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Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in
Molecular Cell Biology presented at Uppsala University in 2002
ABSTRACT
Dikic, I. 2002. Signal Transduction by Proline-Rich Tyrosine Kinase Pyk2.
Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala
Dissertations from The Faculty of Medicine 1154. 66pp. Uppsala. ISBN 91554-5316-3
The proline-rich tyrosine kinase (Pyk2) together with focal adhesion
kinase (FAK) define a family of non-receptor protein tyrosine kinases that
are regulated by diverse stimuli. Activation of Pyk2 has been implicated in
multiple signaling events, including modulation of ion channels, activation of
MAP kinase cascades and apoptotic cell death. This thesis investigates the
role of Pyk2 in the regulation of mitogenic signals and cell cytoskeleton.
We identified a hematopoietic isoform of Pyk2 (designated Pyk2-H)
that is generated by alternative RNA splicing and is mainly expressed in
thymocytes, B cells and natural killer cells. In addition, we demonstrated
that engagement of antigen receptors in lymphocytes leads to rapid tyrosine
phosphorylation of Pyk2-H suggesting a potential role in host immune
responses. These findings were corroborated by defects in B cell-mediated
immune responses of Pyk2-/- mice.
Several reports have previously indicated that Pyk2 acts as an
upstream regulator of ERK and JNK MAP kinase cascades in response to
numerous extracellular signals. Which MAP kinase pathway is activated by
Pyk2 depends on arrays of effector proteins associated with Pyk2. We
proposed a model where the formation of Pyk2-Src complexes results in
phosphorylation of Shc, p130Cas and Pyk2. This creates binding sites for the
SH2 domains of adaptor proteins Grb2 and Crk, which in turn recruit
exchange factors for Ras and Rho GTPases that specifically activate ERK or
JNK.
Integration of signaling pathways initiated by receptor tyrosine
kinases and integrins is essential for growth factor-mediated biological
responses. We described neuronal cellular models where activation of both
growth factor receptors and integrins is required for neurite outgrowth. In
these cells, Pyk2 and FAK associate with integrin-linked complexes
containing EGF receptors via their C- and N-terminal domains. Inhibition of
Pyk2/FAK functions was sufficient to block neurite outgrowth and effectors of
the C-terminal domain of Pyk2/FAK, including paxillin, were shown to
regulate neurite outgrowth independently of ERK/MAP kinase in these cells.
We thus proposed that Pyk2 and FAK play important roles in signal
integration proximal to the integrin-growth factor receptor complexes.
Inga Dikic, Ludwig Institute for Cancer Research, Biomedical Center,
Box 595, SE-751 24 Uppsala, Sweden
 Inga Dikic 2002
ISSN 0282-7476
ISBN 91-554-5316-3
Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002
2
To
Karla,
My Little
Angel
3
This thesis is based on the following papers,
referred to in the text by their Roman numerals:
I
Dikic, I., Dikic, I., and Schlessinger, J. (1998)
Identification of a new Pyk2 isoform implicated in chemokine
and antigen receptor signaling. J. Biol. Chem. 273, 1430114308.
II
Blaukat, A. *, Ivankovic-Dikic, I. *, Grönroos, E., Dolfi, F.,
Tokiwa, G., Vuori, K., and Dikic, I. (1999)
Adaptor proteins Grb2 and Crk couple Pyk2 with activation of
specific mitogen-activated protein kinase cascades. J. Biol.
Chem. 274, 14893-14901. *Equally contributed authors.
III
Ivankovic-Dikic, I., Grönroos, E., Blaukat, A., Barth, B-U. and
Dikic, I. (2000)
Pyk2 and FAK regulate neurite outgrowth induced by growth
factors and integrins. Nat. Cell Biol. 2:574-581.
Imagination is more important than knowledge.”
Albert Einstein
“Om det inte var for sista sekunden skulle inget bli gjort.”
Okänt ursprung
Reprints were made with permission from the publishers
4
TABLE OF CONTENTS
Abbreviations
I
Introduction
Protein tyrosine kinases
Proline-rich tyrosine kinase 2 (Pyk2)
Pyk2/FAK family of protein tyrosine kinases
Src family kinases
The MAP kinase pathway
Integrins and cell adhesion
Signaling components of focal contacts
Co-operation between integrins and growth factor receptors
The organization of actin in growing neurites
II
Present investigation
A
Alternative splicing of Pyk2 in hematopoietic cells (Paper I)
B
Grb2 and Crk link Pyk2 with ERK and JNK activation
(Paper II)
C
Pyk2 and FAK regulate neurite outgrowth induced by growth
factors and integrins (Paper III)
III
Future perspectives
IV
Acknowledgements
V
References
5
Abbreviations
ADT
CAS
Csk
ECM
EGF
EGFR
ERK
FAK
FGF
FGFR
FRNK
GAP
GDI
GPCR
GEF
Grb2
IGF
Jak
JNK
LPA
MAP
MAPK
OS
PDGF
PDGFR
PIP
PI3K
PKC
PKM
PLC
PRNK
PTB
PTK
PTP
Pyk2
Pyk2-H
Pyk2-NT
RTK
SH
SH2
SH3
Shc
Sos
STAT
UV
VEGF
VEGFR
adhesion targeting domain
Crk-associated substrate
C-terminal Src kinase
extracellular matrix
epidermal growth factor
epidermal growth factor receptor
extracellular signal-regulated kinase
focal adhesion kinase
fibroblast growth factor
fibroblast growth factor receptor
FAK-related non-kinase
GTPase-activating protein
guanine dissociation inhibitor
G protein coupled receptor
guanine-nucleotide exchange factor
growth factor receptor bound protein 2
insulin-like growth factor
Janus kinase
c-Jun amino-terminal kinase
lysophosphatidic acid
mitogen-activated protein
mitogen-activated protein kinase
osmotic shock
platelet derived growth factor
platelet derived growth factor receptor
phosphatidyl inositol phosphate
phosphatidyl inositol 3’ kinase
protein kinase C
Pyk2 kinase inactive mutant
phospholipase C
Pyk2-related non-kinase
phosphotyrosine binding
protein tyrosine kinase
protein tyrosine phosphatase
proline-rich tyrosine kinase
proline-rich tyrosine kinase 2-hematopoietic isoform
N-terminal domain of Pyk2
receptor tyrosine kinase
Src homology
Src homology 2
Src homology 3
Src homology and collagen
son of sevenless
signal transducer and activator of transcription
ultraviolet light
vascular endothelial growth factor
vascular endothelial growth factor receptor
6
I
Introduction
In order to provide the exact response in every given situation
all cells receive and respond to signals from their surroundings. It is
in multicellular organisms, that cell communication reaches its
highest level of sophistication. To maintain homeostasis and at the
same time allow desirable changes is an absolute requirement for
organisms to meet their needs as a whole. What reflects complexity
and uniqueness of cell communication is synergy between those at
the first view opposing biological processes that is achieved by
carefully regulated behavior of each individual cell.
Virtually all aspects of cell behavior, including metabolism,
movement, proliferation and differentiation are regulated by external
molecules that bind to specific receptors on the surface of the cell.
The majority of these receptors contain either intrinsic or associated
protein kinase activity, which in turn stimulates intracellular signaling
proteins
thus
controlling
changes
in
gene
expression,
cellular
metabolism, cytoskeletal architecture and cell migration. By far the
most complex stage of signaling processes involves the array of
receptor-proximal proteins that normally receive messages from
ligand-activated
receptors
and
pass
them
along
a
variety
of
pathways.
A critical function of many receptors and signaling proteins is
their enzymatic activity that plays a key role in governing cell
behavior. In many cases these enzymes catalyze posttranslational
changes in their target proteins thus creating diversity in a repertoire
of
intracellular
responses
to
external
stimuli.
Posttranslational
modifications of signaling proteins, as well as their/the ability to
mediate
protein-protein
interactions
have
been
recognized
as
common themes of signal transduction. The latter feature is based on
7
the domain structure of signaling proteins enabling them to bind to
each other and thus form signaling cascades responsible for signal
transmission.
The sequencing of the human genome has provided another
milestone in our understanding of biology (McPherson et al., 2001;
Venter et al., 2001). The surprising finding was that the human
genome contains fewer genes than expected (approximately 30 000 –
40 000 as compared to approximately 20 000 in the C. Elegans),
indicating the presence of alternative means by which development
from lower eukaryotes to humans was accomplished (McPherson et
al., 2001; Venter et al., 2001). One possibility lays in the increasing
complexity of signaling cascades along the evolutionary tree. An
important role for signaling proteins is indicated by the fact that 20%
of the human coding genes encode proteins involved in signal
transduction. The most prominent expansions in the human genome
are found in proteins involved in (i) acquired immune functions; (ii)
neural development; (iii) intercellular and intracellular signaling
pathways in development and homeostasis. In addition, a large
increase in the diversity of domain structures and their widespread
presence among signaling proteins has been found in the human
genome as compared to genome sequences of lower eukaryotes. This
can enable the same proteins to be involved in many signaling
pathways and thus provides an elegant evolutionary mechanism for
multiplication of functions. However, more than 40% of all genes
found in the human genome code for proteins without any currently
known structural domain, indicating the potential existence of yet
unidentified structures or functions in these gene products (Venter et
al., 2001). Even though we can now read almost all human genes,
i.e. the long sought book of life, we are still far away from decoding
its true meaning.
8
PROTEIN TYROSINE KINASES
Protein kinases are enzymes designed to transfer phosphate
groups from ATP (adenosine triphosphate) molecules to the side
chains of specified amino acids in target proteins. The result of this
process is phosphorylation that causes the target protein to gain or
lose a function: to assemble or disassemble in the case of a
structure-building molecule and to turn a biochemical reaction on or
off in the case of a catalytic enzyme (Hunter, 1995). Because each
kinase may modify a variety of proteins, it can elicit a wide range of
responses in the cell simultaneously. Yet another class of enzymes
called protein phosphatases removes phosphate groups from target
proteins by hydrolysis thus exerting tight and reversible control on
protein phosphorylation. The sequencing of the human genome
identified 580 (1.9%) protein kinases and 180 (0.6%) protein
phosphatases (McPherson et al., 2001; Venter et al., 2001). Both of
these enzymes can be subdivided, based on their catalytic specificity,
into tyrosine or serine/threonine kinases and phosphatases (Hunter,
1991; Hunter, 1995). In addition, some possess dual specificity for
both tyrosine and serine/threonine. Based on their topology and
localization, protein tyrosine kinases are classified into receptor and
cytoplasmic PTKs (Neet and Hunter, 1996; Ullrich and Schlessinger,
1990). Receptor type PTKs are transmembrane proteins with an
extracellular domain that specifies the growth factor with which it will
interact, a single transmembrane domain, and an intracellular domain
that has an intrinsic or associated catalytic activity (Ullrich and
Schlessinger, 1990). To date, there are 90 known PTK genes in the
human genome; 58 encode transmembrane receptor PTKs further
distributed into 20 subfamilies, and 32 genes encode non-receptor
PTKs divided in 10 subfamilies (Neet and Hunter, 1996; Ullrich and
Schlessinger, 1990; Venter et al., 2001).
9
The identification that growth factors stimulate intrinsic
tyrosine kinase domains of their receptor led to the identification of
the largest family of ligand-stimulated receptor PTKs (Ullrich and
Schlessinger, 1990). Over the last decade, basic principles of growth
factor receptor signaling have been delineated. In general, the
binding of growth factors to receptor tyrosine kinases induces
receptors
to
dimerize
(Schlessinger,
1988)
allowing
transphosphorylation of residues in the activation loop of the catalytic
domain,
thus
leading
to
enzymatic
activation
and
receptor
autophosphorylation (Plotnikov et al., 1999; Schlessinger, 2000).
Ligand-induced dimerization can result in formation of homo- or
heterodimeric
signaling
complexes,
increasing
the
number
of
activated pathways as well as modulating the action of RPTK through
their
differential
expression
(Heldin,
1995).
Receptor
autophosphorylation creates high affinity binding sites for the SH2
and PTB domains of cytoplasmic signaling proteins (Pawson and Gish,
1992). These proteins are in turn phosphorylated and activated by
growth factor receptors. Examples of proteins that bind to the
phosphotyrosine
sites
on
growth
factor
receptors
include
phospholipase C (PLC), the p85 subunit of PI-3 kinase, adaptor
proteins
Shc,
Nck,
Grb2
and
GTPase-activating
protein
(GAP)
(Pawson and Scott, 1997). The result of binding and phosphorylation
of these signaling proteins is signal propagation to intracellular
cascades and networks that are able to amplify and diversify
incoming stimuli (Pawson, 1995). These signals ultimately control
transcription of both immediate and delayed response genes creating
an appropriate cellular response. In parallel, growth factor-bound
receptors are rapidly internalized and deactivated. Such negative
regulation of PTK receptor activity is accomplished by cytoplasmic
10
dephosphorylation as well as through ubiquitin triggered degradation
in the lysosomes (Waterman and Yarden, 2001).
An alternative mechanism of signal transduction has been
described for receptors that lack intrinsic protein-tyrosine kinase
activity. Upon ligand-binding these receptors bind and recruit
cytoplasmic PTKs at the plasma membrane thus initiating cytoplasmic
signaling cascades (Neet and Hunter, 1996). For example, cytokine
receptors directly bind and activate members of the JAK family of
non-receptor tyrosine kinases (Ihle et al., 1994; Silvennoinen et al.,
1997). Activation of JAK kinases leads to phosphorylation of
cytoplasmic proteins termed signal transducers and activators of
transcription
(STATs).
Tyrosine-phosphorylated
STAT
proteins
dimerize and migrate to the nucleus and regulate expression of their
target genes. Hence, there are multiple distinct pathways by which
PTKs transduce signals from the cell surface to the nucleus.
Deregulation of the PTK-mediated signaling pathways is
commonly associated with development of many human diseases.
The seminal discoveries that PTKs are associated with the activity of
viral oncogenes such as v-Src, v-ErbB2 and v-Abl indicated that
hyperactivation
of
tyrosine
kinases
is
closely
associated
with
oncogenic transformation (Blume-Jensen and Hunter, 2001; Heldin
and Westermark, 1984). PTKs were subsequently shown to be the
largest group of dominant oncogenic proteins; 52% of them have
been shown to be mutated or overexpressed in human cancers
(Blume-Jensen and Hunter, 2001). Protein tyrosine kinases and their
target signaling pathways are thus of interest, not only in the
regulation of normal cells, but also in disease such as cancer.
11
PROLINE-RICH TYROSINE KINASE 2 (PYK2)
Pyk2 is a non-receptor PTK belonging to the focal adhesion
kinase family. It was independently identified by four groups as: Pyk2
isolated from a human brain cDNA library by a PCR screen searching
for a homologous kinase to Pyk1 (Lev et al., 1995); cell adhesion
kinase β (CAK-β) isolated from a rat brain cDNA library (Sasaki et al.,
1995); related adhesion focal tyrosine kinase (RAFTK) cloned by
homology
to
conserved
tyrosine
kinase
domains
from
megakaryocytes (Avraham et al., 1995); and a calcium-dependent
tyrosine kinase (CADTK) by purification and sequencing of a major
autophosphorylated tyrosine kinase following calcium treatment of rat
liver cells (Yu et al., 1996).
N
4.1 band
Kinase domain
Y402
Nir
(rdgB)
Src
family
kinases
PXXP
p130Cas
Graf
Pap
Kv1.2
FAK
p105HEF1
ATD
PXXP
C
Y881
Grb2
Paxillin
EWS
Jak3
Leupaxin
Hic- 5
Pyk2 functions as a protein tyrosine kinase and an adaptor
protein
Pyk2 contains a central catalytic domain flanked by large
amino- and carboxyl-terminal non-catalytic regions (Avraham et al.,
2000). The kinase domain of Pyk2 is most similar to the kinase
domain of FAK. The N-terminal domain contains a tyrosine residue at
position 402, which is the major autophosphorylation site of Pyk2 and
also a direct binding site for the SH2 domain of Src (Dikic et al.,
12
1996). Binding of Src to Y402 is critical not only for Src activation but
also for phosphorylation of several proteins including Pyk2 itself,
p130Cas and paxillin (Blaukat et al., 1999; Hiregowdara et al., 1997;
Lakkakorpi et al., 1999; Li and Earp, 1997; Lipsky et al., 1998;
Ostergaard et al., 1998; Roy et al., 2002). Homology between the Nterminal
part
of
Pyk2
and
a
4.1
band
domain
found
in
ezrin/radixin/myosin proteins was recently described (Girault et al.,
1999b). It was suggested that this domain might be important for
linking Pyk2 to transmembrane receptors. Furthermore, a new class
of proteins called N terminal-interacting receptors (Nirs) was shown
to specifically bind to Pyk2 but not FAK and was implicated in
signaling in neuronal cells (Lev et al., 1999). The C-terminal domain
contains two proline-rich motifs that are important for binding of SH3
domain-containing signaling proteins, such as p130Cas or p105Hef
(Astier et al., 1997b; Lakkakorpi et al., 1999). There is also a binding
site for the SH2 domain of the adaptor protein Grb2 (tyrosine at
position 881-YLNV) and a domain called adhesion-targeting domain
(ATD) (Avraham et al., 2000; Lev et al., 1995). Paxillin was shown to
bind to this domain and was suggested to link Pyk2 with adhesion
sites (Li and Earp, 1997; Ostergaard et al., 1998; Turner, 1998). Yet
another protein termed Pap (Pyk2 C-terminus associated protein)
forms a stable complex with Pyk2 and is tyrosine-phosphorylated
upon Pyk2 activation (Andreev et al., 1999). Pap is a multi-domain
protein shown to exhibit intrinsic GAP activity toward the small G
proteins Arf1 and Arf5. Overexpression of Pap inhibited post-Golgi
vesicle release implicating Pap in the control of vesicular transport
(Andreev et al., 1999). The role of Pyk2 and its interaction with Pap
in these processes remains to be elucidated.
Pyk2 is widely expressed in adult tissues with the most
prominent expression in cells from hematopoietic lineages as well as
13
in the central nervous system (Dikic et al., 1998; Lev et al., 1995).
Immunofluorescence analyses of the cellular distribution of Pyk2 have
shown diverse subcellular localization patterns including diffuse
cytoplasmic staining (Park et al., 2000a; Sabri et al., 1998),
localization to focal adhesions (Fuortes et al., 1999; Matsuya et al.,
1998), enrichment in sites of cell-cell contact (Sasaki et al., 1995), or
cell-matrix attachment (Duong et al., 1998; Duong and Rodan, 2000;
Lakkakorpi et al., 1999). Occasionally, strong Pyk2 staining was
observed in the cell nuclei, with excluding the nucleoli but its
significance is not known (Keogh et al., 2002; Ridyard and Sanders,
2000). The reasons for these variations are not clear but may simply
reflect differences between cell types and the functions of Pyk2 in
different cells.
Two isoforms of Pyk2 have been identified, and they appear to
be differentially expressed and regulated when compared to wild type
Pyk2 (Dikic et al., 1998; Keogh et al., 2002; Li et al., 1998; Xiong et
al., 1998). Hematopoietic isoform of Pyk2 (designated Pyk2-H) is
generated by an alternative RNA splicing and represents the
truncated variant of Pyk2 with a 42-amino-acid deletion within the Cterminal domain (Dikic et al., 1998; Keogh et al., 2002; Li et al.,
1998; Xiong et al., 1998). Pyk2-H is mainly expressed in thymocytes,
B cells and natural killer cells, monocytes and macrophages (Dikic et
al., 1998). Initial characterization showed that engagement of T cell
or B cell antigen receptors leads to rapid tyrosine phosphorylation of
Pyk2-H suggesting a potential role in host immune responses. A
second isoform, referred to as PRNK (Pyk2-related non-kinase), is
derived from mRNAs comprising a unique 5’ leader fused to the Pyk2
sequence that encodes only part of the C-terminal domain of Pyk2
(the C-terminal 238 amino acids of Pyk2) (Xiong et al., 1998). The
mechanism by which PRNK transcripts are generated is not yet clear.
14
The most probable mechanisms are alternative splicing and/or
utilization of alternative transcriptional promoters. PRNK is expressed
in many tissues, at high levels in spleen and cerebellum but poorly in
almost
all
regions
of
the
brain
(Xiong
et
al.,
1998).
Immunofluorescence analysis of ectopically expressed PRNK proteins
showed that sequences within the focal adhesion targeting domain
are critical for its localization to focal contacts (Xiong et al., 1998).
Moreover, both Pyk2 and PRNK were shown to bind strongly to
paxillin (Xiong et al., 1998). In conclusion, the functional diversity
mediated by Pyk2 may be in part governed by expression of its
alternatively spliced isoforms.
Pyk2
is
rapidly
phosphorylated
on
tyrosine
residues
in
response to agonists that increase intracellular calcium concentrations
or activate protein kinase C (Brinson et al., 1998; Lev et al., 1995;
Yu et al., 1996), as well as cell-cell contacts or cell adhesion
(Avraham et al., 2000).
Growth factor receptors
(PDGF, EGF, FGF)
Ion channels
Antigen receptors
GPCRs
Integrins
Cytokine receptors
Stress stimuli (UV, OS)
Pyk2/FAK
Actin
Ion channel
MAPK
Cell
Cell
function cytoskeleton cascades proliferation apoptosis
Diverse cellular stimuli activate Pyk2 and FAK to regulate multiple
biological functions
15
To this day, a large number of external stimuli have been
shown to cause phosphorylation and activation of Pyk2, including
cytokines (Benbernou et al., 2000; Hatch et al., 1998; Liu et al.,
1997; Miyazaki et al., 1998; Yan and Novak, 1999), growth factors
(Ivankovic-Dikic et al., 2000; Park et al., 2000b; Soltoff, 1998),
integrin ligation (Astier et al., 1997a; Duong et al., 1998; Sanjay et
al., 2001), crosslinking of T cell receptors (Dikic et al., 1998; Qian et
al., 1997), HIV envelope proteins (Davis et al., 1997), stress stimuli
(Koh et al., 2001; Tian et al., 2000; Tokiwa et al., 1996) and Gprotein-coupled receptor agonists (Bajetto et al., 2001; Cazaubon et
al., 1997; Del Corno et al., 2001; Dikic et al., 1996; Tapia et al.,
1999; Wang et al., 1999). Although many stimuli can initiate
phosphorylation and activation of Pyk2, little is known about the
actual molecular mechanisms by which Pyk2 is activated. Upon
activation Pyk2 is able to trigger multiple intracellular pathways
leading to modulation of ion channels (Lev et al., 1995), regulation of
vesicular transport (Andreev et al., 2001), cytoskeletal reorganization
(Ivankovic-Dikic et al., 2000; Salgia et al., 1996) apoptotic cell death
(Xiong and Parsons, 1997), cell attachment, spreading and motility
(Duong et al., 2001; Duong and Rodan, 1998; Watson et al., 2001),
neurotransmission and neuroplasticity (Girault et al., 1999a). These
diverse functions are mediated by a large panel of effector proteins
that bind and/or are phosphorylated by Pyk2.
PYK2/FAK FAMILY OF PROTEIN TYROSINE KINASES
Due to their structural similarities and high sequence identity
Pyk2 together with FAK constitute a distinct family of non-receptor
protein tyrosine kinases called Pyk2/FAK family (Neet and Hunter,
1996). Currently there are only these two members identified in
16
mammals, while in fruit fly there is only one gene coding for dFAK
(Palmer et al., 1999).
Pyk2 and FAK share 60% identity in the catalytic domain, 31%
in the amino terminal part and 45% in their carboxyl termini
(Avraham et al., 2000). Several tyrosine residues are conserved
between FAK and Pyk2, including the binding site for the SH2
domains of Src and Fyn (Y397 in FAK, Y402 in Pyk2) and the putative
binding site for the SH2 domain of Grb2 (Y925 in FAK, Y881 in Pyk2).
In addition, FAK and Pyk2 also contain the proline-rich sequences
responsible for mediating the binding of p130Cas and Graf. The
paxillin binding region in the C-terminal domain of FAK is also highly
conserved within Pyk2. At last, Pyk2 interacts with many of the FAK
binding partners in a manner similar to that of FAK (Avraham et al.,
2000).
Pyk2
N
Y
402
Kinase domain
Pro-rich
Y
397
Kinase domain
Pro-rich
Y
881
C
FAK
N
Identity:
42%
61%
Y
925
C
36%
Homology comparison between Pyk2 and FAK
In addition to their structural characteristics, Pyk2 and FAK
share many functional similarities. They are both activated in
response to a variety of extracellular stimuli in different cell types
including GPCRs, integrin receptors, growth factor receptors, antigen
receptors, cytokine receptors, ion channels, stress stimuli and
17
calcium mobilization (Avraham et al., 2000; Girault et al., 1999a;
Hanks and Polte, 1997; Schaller, 2001; Schlaepfer et al., 1994). This
variety of extracellular signals that activate Pyk2 and FAK indicate
that these kinases might have a broad and important role in
regulation of cell functions.
Nevertheless, there are also differences in their signaling
potentials. Adherence of fibroblast to extracellular matrix activates
the focal adhesion kinase (Schaller et al., 1992). Unlike FAK, Pyk2 is
not potently tyrosine phosphorylated following cell adhesion of
epithelial (Yu et al., 1996), neuronal (Lev et al., 1995) and smooth
muscle cells (Brinson et al., 1998). Rather Pyk2 is rapidly activated
and tyrosine phosphorylated when an intracellular calcium or protein
kinase C signal is generated in these cells [Yu, 1996 #543; (Brinson
et al., 1998; Lev et al., 1995). Therefore it is believed that each of
them has the capacity to mediate distinct signaling responses, Pyk2
being more involved in signaling by different soluble factors while FAK
is strongly activated by cell adhesion and integrin engagement.
However, Pyk2 can also be activated by cell adhesion, particularly in
cells that lack FAK. In such cells Pyk2 appears to act as an adhesiondependent kinase and can substitute functions of FAK in focal
contacts. For example, in freshly isolated human monocytes Pyk2
plays an early role in post-adherence signaling. Its activation is
apparently a two stage process involving a cytoskeletal engagement
which is the permissive step required for Pyk2 activation, and an
additional intracellular calcium or PKC signal (Li et al., 1998). Yet
another example is activation of Pyk2 in peritoneal macrophages and
osteoclasts where Pyk2 is functionally linked to the formation of
podosomes and regulates cell spreading and migration (Duong et al.,
2001; Duong and Rodan, 1998; Watson et al., 2001),.
18
An additional striking difference between Pyk2 and FAK is their
expression
patterns.
While
FAK
is
widely
expressed
during
development (Furuta et al., 1995; Ilic et al., 1995; Ilic et al., 1996;
MacPhee et al., 2001) and in the majority of mature cell types
(Girault et al., 1999a; Martin, 1996), expression of Pyk2 appears
later in development than FAK (Sieg et al., 1998) and appears to be
more restricted to mature specialized cells, such as neuronal,
hematopoietic and bone cells (Avraham et al., 2000; Dikic et al.,
1998; Duong et al., 1998; Lev et al., 1995). These characteristics are
also reflected in the phenotypes of Pyk2- and FAK-deficient mice.
FAK-/- mice die at approximately day 9 of embryonic development
(Furuta et al., 1995; Ilic et al., 1995). They show gross defects in
mesoderm development, defects in cell migration, but surprisingly
enhanced focal adhesions (Furuta et al., 1995; Ilic et al., 1995). In
addition, fak-/- fibroblasts appear to have elevated expression of
Pyk2, which together with Src can compensate for the loss of FAK in
integrin signaling measured by MAP kinase activation, but do not
rescue migratory defects (Sieg et al., 1998). In contrast, Pyk2
deficient mice are alive and fertile (Guinamard et al., 2000). Primary
phenotypes are observed in the spleen, which lacks marginal zone B
cells due to their inability to migrate in the peripheral zone of the
spleen. The osteoclast function was also impaired and bones were
more fragile (Sanjay et al., 2001). These studies indicated that Pyk2
and FAK have overlapping as well as unique functions in different
cells and at different times during development.
SRC FAMILY KINASES
Src was initially identified as the oncogenic protein of Rous
sarcoma virus and was the first protein shown to possess proteintyrosine kinase activity, thus it has played a pivotal role in
19
experiments leading to our current understanding of cell signaling
(Courtneidge et al., 1980; Czernilofsky et al., 1980; Hunter and
Sefton, 1980). The cellular counterpart of v-Src, c-Src, is widely
expressed in mammalian cells, with particularly high concentrations
in brain, platelets and bone-resorbing osteoclasts (Courtneidge et al.,
1993b). In contrast to receptor tyrosine kinases, which are integral
plasma membrane proteins, Src is anchored in the inner leaflet of the
plasma membrane and belongs to the non-receptor class of tyrosine
kinases (Neet and Hunter, 1996). The Src family of PTKs comprises
eight members in vertebrates, namely: Src, Fyn, Yes, Fgr, Hck, Lyn,
Lck and Blk [(Courtneidge et al., 1993b; Neet and Hunter, 1996).
The structure of Src and its regulation are well understood as
a result of both mutational and structural studies (Superti-Furga and
Courtneidge, 1995). Src consists of 4 well-characterized protein
domains: the catalytic (SH1), and the SH2, SH3 and SH4 Src
homology domains. The amino terminal region contains a consensus
sequence for attachment of a fatty acid, myristate; myristylation is
required for both membrane localization and biological activity of Src
(Resh, 1994). The SH2 and SH3 domains are adjacent to the Nterminus, and are involved in protein–protein interactions. The
tyrosine kinase domain has a two-lobe structure that is common to
other protein kinases (Xu et al., 1997). Several mechanisms are
involved in Src activation and inactivation (Superti-Furga and
Courtneidge, 1995; Williams et al., 1998). An interaction between the
SH2/SH3-domains keeps the protein turned off. Two competing
tyrosine residues are crucial for the activation. Phosphorylation of
Tyr418 activates Src, whereas interaction of the SH2 domain with
phosphorylated Tyr527 keeps the protein in an inactive conformation.
Src
also
undergoes
a
conformational
20
change
when
activated.
Activation of Src can also take place by dephosphorylation of Tyr527
by tyrosine phosphatases.
Src family kinases are involved in a number of signal
transduction pathways, including those that result in cell proliferation,
differentiation, motility and cytoskeletal rearrangements (Thomas and
Brugge, 1997). Src family kinases bind directly to activated RTKs and
are activated in these complexes (Parsons and Parsons, 1997). The
catalytic activity of Src, Fyn and Yes is increased upon growth factor
stimulation (Courtneidge et al., 1993a; Mori et al., 1994; Osherov
and Levitzki, 1994; Roche et al., 1995). The Src family kinases
associate with the juxtamembrane region of the PDGF receptor via
their SH2 domains (Kypta et al., 1990; Mori et al., 1994; Ralston and
Bishop, 1985). Activation of Src kinases appears to be required for
DNA synthesis induced by PDGF, EGF and colony stimulating factor-1
but is not involved in the mitogenic responses to ligands that activate
G proteins, such as lysophosphatidic acid and bombesin (Roche et al.,
1995). Src family kinases also mediate important functions in
neuronal cells: microinjection of an antibody specific for Src inhibits
neurite outgrowth in response to nerve growth factor and fibroblast
growth factor in PC12 cells (Kremer et al., 1991), and furthermore,
mice lacking catalytically active Fyn show impairment of long-term
potentiation and myelination (Grant et al., 1992; Umemori et al.,
1994). Despite the high level of expression of Src in brain and
platelets, mice that are deficient in Src do not show major
abnormalities in brain function and blood clotting (Soriano et al.,
1991). Instead, the phenotype of these animals is restricted to
defective bone resorption, which results in excessive bone mass and
osteopetrosis (Soriano et al., 1991). This isolated phenotype in Src
deficient mice can be due to compensatory mechanisms by other
members of the Src family that are expressed in a given cell type.
21
Consistent with this hypothesis, mice lacking more than one member
of the Src family usually show phenotypes that are more severe than
the sum of the separate phenotypes (Lowell et al., 1994; Stein et al.,
1994).
THE MAP KINASE PATHWAY
The MAP kinase pathway refers to a cascade of protein kinases
that are highly conserved in evolution and play central roles in signal
transduction in all eukaryotic cells. The central element in the
pathway is a family of protein serine/threonine kinases called the MAP
kinases (for mitogen-activated protein kinases). The cascade is
composed
of
two
additional
upstream
protein
kinases,
where
serine/threonine MAPKK kinase phosphorylates and activates dual
specificity MAPK kinase, which in turn activates MAP kinase by
phosphorylation of the conserved TEY motif (Chang and Karin, 2001;
Marshall, 1994). In yeast, MAP kinase pathways control a variety of
cellular responses, including mating, cell shape, and sporulation
(Posas et al., 1998). In higher eukaryotes MAP kinases are ubiquitous
regulators of cell growth, proliferation and differentiation, as well as
stress
and
inflammatory
responses
(Chang
and
Karin,
2001;
Waskiewicz and Cooper, 1995). In mammalian cells, there are three
distinct MAP kinases: ERK (extracellular signal-regulated kinase),
JNK/ SAPK (c- Jun amino-terminal kinase/ stress activated protein
kinase) and p38 MAP kinase (Chang and Karin, 2001). Upstream of
MAP kinases are distinct small GTP-binding proteins and cytoplasmic
cascades composed of MAPKK and MAPKKK protein kinases (Marshall,
1994). In most cases, the rate-limiting step in activation of the MAP
kinase pathway is the conversion of small GTP-binding proteins from
the inactive GDP-bound form to their active GTP-bound state. The
GDP/GTP exchange is modulated by GEFs (guanine nucleotide
exchange factors), which promote formation of the GTP-bound form,
22
and by GAPs (GTPase activating proteins), which stimulate the rate of
intrinsic GTP hydrolysis of G-proteins.
Perhaps
the
best-characterized
signaling
pathway
in
mammalian cells is growth factor-stimulated and Ras-dependent
activation of the ERK/MAP kinase pathway (Schlessinger, 1993). The
adaptor protein Grb2 binds to tyrosine-phosphorylated growth factor
receptors such as the EGF receptors through its SH2 domain. The Ras
guanine-nucleotide exchange factor, Sos, also binds to Grb2 via the
Grb2 SH3 domain. The Grb2/Sos protein complex is brought to the
receptor at the cytoplasmic surface of the plasma membrane, where
Sos catalyzes GTP loading and formation of the activated Ras–GTP
complex. The serine-threonine protein kinase Raf (MAPKKK) is an
effector for Ras– GTP. Interaction of Raf-1 with Ras–GTP allows Raf
activation, which in turn phosphorylates and activates MEK (MAPKK),
a specific threonine/tyrosine-directed protein kinase (Chang and
Karin, 2001; Marshall, 1994). MEK in turn phosphorylates MAPK on
both a tyrosine and threonine, resulting in activation of ERK (Chang
and Karin, 2001; Waskiewicz and Cooper, 1995). Once activated, ERK
is translocated to the nucleus, where it phosphorylates several
transcription factors implicated in mitogenic responses, including Fos,
Jun, AP-1, TCF (Treisman, 1996). In addition, MAPK phosphorylates
and regulates the activity of a number of cytoplasmic proteins,
including additional serine-threonine protein kinases that contribute
to responses controlled by the MAPK signal transduction pathway.
INTEGRINS AND CELL ADHESION
Integrins are heterodimeric transmembrane receptors that
mediate two types of stable junctions in which the cytoskeleton is
linked to the extracellular matrix (Ruoslahti and Pierschbacher,
1987). In focal adhesions, bundles of actin filaments called stress
23
fibers are anchored to the β subunits of most integrins via association
with a number of other proteins including α-actinin, talin and vinculin
(Geiger et al., 2001). In hemidesmosomes, α6β4 integrin links the
basal lamina to the intermediate filaments, which are attached to a
dense plaque of intracellular proteins (Nievers et al., 1999). In
addition to this structural role, integrins serve as receptors that
activate intracellular signaling pathways, thereby controlling gene
expression and other aspect of cell behavior in response to adhesive
interactions (Clark and Brugge, 1995; Geiger et al., 2001; Giancotti
and Ruoslahti, 1999).
Integrins comprise 24 α and 9 ß subunits in mammals leading
to differential combinations of single α and ß subunits to form at least
24 different receptors with distinct and often overlapping specificity
for ECM proteins. The ECM-binding region is composed of portions of
both subunits that bind to distinct peptide sequences present in
multiple components of the extracellular matrix, including collagen,
fibronectin, and laminin (Johansson et al., 1997; Ruoslahti and
Pierschbacher, 1987). The integrins have short cytoplasmic tails that
lack
any
intrinsic
enzymatic
activity.
However,
tyrosine
phosphorylation of proteins is an early response to the interaction of
integrins with extracellular matrix components, suggesting that the
integrins are functionally linked to protein tyrosine kinases (Clark and
Brugge, 1995).
Integrin-mediated signals regulate a variety of important
events contributing to regulation of normal cell growth and motility,
and, in cancer cells, to uncontrolled cell proliferation and metastasis
(Ruoslahti, 1999). Integrins also play a role in survival pathways in
specific cell types. Fibroblastic, epithelial, and endothelial cells must
be attached to appropriate extracellular matrix in order to survive - a
phenomenon termed anchorage dependence. The capacity of cells to
24
survive and proliferate in the absence of integrin-mediated adhesion
in vitro (anchorage independence) correlates with tumorigenesis in
vivo (Schwartz, 2001). Many of these functions are modified by
actions of growth factors and a significant cross talk in signaling by
integrins and RTKs has been observed. This cooperation may be
particularly relevant in processes such as development and wound
healing, where cells use integrins to interact with the matrix, which
may augment the response to low levels of growth factors (Eliceiri
and Cheresh, 2001).
SIGNALING COMPONENTS OF FOCAL CONTACTS
Integrin
ligation
is
known
to
induce
a
wide
range
of
intracellular signaling events, including the activation of several
protein kinases. Integrin-mediated cell adhesion can activate tyrosine
protein kinases (e.g. FAK and Src) and serine/threonine protein
kinases (e.g. protein kinase C (PKC), MAP kinase/ERK kinase).
Moreover, it regulates the activity of Rho family small GTPases, and it
activates inositol lipid metabolism (Clark and Brugge, 1995; Giancotti
and Ruoslahti, 1999; Parsons and Parsons, 1997; Schlaepfer and
Hunter, 1998). Since integrins have very short cytoplasmic tails that
lack any intrinsic enzymatic activity, they activate these pathways via
recruitment of other signaling and adaptor proteins into focal
contacts. Furthermore, adapter proteins including p130Cas/Crk and
Shc are important for coordinating intracellular signals during
integrin-mediated cell migration.
In 1992, focal adhesion kinase (FAK) was identified as a
protein tyrosine kinase activated by integrin-mediated adhesion and
localized to sites of adhesion (Schaller et al., 1992). FAK has since
emerged as a pivotal molecule controlling signaling from integrins as
well as maintaining the structure of focal contacts (Schlaepfer and
25
Hunter, 1998). Multiple associations of FAK with other signaling
molecules in focal contacts appear to be critical for these functions
(Combs et al., 1999; Hildebrand et al., 1993). Phosphorylation of
many proteins in focal contacts is mediated by FAK-associated Src
family kinases (Schlaepfer and Hunter, 1998). More recently similar
findings were demonstrated for the related kinase Pyk2 in specific cell
types (see B). It appears that the major functional role for FAK and
Pyk2 is to act as adaptor proteins with intrinsic tyrosine kinase
activity.
There is a significant body of evidence suggesting a potential
role for Src tyrosine kinases in signaling mediated by cell adhesion
(Parsons and Parsons, 1997). Upon integrin engagement, FAK
becomes tyrosine phosphorylated, creating a binding site for the SH2
domains of Fyn and Src (Combs et al., 1999). In addition, in Srctransformed cells, most FAK is associated with Src in focal adhesions
(Xing et al., 1994). Src in turn phosphorylates several sites in FAK
(Calalb et al., 1995; Calalb et al., 1996), thus controlling kinase
activity of FAK and creating binding sites for other proteins containing
SH2 domains, including Grb2 that leads to Ras-dependent activation
of the MAP kinase pathway. Src also phosphorylates the adaptor Shc,
which provides a separate link to the Ras/MAP kinase pathway
(Barberis et al., 2000; Wary et al., 1996). Furthermore, the
repressed form of Src is found mostly in an early endosome
compartment, whereas activated forms are associated with focal
adhesions (Kaplan et al., 1994). Thus, recruitment of Src to focal
adhesions
may
result
in
its
activation
through
intermolecular
association with FAK. In addition to their role as cofactors for FAKdependent responses, Src tyrosine kinases can also be activated
independently of FAK and contribute to a distinct set of responses.
Src interactions with c-Cbl are important for the motility of
26
osteoclasts and bone resorption (Sanjay et al., 2001; Tanaka et al.,
1996), as well as for the recruitment of vascular smooth muscle cells
in response to PDGF during blood vessel formation (Mureebe et al.,
1997).
Paxillin is a multi-domain protein that localizes in cultured
cells primarily to focal adhesions (Turner, 2000; Turner et al., 1990).
Paxillin localizes to focal adhesions through its LIM domains, possibly
through a direct association with ß-integrin tails or an intermediate
protein (Brown et al., 1996; Turner and Miller, 1994). Its primary
function is to provide multiple docking sites at the plasma membrane
for an array of signaling and structural proteins. For example, it
provides a platform for protein tyrosine kinases such as FAK/Pyk2
and Src, which are activated as a result of adhesion or growth factor
stimulation (Turner, 2000). Phosphorylation of residues in the Nterminus
of
paxillin
by
these
kinases
permits
the
regulated
recruitment of downstream effector molecules such as Crk, which (via
association with p130Cas) is important for transduction of external
signals into changes in cell motility and for modulation of gene
expression by the various MAP kinase cascades (Petit et al., 2000;
Shaw et al., 1995; Turner et al., 1999). LIM-domain-associated
kinases regulate recruitment of paxillin to focal adhesions (Brown et
al., 1998). Paxillin also binds structural proteins such as vinculin and
actopaxin that bind actin directly, as well as regulators of actin
cytoskeletal dynamics such as the ARF GAP, PKL, the exchange factor
PIX and the p21-activated kinase, PAK (Turner et al., 1999). These
proteins serve as modulators of the ARF and Rho GTPase families.
Adhesion of fibroblasts to fibronectin also promotes the
tyrosine phosphorylation of another focal adhesion protein, p130Cas,
a protein originally identified as a substrate for phosphorylation by
the oncoproteins Src and v-Crk (Harte et al., 1996; Nojima et al.,
27
1995; Sakai et al., 1994; Vuori et al., 1996). p130Cas is localized to
focal adhesion complexes and directly binds to FAK (Harte et al.,
1996). The multidomain nature of p130Cas, coupled with the capacity
to interact in a tyrosine phosphorylation-dependent manner with
several SH2-domain-containing proteins, enables it to play a role in
stabilizing the assembly of focal adhesions.
Activated Rho and structurally related members of the Rho
family of small GTP-binding proteins, Rac and Cdc42, also play a
prominent role in regulating the formation of focal adhesions and the
actin cytoskeleton (Hall, 1998). These members of Rho GTPases act
at different levels to organize actin structures. Activation of Rac
stimulates the formation of the broad, actin-rich cellular protrusions
known as lamellipodia (Ridley et al., 1992). Activation of Cdc42
causes the extension of more elongated cellular protrusions known as
filopodia and activation of RhoA yields the formation of contractile
actin bundles or stress fibers (Nobes and Hall, 1995; Ridley and Hall,
1992). Cdc42 and Rac are also required for the assembly of adhesion
sites to the extracellular matrix (Allen et al., 1997). Together, these
observations provide support for a role of Rho family members in the
control of different actin-based cytoskeletal structures.
COOPERATION BETWEEN INTEGRINS AND GFR
Integrins were shown to act in concert with growth factor
receptors to regulate survival and differentiation. Integrin-mediated
cell adhesion to fibronectin is particularly efficient in supporting
growth factor-dependent proliferation of fibroblastic, epithelial, and
endothelial cells. For example, optimal cell stimulation with EGF,
PDGF, insulin, or VEGF depends on integrin-mediated cell adhesion
(Guilherme et al., 1998; Moro et al., 1998; Soldi et al., 1999; Vuori
and Ruoslahti, 1994). Moreover, integrins are also important for
28
promoting growth factor–induced kinase activation of VEGFR (Soldi et
al., 1999). In addition, insulin potently activates integrin alpha5beta1
mediated cell adhesion, while integrin signaling in turn enhances
insulin receptor kinase activity and formation of complexes containing
IRS-1 and PI 3-kinase (Guilherme et al., 1998). These findings raise
the hypothesis that RTK and integrin signaling act synergistically to
enhance cell adhesion. The coordinated action of growth factors and
vascular cell adhesion events is also critical for angiogenesis (Eliceiri
and Cheresh, 2001). Integrins physically associate with RTKs (Falcioni
et al., 1997; Schneller et al., 1997; Sundberg and Rubin, 1996) and
are able to promote growth factor-independent activation of PDGF,
EGF and VEGF receptors (Moro et al., 1998; Soldi et al., 1999;
Sundberg and Rubin, 1996). Co-immunoprecipitation of growth factor
receptors with integrins has been an important approach to identify
biochemical interactions between these receptors in cultured cells.
For example, αvß3 has been found to associate with the PDGF
receptor (Schneller et al., 1997) or the VEGF receptor (Soldi et al.,
1999), as well as IRS-1, a cytoplasmic signal transduction mediator
of insulin and IGF receptors (Guilherme et al., 1998; Vuori and
Ruoslahti, 1994; Zheng and Clemmons, 1998). The capacity for
growth factor stimulation to synergize with extracellular matrix and
promote a crosstalk between integrins and growth factor receptors in
multiple cell types may be the result of co-clustering of these
receptors on the surface of the cell in focal adhesions.
Many of the signaling pathways and effectors, which are
activated by integrin ligation are also activated after growth factor
stimulation (Clark and Brugge, 1995; Guilherme and Czech, 1998;
Guilherme et al., 1998; Schwartz and Baron, 1999; Vuori and
Ruoslahti, 1994). The ERK MAP kinase, Rho GTPases and G1-phase
cyclin-dependent kinases are all regulated jointly by growth-factor
29
receptors and integrins (Assoian and Schwartz, 2001; Roovers et al.,
1999). For example, efficient activation of the ERK MAP kinase
cascade induced by growth factor receptors is modulated by cell
adhesion. Integration of intracellular pathways initiated by RTKs and
integrins via the Ras pathway seems important for sustained MAP
kinase activation and regulation of cell cycle progression (Assoian and
Schwartz, 2001; Renshaw et al., 1997). Another example is the
ability of integrins to activate small Rho GTPases as well as coupling
of active GTPases to their effectors (Hall, 1998). Given that integrinmediated adhesion occurs at specific cellular locations, it was
suggested that adhesion could direct the subcellular localization of
Rho GTPase activity triggered by growth factors as well as to define a
specific repertoire of Rho-effectors in focal contacts (Schwartz and
Shattil, 2000). Such a mechanism represents a highly effective
means to coordinate cell migration, which is dependent on both
soluble factors and cell adhesion. Therefore, integrin- and growth
factor–mediated cellular responses function to coordinate biochemical
responses (Assoian and Schwartz, 2001; Schwartz and Baron, 1999).
One of the challenges is to elucidate how signaling specificity is
achieved in these signaling networks.
THE ORGANIZATION OF ACTIN IN GROWING NEURITES
During the formation of the nervous system neurons extend
axons and dendrites, collectively referred to as neurites, in order to
form precise patterns of neuronal connectivity. The neurons recognize
precisely their target cells and form synapses, and these steps are
complex, but well organized spatially and temporally. The dynamic
structures at the top of neurites known as growth cones rapidly
remodel the actin cytoskeleton through cyclic extension of filopodia
and lamellipodia (Hall, 1998; Pollard et al., 2000). Numerous studies
30
have provided evidence that this process relies on the interplay
between receptor tyrosine kinases and adhesion receptors (Huang
and Reichardt, 2001; Marshall, 1995; Rossino et al., 1990; Viollet
and Doherty, 1997)]. It is well known that extracellular matrix
proteins such as laminin and their receptors, and cell adhesion
molecules such as NCAM are required for cell adhesion, neurite
growth and synaptic formation (Doherty and Walsh, 1996; Kolkova et
al., 2000; Taira et al., 1993; Viollet and Doherty, 1997; Yip and Siu,
2001). The concept that adhesion molecules work mainly by
providing an adhesive surface for neuronal growth cones has been
challenged by evidence that growth cones integrate a variety of
growth-promoting and inhibitory signals and translate them into
directed locomotion. Cell adhesion molecules can activate novel
signaling pathways in the growth cone by the recruitment of growth
factor receptors leading to neurite outgrowth (Ivankovic-Dikic et al.,
2000; Viollet and Doherty, 1997). On the other hand, nerve growth
factor receptors, epidermal growth factor receptors and insulin
receptors have also been shown to promote neurite outgrowth in
PC12 cells via sustained activation and nuclear translocation of the
ERK MAP kinase (Dikic et al., 1994; Marshall, 1995; Traverse et al.,
1994). Integrin binding to extracellular matrix enhances growth
factor-induced differentiation in these cells (Fujii et al., 1982;
Tomaselli et al., 1987). Studies of integrin regulation of the ERK MAP
kinase illustrate a critical distinction between transient and sustained
signals. Integrin engagement in the absence of growth factors
induces a transient induction of ERK activity that is not sufficient to
induce neurite formation or cell cycle progression (Ivankovic-Dikic et
al., 2000; Roovers et al., 1999). Integrin engagement also stimulates
other signaling pathways including Rho family GTPases and their
effectors (Assoian and Schwartz, 2001), that could be the direct link
31
between incoming signals and the regulation of actin dynamics and
cell adhesion in the neuronal growth cone (Kozma et al., 1997;
Lamoureux et al., 1997; Nishiki et al., 1990; Sebok et al., 1999; Tigyi
et al., 1996)]. Consistent with this hypothesis, the expression of
dominant-negative mutant Rac inhibited neurite growth induced by
NGF, whereas Cdc42 was shown to act upstream of Rac1 and
promotes neurite outgrowth by regulating the formation of growth
cone filopodia and lamellae (Kozma et al., 1997; Lamoureux et al.,
1997). Inactivation of Rho by C3 also blocks growth cone collapse
induced by lysophosphatidic acid, whereas microinjection of Rho or a
constitutively active form of Rho causes growth cone collapse
(Mackay et al., 1995). Thus, neurite outgrowth may be regulated as
the Rho and Cdc42/Rac pathways within growth cones compete,
perhaps for limited resources, resulting in either the establishment of
growth cone lamellar and filopodial structures (via Cdc42/Rac) or the
loss of these structures (via the Rho pathway).
32
II
A.
Present investigation
Alternative splicing of Pyk2 in hematopoietic cells
(Paper I)
This
project
evolved
from
an
initial
observation
that
in
hematopoietic tissues Pyk2 runs as a doublet with an apparent
molecular weight of 106,000 and 110,000 when analyzed by SDSPAGE (Dikic et al., 1998). Such finding suggested that different Pyk2
isoforms exist in hematopoietic cells.
In order to determine the expression profile of these two forms
of Pyk2, we collected different tissue lysates including spleen,
thymus, brain, lever, heart and lung, and subjected them to
immunoprecipitation with Pyk2 antibodies. The presence of the two
forms of Pyk2 was observed in spleen and thymus, and to lesser
extent in liver, heart and lung. We ruled out the possibility that the
observed Pyk2 doublets are degradation products due to proteolysis
or due to differences in phosphorylation state of Pyk2, by using
specific inhibitors of proteases and phosphatases. These treatments
did not affect the pattern of Pyk2 migration in SDS-PAGE gels
suggesting that the two forms of Pyk2 are different Pyk2 isoforms
present in those tissues.
For further identification of Pyk2 isoforms we employed a PCR
approach. cDNAs prepared from mouse brain and thymus mRNAs
were used as templates and primed with pairs of oligonucleotides
corresponding to the non-catalytic amino- and carboxyl-terminal
regions of Pyk2. Nucleotide sequence analysis of PCR products
revealed that the shorter product obtained from the carboxyl
terminus of Pyk2 in spleen represented an in-frame deletion of 126
base pairs. This led to the removal of 42 amino acid residues from
the first proline-rich region of the carboxyl terminus of Pyk2. Since
33
this isoform was preferentially expressed in hematopoietic cells we
named it Pyk2-H. In order to find out intron-exon boundaries, long
range PCR was performed using a genomic clone of Pyk2 as a
template and oligonucleotide primers corresponding to the adjacent
exons. Nucleotide sequence analysis revealed that the spliced exon is
preceded by a 2.2-kilobase intron and followed by a 4.8-kilobase
intron. In addition, a consensus sequence for RNA splicing (5’GT- and
-AG3’) was identified surrounding the spliced exon of Pyk2. Taken
together, these results pointed out that alternative RNA splicing in
the carboxyl terminus of Pyk2 generates the Pyk2-H isoform. Raising
isoform-specific anti-Pyk2 antibodies that recognize only the spliced
exon and therefore could distinguish Pyk2 from spliced form of Pyk2
at the protein level, enabled us to further explore expression pattern
of Pyk2-H versus Pyk2.
Pyk2-H was found to be mainly expressed in hematopoietic
tissues and cells, with the highest expression in the spleen and
thymus. Furthermore, expression of Pyk2-H appears to be restricted
to specific hematopoietic cells, such as thymocytes, B cells, and
natural
killer
cells,
while
Pyk2
is
present
in
platelets,
megakaryocytes, mast cells, and neuronal cells. Such tissue and cell
specific expression of Pyk2 suggests that an alternative splicing event
might provide specificity in signaling between Pyk2 and Pyk2-H in
hematopoietic versus neuronal cells. Moreover, we found that Pyk2-H
and not Pyk2 is the main isoform that is phosphorylated in response
to antigens and inflammatory cytokines in primary hematopoietic
cells
The spliced exon of Pyk2 contains 42 amino acids enriched in
proline, serine, and threonine residues, suggesting that this region
may mediate protein-protein interactions, be a target of proline
directed serine/threonine kinases, or its removal might lead to
34
changes in the intrinsic tyrosine kinase of Pyk2. We tested these
hypotheses and could show that the structural changes in the
carboxyl terminus of Pyk2-H did not influence its in vitro kinase
activity. However, the carboxyl termini of Pyk2 and Pyk2-H may
mediate both common and differential interactions with cellular
proteins. We found that paxillin binding to Pyk2-H is not affected by
the splicing event and is very similar to that of Pyk2. However, we
observed a p115 tyrosine phosphorylated protein that preferentially
binds to the carboxyl terminus of Pyk2 and not Pyk2-H.
Further functional characterization of the Pyk2-H isoform, as
well as comparing and analyzing the properties of Pyk2 and Pyk2-H
could in turn explain the functional diversity mediated by Pyk2.
Moreover, two additional groups have confirmed the identical splicing
event in CADTK/Pyk2 (Li et al., 1998; Xiong et al., 1998).
B. Grb2 and Crk link Pyk2 with ERK and JNK
activation (Paper II)
At the time when this study was initiated, there were already
reports describing the role of Pyk2 as an upstream regulator of MAP
kinase cascades, including ERK, JNK, and p38MAPK (Della Rocca et
al., 1999; Dikic et al., 1996; Ganju et al., 1998; Tokiwa et al., 1996;
Yu et al., 1996). However, very little was known about the precise
molecular mechanisms by which Pyk2 regulates specific MAP kinase
signaling pathways in response to different stimuli or in different
cells.
To address these questions, we used overexpression of Pyk2
in HEK293T cells that leads to activation of Pyk2 kinase activity as
well as activation of both ERK and JNK MAP kinases. The first step
was to generate a series of expression vectors encoding for different
Pyk2 mutants and to characterize their ability to phosphorylate
35
cellular proteins and to activate ERK and JNK pathways. Following
transient
transfections,
cell
lysates
were
subjected
to
immunoprecipitation with specific antibodies and immunoblotting, or
to in vitro kinase assays. JNK activity was determined by in vitro
phosphorylation of glutathione S-transferase (GST) c-Jun (1-79)
fusion protein. ERK2 activation was measured by immunoblotting
with antibodies specific for activated ERK2 or by in vitro kinase
reaction using myelin basic protein (MBP) as a substrate.
We found that autophosphorylation of Pyk2 on tyrosine 402 as
well as Tyr-402-linked activation of Src kinases are critical steps in
mediating phosphorylation of a broad panel of cellular proteins and
activation of both ERK and JNK pathways. The formation of a Pyk2Src complex via direct binding of the SH2 domain of Src to Tyr-402
might have a dual function. On one hand, it leads to opening of Src
structure and subsequent activation of Src kinase domain. On the
other hand, it enables Src to phosphorylate Pyk2 within the carboxyl
terminus (Tyr-881) which promotes Grb2 binding, and probably also
within the catalytic domain (Tyr-579 and Tyr-580) as a prerequisite
for full kinase activity of Pyk2. In addition, activated Src bound to
Pyk2 might directly phosphorylate adjacent cellular proteins, such as
Shc and p130Cas, and thus propagate signals downstream of Pyk2.
In the second part we focused on identification of downstream
proteins that link Pyk2 with the activation of distinct MAP kinases.
We used dominant interfering forms of adaptor and docking proteins
Grb2, Sos, p130Cas and Crk, that were previously shown to be
involved in Pyk2-induced MAP kinase signaling. Pyk2 was coexpressed with dominant interfering mutants of these proteins and
ERK and JNK activities were assayed as previously described. The
results from these studies as well as use of different Pyk2 mutants
indicated that the Grb2/Sos complex was essential in coupling Pyk2
36
with ERK. Downstream signal is transmitted directly by binding of the
Grb2 SH2 domain to phosphorylated tyrosine 881, and indirectly via
Grb2 binding to tyrosine-phosphorylated Shc proteins. It seems that
an Shc-linked Grb2/Sos pathway is largely sufficient to substitute for
the loss of the direct Grb2 binding to Pyk2 when compared with the
ability of Pyk2 and Pyk2-Y881F mutant to activate ERK. Surprisingly,
Grb2 with a deletion of the carboxyl-terminal SH3 domain was also
found to inhibit Pyk2-induced JNK activity. This suggests the
existence of additional effectors bound to the SH3 domain of Grb2
that are involved in Pyk2-induced activation of JNK. In addition,
recent findings propose that the carboxyl-terminal SH3 domain of
Grb2 can directly bind to cytoplasmic serine/threonine kinases and
thus directly activate JNK.
Regarding the p130Cas/Crk complex, this study has shown that
Src, and not Pyk2 itself, mediate phosphorylation of p130Cas and
that formation of a p130Cas/Crk complex specifically links Pyk2 with
the activation of JNK. The fact that dominant interfering mutants of
p130Cas and Crk only partially inhibited (up to 50-60%) Pyk2induced JNK activation, indicated the presence of redundant signaling
pathways involved in Pyk2-mediated JNK activation. Other cellular
proteins that constitutively bind to Pyk2 might therefore be involved
in regulation of the JNK cascade (Pax, Graf, Nirs, Pap). From our data
paxillin appears not to be necessary for coupling Pyk2 with Crkmediated JNK activation. Crk can further recruit different effector
proteins, such as C3G and DOCK180 to the Pyk2 complex, leading to
JNK activation, while Grb2 translocates the guanine exchange factor
Sos, which is necessary for Pyk2-induced ERK activation.
In a simplified scenario Pyk2 activates different MAP kinases as
follows:
37
Pyk2 is activated by various extracellular signals via not yet fully
understood mechanisms. However, an increase in intracellular Ca2+
and the activation of PKC seem to be common pathways involved in
activation of Pyk2 in response to a wide range of stimuli. Following
activation, Pyk2 is autophosphorylated on the tyrosine at position
402, which in turn creates a direct binding site for the SH2 domain of
Src. Binding of the SH2 domain of Src to autophosphorylated Tyr-402
leads to a Src activation, which is critical for
- phosphorylation of Pyk2 itself , therefore promoting Grb2 binding as
well as enhancing Pyk2 kinase activity
- phosphorylation of several adjacent cellular proteins, such as Shc
and p130Cas, thus creating direct binding sites for SH2 domains of
adaptor proteins Grb2 and Crk, and transmitting signals downstream
of Pyk2 toward different MAP kinase pathways.
Calcium
PKC
Src
P
Pyk2
P
p130Cas
P
Pyk2
P
Src
Crk
Crk effectors
e.g. DOCK180, C3G
P
Shc
Grb2
Sos
Grb2
Sos
JNK
cascade
ERK/MAPK
cascade
Pathways linking Pyk2 with ERK/MAPK and JNK cascades
38
Nevertheless, the most interesting aspect of the present work is
perhaps to reveal the biology underlying Pyk2-mediated selective
activation of the ERK pathway versus the JNK pathway.
C. Pyk2 and FAK regulate neurite outgrowth induced
by growth factors and integrins (Paper III)
Integration
of
signaling
pathways
triggered
by
receptor
tyrosine kinases and integrins is essential for regulation of various
biological processes, including cell proliferation, differentiation and
migration (Assoian and Schwartz, 2001; Eliceiri and Cheresh, 2001;
Ivankovic-Dikic et al., 2000; Renshaw et al., 1997; Schneller et al.,
1997). However, very little is known about the molecular mechanisms
that link integrins to RTKs and further how signal is propagated
downstream to achieve such a variety of biological outcomes.
In this project we were interested to study the role of Pyk2
and FAK in signaling by integrins and growth factor receptors in
neuronal cells. Rat pheochromocytoma PC12 cells and human
neuroblastoma SH-SY5Y cells were selected to establish a cellular
model (designated serum-free differentiation assay) in which both
growth factor receptors and integrins are necessary for activation of
Pyk2/FAK and for regulation of neurite outgrowth. Cells were
resuspended in serum free medium and plated on plastic tissueculture dishes or on dishes coated with different ECM components
(laminin or collagen I), in the presence or absence of corresponding
growth factors. Although EGF and IGF-1 were not able to mediate the
entire process of neuronal differentiation in PC12 and SH-SY5Y cells
plated on extracellular matrix, co-stimulation of growth factor
receptors and integrins, was both sufficient and necessary for the
induction of neurite outgrowth in these cells.
39
Since ERK/MAP kinases are known to have a critical role in
growth factor-induced neuronal differentiation (Marshall, 1995), we
were interested to test their kinetics and activation in our cellular
model. The results showed that EGF and IGF-1 activate ERK to a
similar extent in PC12 or SH-SY5Y cells regardless of integrin
engagement, indicating that further signaling pathways may be
necessary to induce formation of neurites in these cells.
We next searched for proteins that might link integrins and
growth factor receptors with the ERK-independent regulation of
neurite outgrowth. Previous studies have identified adhesion kinases
Pyk2 and FAK as potential common mediators in signaling by these
receptor systems (Avraham et al., 2000; Clark and Brugge, 1995). In
PC12 cells, Pyk2 was weakly phosphorylated by plating cells on
laminin or collagen and additionally activated by EGF stimulation,
indicating that engagement of both growth-factor receptors and
integrins is necessary for Pyk2 activation in this cell type. On the
other hand, FAK was already maximally tyrosine phosphorylated
during cell adhesion in PC12 cells, whereas in SH-SY5Y cells FAK, in
the absence of Pyk2, was involved in signaling by both growth factors
and integrins.
In
addition,
we
observed
that
paxillin
and
a
tyrosine
phosphorylated protein of relative molecular weight 180,000 (Mr
180K, termed p180) co-immunoprecipitated with Pyk2 and FAK in
EGF-treated cells. Paxillin preferentially bound to Pyk2 as compared
to FAK, and tyrosine phosphorylation of Pyk2-associated paxillin was
slightly increased upon stimulation with EGF. We next investigated
whether the tyrosine-phosphorylated p180 represents the activated
EGF receptor. Eventually, Pyk2 was found in complex with small
amounts of tyrosine-phosphorylated EGF receptors in EGF-stimulated
cells. Upon longer exposure we detected EGF receptors associated
40
with Pyk2 in unstimulated cells suggesting that in cells deprived of
growth factors Pyk2 interacts with small amounts of EGF receptors,
whereas stimulation with growth factors leads to Pyk2 activation and
a
more
efficient
complex
formation
with
phosphorylated
EGF
receptors.
The nature of the interaction between Pyk2 and EGF receptors
was assessed by expressing different mutants of Pyk2 and analyzing
their abilities to associate with activated EGF receptors. We used a
kinase-inactive Pyk2 mutant (PKM), an alternatively spliced form of
Pyk2 containing only the C-terminal tail of Pyk2 (PRNK), and the Nterminal domain of Pyk2 (Pyk2(NT)). All Pyk2 mutants were able to
co-immunoprecipitate with tyrosine phosphorylated EGF receptor,
more in stimulated than in unstimulated cells, PRNK being more
potent than PKM and Pyk2-NT. These results indicate that active Pyk2
binds more readily to EGF receptors than does a Pyk2 kinase-inactive
mutant, and that both the N- and C-terminal domains of Pyk2
mediate this association. Interactions are presumably mediated via
accessory proteins as isolated N- or C-terminal domains of Pyk2 and
FAK could not bind directly to EGF receptors in an overlay assay.
Recent work (Sieg et al) suggested that FAK is one of the linkers
between integrins and EGF receptors by its ability to bind integrin
complexes via its C-terminal tail and EGF receptor complexes via its
N-terminal domain. It is important to say that neither interaction is
direct. In this way FAK acts as an indirect bridge between these
receptors. Therefore, both Pyk2 and FAK may act as proximal linkers
between integrins and EGF receptors in different cell types.
To further investigate whether Pyk2 and FAK are necessary for
growth factor-induced neurite outgrowth in PC12 and SH-SY5Y cells
plated on laminin and collagen we tested if dominant interfering
forms of Pyk2 e.g. PKM and PRNK, can block the formation of
41
neurites. PKM and PRNK are thought to inhibit neurite outgrowth by
competing with endogenous Pyk2 for interactions with downstream
effectors and thereby interrupting the transmission of signals from
integrin and growth factor receptors. Inhibition of neurite formation
was observed in cells transfected with PKM and PRNK and stimulated
with EGF, while expression of the same dominant interfering forms
did not influence activation of MAP kinases following EGF stimulation.
Our data thus indicate that Pyk2/FAK-mediated signals primarily
regulate morphological changes associated with neurite formation and
are less critical for signaling cascades that control gene transcription
in the nucleus.
At
last,
we
focused
on
understanding
the
downstream
mechanisms by which the carboxyl terminus of Pyk2/FAK may
regulate neurite formation. For that purpose we introduced different
dominant interfering mutants of paxillin, p130Cas and Crk and also
mutant or deleted forms of PKM unable to bind to either paxillin or
p130Cas. Expression of PKM lacking the adhesion targeting domain
(ATD) did not affect neurite outgrowth upon EGF stimulation, whereas
PKM containing point mutations in the proline-rich sequences was as
effective as PKM in blocking formation of neurites. These results
indicate that PKM requires the ATD to efficiently interfere with
endogenous
Pyk2,
whereas
interactions
involving
SH3-domain-
containing proteins seem to be less important in this regard. This was
consistent with the lack of inhibitory effect of dominant interfering
forms of p130Cas and Crk on neurite formation. On the other hand,
expression of a paxillin-LD4 deletion mutant (Paxillin-∆LD4) led to a
similar inhibition of neurite formation as it has been observed with
PKM, suggesting that effectors of LD4 domain are necessary for the
control of neurite formation. Previous studies have shown that the
LD4 domain forms a multiprotein complex containing PAK, the PAK
42
exchange factor PIX and the paxillin-kinase linker with ARF-GAP
activity (p95PKL) to control actin reorganization and lamellipodia
formation (Turner et al., 1999). Taken together our data indicated
that paxillin may act as an intermediate between Pyk2/FAK and the
regulation of neurite formation.
Actin
Integrins
EGF
receptor
α
β
K
xi
lli
n
k2
Py
Pa
K
A
/F
PA
X
PI
PKL
p95
Rho GTPases
WASP
Arp2/3
Growing
Neurite
Hypothetical model of Pyk2/FAK involvement in the regulation of
cytoskeletal changes associated with neurite outgrowth
Taken together, our results suggest that Pyk2 and FAK have
overlapping and distinct roles in the regulation of integrin/growthfactor receptor signaling in neuronal cells. Specific growth-factor
receptors associate with integrin-linked complexes, leading to the
activation of Pyk2 and FAK and subsequent paxillin-dependent
propagation of signals that are responsible for neurite formation. It is
possible that activated FAK act as a proximal linker between integrins
and EGF receptors in growth-factor-deprived cells, whereas activated
43
Pyk2 additionally contributes to complex formation upon growth
factor stimulation. The functional significance of paxillin effectors
associated with neurite outgrowth and the molecular interactions
involved remain to be investigated.
In conclusion, this thesis work has made a contribution to the
understanding of molecular mechanisms underlying the role of Pyk2
in intracellular signaling with the emphasis on the regulation of
mitogenic signals and cell cytoskeleton. However, along this way and
as a consequence of our findings, new scientific questions have
emerged leaving us with exciting challenges for future investigations.
44
III Future perspectives
While working on this thesis I became entirely aware of the
simple and not written, but rather general scientific rule that every
single finding gives rise to many new questions, which will in turn
make many new reasons to continue the research work.
Identification and cloning of a novel isoform of protein tyrosine
kinase
Pyk2
(Pyk2-H)
initiated
further
studies
regarding
the
characterization and potential biological role of Pyk2-H (Dikic et al.,
1998). Given the fact that the splicing event in Pyk2-H does not
change its catalytic activity, the identification of the proteins that
associate specifically with the residues of the spliced exon of Pyk2 or
that bind specifically to the shorter carboxyl terminus of Pyk2-H could
be an approach to enlighten the role of this region in the control of
Pyk2 and Pyk2-H functions. Perhaps the two-hybrid system and
biochemical purification are methods to consider in such efforts.
Elucidating the potential biological role of Pyk2-H in comparison to
Pyk2, meaning primarily the molecular mechanisms by which Pyk2
and Pyk2-H may signal differently, still remains a challenge. In
addition, we demonstrated that Pyk2-H, and not Pyk-2, is the main
isoform that is activated by antigen or chemokine receptors in
primary hematopoietic cells (Dikic et al., 1998). This finding suggests
a potential role of Pyk2-H in host immune responses as well as raises
yet more new questions regarding the signaling pathways involved in
such a cell type specific response. A recent report indicated that a
deficiency of Pyk-2/Pyk2-H in mice results in a cell autonomous
defect of marginal zone B cells in spleen (Guinamard et al., 2000).
Because of their position adjacent to the marginal sinuses, marginal
zone B cells are amongst the first population of cells seen by bloodborn antigens and are presumed to have a critical role in host
45
defense against bacterial pathogens. It is not yet clear how
Pyk2/Pyk2-H may control migration of B cells from spleen pulp to the
marginal zone. One hypothesis is that B cells are attracted by the
action
of
specific
chemokines
including
SDF-1,
BLC
and
SLC
(Guinamard et al., 2000). This finding is consistent with our results
showing that chemokines SDF-1, RANTES and MIP-1b activate Pyk2H in hematopoietic cells (Davis et al., 1997), suggesting that Pyk2-H
may also play an essential role for migration of B cells in response to
chemokines in spleen. Yet these interesting possibilities have not yet
been confirmed in vivo.
Our data also demonstrated that the majority of biological
functions of Pyk2 are dependent on its ability to regulate transcription
largely via activation of MAP kinase cascades as well as regulation of
dynamic structures of adhesion sites and the actin cytoskeleton.
These functions are deregulated during pathogenic conditions such as
tumor development. Pyk2 and FAK were suggested to contribute to
cell transformation by coordinating signaling networks induced by
adhesion, mitogenic signals and oncogene products. This is at least in
part
controlled
by
the
ability
of
Pyk2/FAK
to
regulate
gene
transcription via activation of cytoplasmic kinase cascades. However
little is still known about gene targets of Pyk2/FAK signals that may
be of relevance when comparing normal versus transformed cells. By
using gene microarrays one could identify targets induced by
activation of Pyk2/FAK in normal and transformed cell. Yet another
interesting question is what are the downstream signaling cascades
responsible for the ability of Pyk2 to trigger gene expression. In other
words whether differences we observed in the effectors coupling Pyk2
with ERK versus JNK pathway are also reflected in the profile of genes
expressed. This could be tested by expressing Pyk2 together with
dominant interfering mutants of Grb2 or Crk followed by microarray
46
analysis. One could anticipate that mutual interactions between
components of ERK and JNK pathways will correlate well with the
majority of genes triggered while a minor proportion of genes may be
specific for each pathway in a particular cell type and in response to
specific stimuli.
We have also shown that FAK and Pyk2 act as proximal
effectors of integrin and growth factor receptors important for the
regulation of cell migration and neurite outgrowth (Ivankovic-Dikic et
al., 2000; Sieg et al., 2000). Moreover, a sufficient body of literature
demonstrates an involvement of Pyk2 and FAK in invasion and
metastasis of breast, brain, colon, and prostate cancers, cerebral
metastases and migration of breast cancer cell lines (Agochiya et al.,
1999; Blume-Jensen and Hunter, 2001; Kornberg, 1998; Ludwig et
al., 2000; Stanzione et al., 2001; Zheng et al., 1999; Zhu et al.,
1999; Zrihan-Licht et al., 2000). Inhibition of Pyk2/FAK functions in
vivo
could
thus
prove
useful
in
blocking
tumor
growth
and
metastases. Development of inhibitors of Pyk2/FAK functions in vivo
by using a Pyk2/FAK antisense oligonucleotide-based approach or
small inhibitory RNA molecules could prove useful in inhibiting tumor
progression and metastases.
47
IV
Acknowledgements
This study was carried out at the Ludwig Institute for Cancer
Research in Uppsala. Behind this name there are many people who
have contributed significantly to this work by giving a valuable input
and advices, challenges, understanding, support and love. Ludwigs
were my second every-day family, circle people and fellow travelers:
I thank you all, who in one way or another have contributed to this
thesis, for the energy exchanged and the synergy it has created!
In particular I would like to extend my gratitude to:
Ivan Dikic, who has bravely chosen to ride this wave with me and
who has generously put his enormous energy and enthusiasm,
dreams and passion behind all projects. Infallibly seeing the complete
picture, he gave me the opportunity that I always dreamed of and
somehow, yet only he knows how, made me make it possible. By
putting the two together, maintaining my state of mind in troubled
times
and
providing
me
with
the
necessary
inspiration
and
excitement to continue my journey, he was a teacher unlike any
before or after.
Professor
Carl-Henrik
Heldin,
for
his
respect,
kindness
and
supervision over the years. For running the institute in a way that
creates a vibrant and international yet open and friendly scientific
environment.
All former and present members of the LICR for fruitful and enjoyable
collaborations, stimulating scientific chats, cheerful conversations and
honest friendship in and out of the lab.
48
Andree Blaukat for being skillful and knowledgeable yet endlessly
patient when answering my questions, as well as for sharing some
unforgettable moments while putting wallpapers, buying our first car
and participating in the 1st ICST conference in Dubrovnik.
Eva Grönroos for uplifting conversations and reflections.
Kaisa Haglund for her kindness, for making me less homesick and
especially for her hard work and persistent efforts in finishing our last
project.
Iwona Szymkiewicz for many nice lunches and honest friendship.
Noriaki Shimokawa, Philippe Soubeyran, Lucie Hajkova, Kaska
and Marcin Kowanetz for making every day in a lab stimulating and
enjoyable.
Countless Ivankovic & Dikic families for reminding me about the
most important things in life. My beloved parents for being an endless
source of love and support; mama Danica, who has never let me
down and taught me kindness along with persistence; tata Branko,
for being so forward, knowing me so well, thus humoring my
struggling efforts to be the one who is easy to deal with.
My dear mother-in-law Ankica who has been tirelessly taking care of
us, never asking one single thing in return, accepting me as I am and
making it possible for me to live a complete life including career,
family and a lot of fun.
At last, to Karla my guardian angel who has graced me with her
unconditional love, to my beloved son Lovro and to the same Ivan
from the first paragraph for giving me a full happiness in life.
49
V
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