of the 3’UTR of nekl-3
in MR931 worms yielded a similar hyperplasia intestinal count to the MR1619 mutant which
was in the upper 20 intestinal cell range. This validates the effectiveness of this 3’UTR injection
strategy given that the supernumerary intestinal cells in the eggs of the homozygous MR1619
null mutants would have no nekl-3 expression.
More questions can be raised about the
possibility that there may be several kinases that have similar roles in triggering CDC-25.1
polyubiquination due to the partial hyperplasia, observed in the 3’UTR injections nekl-3
injections and the MR1619 strain, when compared to the loss-of-function cdc-25.1(rr31). More
RNAi experiments with combinations of target kinases will allow further investigation into this
hypothesis.
Furthermore, this study leads us to believe that NEKL-3 is possibly involved in the regulation of
CDC-25.1 by directly phosphorylating the critical serine residue(s) located within the DSGX4S
destruction box, however, further phosphorylation assay will confirm this hypothesis. Evidence
that overexpression of NEKL-3 in mammalian cells has the ability to possibly target CDC25A
suggests that NEK11 and NEKL-3 share homologous functions. Confirmation of NEKL-3
ability to phosphorylate the critical serine residues of the GST tagged N-terminal of CDC-25.1
and the peptide containing the phosphodegron will further verify if NEKL-3 is the kinase
responsible for regulating CDC-25.1 in C. elegans.
63
Future experiments designed to follow up on additional defects observed in nekl-2(gk839) and
nekl-3(gk506) arrested eggs will allow the identification of their essential role during
embryogenesis in regards to their function during the early cell cycles. In addition, there are
numerous mammalian studies that describe NIMA-Related kinases roles in the S and M phase
cell cycle phase progression and checkpoints, therefore, the further characterization of the nekl-2
and nekl-3 role, by the utilization of C. elegans genetics, will help to identify many different
pathways that impinge on the NEKL family.
64
Table 3: L1 Intestinal cell number of bacteria feed nek-like RNAi clones
Strain
MR156
MR931
RNAi
Intestinal cells
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
20 (n=20)
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
21.73± 1.28(n=15)
Each RNAi clones was grown from the original cosmids found in the Ahringer library and
fed to L4-adult hermaphrodites. The nekl RNAi clones seem to have little effect on the
intestinal cell count except for a very small increase in nekl-3 RNAi.
65
Table 4: L1 Intestinal cell number of bacteria feed nek-like 3’UTR RNAi clones
Strain
MR156
MR931
RNAi
Intestinal cells
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
20 (n=20)
nekl-4
20 (n=20)
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
23.5±1.7 (n=20)
nekl-4
20 (n=20)
Each RNAi 3’UTR clones was fed to L4-adult hermaphrodites. The nekl 3’UTR RNAi
clones seem to have little effect on the intestinal cell count except for slightly increasing the
MR931 hyperplasia when compared to the Ahringer RNAi clone.
66
Figure 6: L4440 expression vector and 3’UTR cloning
RED: Stop codon
Underlined: 3’UTR
3’UTR nekl-1
gtttccagaaagggagaatacgagtacaacatgtgttattttgtaaTTTTTTTTTGTAATTTGTCTATTGATTTAAATAAA
3’UTR nekl-1primers
XbaI 5’ actgTCTAGA GGAATGTCACGTATAACAAA
(rr1452)(tm49.5)
XhoI 3’ actgCTCGAG TTTATTTAAATCAATAGACAAATTA
(rr1453)(tm48.75)
3’UTR nekl-2
atcagagaacacaatcaagaagtcaagtgcattcaaagtattaaCTCCTGTATTAATTGTTTGTTTCTTTAAAATTAAAATTGAAGTTATTTTTTCACATAAAA
ACCAATCATTCG
3’ UTR nekl-2 primers
XbaI 5’ acgtTCTAGA CTCCTGTATTAATTGTTTGTTTC
(rr1454)(tm51.25)
XhoI 3’actgCTCGAG CGAATGATTGGTTTTTATGT
(rr1455)(tm51.25)
3’UTR nekl-3
cgccttccggggaccaatcaacaactccttcaacgcaattctaaAAAAAGCTATAACATTTCAATTTCAAACATTTTCTTTAAAACGTAGTGTTCTTGTATTTTC
AAAAGGTGGAAACATTCGTCAATGACCACGTGAATCCGTGATGTGCTAAATTTTACTCTCATACTGTCTCTTAATTCATGTAGATTATAAATGT
TTTATTTT
3’ UTR nekl-3 primers
XbaI 5’ aaaaTCTAGA aaaaagctataacatttcaattc
(rr1420) (Tm:55.59)
XhoI 3’ aaaaCTCGAG aaaataaaacatttataatctacatga
(rr1421)( Tm:55.72)
3’UTR nekl-4
cgtgcattgaatgtttgattgcagaaaatccagcagcgaagtgaCATATGCTAAATTGTATATACTGTTTTTTTGTATATTATTGATTGATTACTGTCATAATTT
GTTTTAGTGGTGGAAGCATATTTAATGAAAATAAGGGAAAATCCAAGATGTGGGCGGAGCCTATTAGCAATAGTATTTCAATAGTACATCAG
GTACCTTTCTTCATGCCTATCTATAAGTTTAAATAATACTT
3’ UTR nekl-3 primers
XbaI 5’ acgtTCTAGA CATATGCTAAATTGTATATA
(rr1456)(tm39)
XhoI 3’ acgtCTCGAG AAGTATTATTTAAACTTATAG
(rr1457)(tm37)
67
Table 5: Intestinal cell number of young adult injected nek-like 3’UTR dsRNA
Strain
MR156
MR931
RNAi
Intestinal cells
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
20 (n=20)
nekl-4
20 (n=20)
nekl-1
20 (n=20)
nekl-2
20 (n=20)
nekl-3
26±3.65 (n=16)
nekl-4
20 (n=20)
Each dsRNA was injected into young adult hermaphrodites. The nekl-3 3’UTR dsRNAi
seems to have affected the intestinal cell count of the specific intestinal RNAi sensitive
worms.
68
Figure 7: Microscopy of nekl-3 3’UTR injected worms
A
i)
white light
UV
ii)
Different embryonic lethal MR156 (elt-2::gfp) eggs of dsRNA nekl-3 3’UTR injected
young adult in white light and UV(GFP).
B
GFP imaging of MR931 injected with nekl-3 3’UTR dsRNA, solid green pharynx (myo-2::gfp) and individual
intestinal cells (elt-2::gfp). i) L1 hatching with 26 intestinal cells ii) hyperplasia seen in the developing egg.
Scale Bar = 10 microns
69
Table 6: Intestinal count of nekl mutants strain with integrated elt-2::gfp
Strain
Genotype
L1 Intestinal Cells
MR1617
ok1662; elt-2::gfp
20 (n=20)
MR1618
gk838; elt-2::gfp
20.38±2.05 (n=20)
MR1619
vc1155; elt-2::gfp
28.25±2.27 (n=20)
70
Figure 8: Various MR1619 embryonic lethal egg phenotypes
A-B: intestinal hyperplasia seen in MR1619 mutants
C: embryos that failed before entering comma stage with wt number of intestinal cells
D: embryos that failed at the 2 cell stage
71
Figure 9: NEKL-3 amino acid sequences alignment
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
MLKFQEAAKCVSGSTAISTYPKTLIARRYVLQQKLGSGSFGTVYLVSDKKAKRGEE-LKV 59
MPNIQECERMCHAG-PLSACTDTLIAKRYLLQQRLGKGSFGTVYLVIDKKAKDEQESLKV 59
MDKISNIYNFDDPP------PDKLSLELFIIEKKIGKGQFSEVFRA---QCTWVDLHVAL 51
* ::.: .
...* . :::::::*.*.*. *: .
:.. : : :
CHK-1____
LKEISVGELNPNETVQANLEAQLLSKLDHPAIVKFHASFVEQDNFCIITEYCEGRDLDDK 119
LKEIPVGELNPNETVQANVEAQLLSKLDHPAIVKFHASFLENESFCIITEYCEGRDLDFK 119
KKIQVFEMVDQKARQDCLKEIDLLKQLNHVNVIRYYASFIDNNQLNIVLELAEAGDMSRM 111
*
. :: :
:. * :**.:*:* :::::***::::.: *: * .*. *:.
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
IQEYKQAGKIFPENQIIEWFIQLLLGVDYMHERRILHRDLKSKNVFLKNN-LLKIGDFGV 178
VRECKKNEEKIAENQVVEWFIQLLLGVNYMHVRRILHRDLKAKNIFLKNN-LLKIGDFGV 178
IKHFKKGGRLIPEKTIWKYFVQLARALAHMHSKRIMHRDIKPANVFITGNGIVKLGDLGL 171
::. *: . :.*: : ::*:** .: :** :**:***:*. *:*:..* ::*:**:*:
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
SRLLMGSCDLATTLTGTPHYMSPEALKHQGYDTKSDIWSLACILYEMCCMNHAFAG--SN 236
SRLLMGSCDLATTFTGTPYYMSPEALKHQGYDSKSDIWSLGCILHEMCCLEHAFIG--YN 236
GRFFSSKTTAAHSLVGTPYYMSPERIQESGYNFKSDLWSTGCLLYEMAALQSPFYGDKMN 231
.*:: ..
* ::.***:***** ::..**: ***:** .*:*:**..:: .* *
*
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
FLSIVLKIVEGDTPSLP-ERYPKELNAIMESMLNKNPSLRPSAIEILKIPYLDEQLQNLM 295
FLSVVISIVEGETPSLP-DCYSSSLNLIMNRMLNKDPALRPSAGEILQDPFINEQLKQVK 295
NEK11_Hsapiens
NEK11_Xtropicalis
NEKL-3_Celegans
CRYSEMTLEDKNLDCQKEAAHIINAMQKRIHLQTLRALSEVQKMTPRERMRLRKLQAADE 355
WEVYGAEVKDKTTICKKEADQIRNVVQRKFHLQTLRELSEVQKMTPRERMRLRKLNAADE 355
----------------------------AEHMNNYFSPSGDQSTTPSTQF---------- 302
*::.
* *. ** ::
LYSLCKKIENCEYPPLPADIYSTQLRDLVSRCILPEASKRPETSEVLQV----------- 280
: *: .* : : *.** : *...*. ::. : :.: **.: *:*:
Protein sequence alignment of NEKL-3 with NEK11 from Homo Sapiens and Xenopus Tropicalis.
Residue
Colour
Property
AVFPMILW
DE
RK
STYHCNGQ
Others
RED
BLUE
MAGENTA
GREEN
Grey
Small (small+ hydrophobic (incl.aromatic -Y))
Acidic
Basic - H
Hydroxyl + sulfhydryl + amine + G
Unusual amino/imino acids etc
* indicate conserved residues.
: indicate semi-conserved residues.
indicates CHK phosphorylation site
72
GENERAL DISCUSSION
Proper development of an organism is regulated by precise control of pathways that are involved
in growth and differentiation of cells. Vital cell cycle mechanisms along with specific gene
expression ensure accurate determination of cellular fate during development of various tissues
and organs.
Improper control of growth and differentiation during these initial stages of
development can cause various detrimental outcomes. The overall focus of my research was to
identify and characterize upstream kinase(s) that target CDC-25.1 for degradation. Using C.
elegans as a genetic model with the use our kinome RNAi library, we expected to uncover many
novel kinases that play a role in maintaining proper embryo development in a multicellular
organism.
NEKL-3 involvement in CDC25-1 regulation
The study of the potential role of the NEKL family’ as the upstream kinases responsible for
triggering CDC-25.1 degradation has raised many new questions. The previously characterized
cdc-25.1(rr31)gf L1 larvae has been shown to hatch with a supernumerary number of intestinal
cells (41cells) [119].
A single mutation (G47D) in an amino acid sequences that represents a
highly conserved DSGX4S destruction motif in the N-terminal domain causes the stabilization of
the CDC-25.1(rr31) that affects a critical developmental window of the C. elegans intestine and
causes improper regulation (extra cells at the end of embryogenesis due to prolonged activity of
CDC-25.1 past the E8 stage). We predicted that knockdown of the kinase responsible for
phosphorylating this critical domain by RNAi would be enough to mimic the intestinal
hyperplasia seen in the cdc-25.1(rr31) worms.
Halfway through my research, Melixetan et al. reported that mammalian NIMA-related kinase
NEK11 was the sought after kinase responsible for phosphorylating S82 of CDC25A DSG motif
during G2/M downstream of the ATR/ATM pathway and responsible for its recognition for
CDC25A degradation via β-TrCP. This brought our attention toward the four members of the C.
elegans NEKL family. However, after many different RNAi methods to study to the effects of
nekl-1/2/3/4 RNAi and null mutant analysis, we could not reproduce the expected full phenotype
seen in the cdc-25.1(rr31)(gf) mutant. It is possible that the feeding RNAi experiments were not
73
successful in downregulating all the individual target nekl genes and perhaps RT-PCR
experiments will confirm the efficiency RNAi experiments.
Since it has been shown that
NEK11 phosphorylates the DSGX4S downstream during DNA damage and we were not able to
reproduce the full 41 cell hyperplasia phenotype with any combination of nekl RNAi, we can
conclude that there must be other pathways with diverse upstream kinases that impinge on CDC25.1 during embryogenesis and/or phosphorylates CDC-25.1 during S phase.
Further
experimental evidence to support this statement can be explained by the observed partial
hyperplasia phenotype in the eggs of the nekl-3 null mutants of the MR1619 strain. This partial
phenotype was also observed in experiments involving injecting the dsRNA of the 3’UTR of
nekl-3 into young MR931 adults. If this mutant MR1619 strain and 3’UTR nekl-3 RNAi injected
worms have no nekl-3 expression that results in a partial hyperplasia phenotype of approximately
28 intestinal cells, then one can assume that there are other kinases that are involved in this
critical function.
Since most of these evolutionary mechanisms have been conserved throughout time, having
multiple pathways that use kinases to regulate the phosphorylation of the CDC-25.1
phosphodegron in C. elegans is very likely conserved. This possibility of redundant kinase
function has been documented, in mammals, for the initiation of the phosphorylation required for
CDC25A recognition by the E3 ligase. CK1α, CK1ε, GSK-3β and CHK1 were shown to
phosphorylate the destruction motif [189, 191]. It is highly possible that various levels of these
kinases are required to collectively to control the phosphorylation status and degradation of
CDC-25.1. This can explain the partial intestinal hyperplasia observed in nekl-3 RNAi and in
the analysis of MR1619 eggs. Further investigation using the MR931 with a combination RNAi
approach may cause a more robust hyperplasia phenotype in the L1 larvae.
When considering the development of multicellular organisms, we can postulate that having
several kinases and an assortment of phosphorylation events on CDC-25.1, intrinsically
controlled by several different pathways, would be valuable in cell cycle control. The various
external and internal environmental cues that regulate CDC-25.1 would help control the levels of
CDK activity and govern the diverse events of cell cycle lineage progression during
development. Furthermore, it has been accepted that during embryogenesis different rates of
proliferation are characteristic of each individual cell lineage [130]. This can account for the
74
potential differences in activity levels of each kinase critical to the regulation of CDC-25.1
activity. For example, Melixetian et al. reported that when NEK11 phosphorylated by CHK1,
NEK11 can target CDC25A for degradation and Honaker et al. showed that canonical CK1α
activity can target the destruction motif in U2OS (human osteosarcoma cells). However, in
HEK293 (human embryonic kidney) cells, knockdown of CK1ε seem to increase CDC25A
stability but in HeLa cells, knockdown of endogenous CK1ε failed lead to the destruction of
CDC25A.
Additionally, CK1α plays a more important role in phosphorylating CDC25A then
CK1ε in HCT116 (human colon carcinoma) cells [189, 191]. Taken from the above examples,
we can conclude different cell types found in various lineages may have to coordinate a variation
of basal levels of these kinases that lead to the regulation of the master phosphatase levels and
the overall rate of cellular proliferation. These kinase levels, regulated by environmental factors,
may ultimately be responsible for maintaining proper lineage segregation among other wellknown factors during embryogenesis.
Given that mammalian studies have shown various kinases are responsible for targeting
CDC25A in different cell lines and under different conditions, future experiments will have to
focus on the diverse roles that these kinases play in C. elegans CDC-25.1 cell cycle regulation.
It would be interesting to study the RNAi combination effect using the individual nekl family
members with the C. elegans casein kinase homologues to determine if they act together in
targeting CDC-25.1 in the E lineage causing a more robust hyperplasia phenotype similar to the
cdc-25.1(rr31)(gf) mutant. Since single RNAi of individual casein kinase homologues did not
phenocopy the supernumerary cellular divisions of the rr31 mutant intestine, an RNAi
combination approach using our various elt-2::gfp strains will enable us to distinguish if these
kinases are dually responsible for regulating CDC-25.1 in the intestine.
Further investigation, following a successful RNAi combination approach resulting in the desired
hyperplasia phenotype, should be performed to substantiate that the supernumerary cell divisions
are triggered by CDC-25.1 inability to be phosphorylated by the candidate kinases. To confirm
that the target kinases are responsible for phosphorylating the CDC-25.1 motif, in vitro kinase
phosphorylation assays using GST tagged N-terminal of CDC-25.1 and a synthetic peptide
representing the F-box motif (S46 and S52) will validate their ability to target the specific motif.
75
In conjunction, to determining the candidate kinases’ ability to phosphorylate the CDC-25.1
destruction motif, a closer analysis of CDC-25.1 cellular dynamics by the use of a specific
intestinal GATA transcription factor (end-3), that expresses the CDC-25.1::GFP during E2 to E8
of embryogenesis, should show the stabilization of CDC-25.1 through RNAi of candidate
kinases during this critical developmental stage where the supernumerary cell division occurs in
the intestine. This powerful technique will prove to be very valuable in studying our candidate
kinases role in the intestine development. Analysis of the proliferation rates of other lineages
due to combinational candidate RNAi can also be studied in the D (muscles) and P4 (germline)
that are already known to be sensitive to the effects of CDC-25.1 gain of function mutant
stability [130]. This will undoubtedly further reveal more unanswered questions on how the
developing embryo coordinates various signaling pathways impinging on these candidate kinases
to control CDC-25.1 activity levels.
76
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