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NEOPLASIA
Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate
B-myb at functionally important serine and threonine residues:
tissue-specific regulation of B-myb function
Carsten Müller-Tidow, Wenbing Wang, Gregory E. Idos, Sven Diederichs, Rong Yang, Carol Readhead,
Wolfgang E. Berdel, Hubert Serve, Mark Saville, Roger Watson, and H. Phillip Koeffler
Cyclin A1 is tissue-specifically expressed
during spermatogenesis, but it is also
highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML
and in the cell cycle of leukemic blasts is
unknown. B-myb is essential for G1/S
transition and has been shown to be
phosphorylated by the cyclin A2/cdk2
complex. Here it is demonstrated that
cyclin A1 interacts with the C-terminal
portion of B-myb as shown by glutathione S-transferase (GST) precipitation.
This interaction is confined to cyclin A1
because binding could not be detected
between cyclin A2 and B-myb. Also, cdk2
was not pulled down by GST–B-myb from
U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in
vivo. Baculovirus-expressed cyclin A1/
cdk2 complexes were able to phosphorylate human as well as murine B-myb in
vitro. Tryptic phosphopeptide mapping
revealed that cyclin A1/cdk2 complexes
phosphorylated the C-terminal part of Bmyb at several sites including threonine
447, 490, and 497 and serine 581. These
phosphorylation sites have been demon-
strated to be important for the enhancement of B-myb transcriptional activity.
Further studies showed that cyclin A1
cooperated with B-myb to transactivate
myb binding site containing promoters
including the promoter of the human cyclin A1 gene. Taken together, the data
suggest that cyclin A1 is a tissue-specific
regulator of B-myb function and activates
B-myb in leukemic blasts. (Blood. 2001;
97:2091-2097)
© 2001 by The American Society of Hematology
Introduction
Important differences in the cell cycle machinery are obviously
necessary to direct processes as different as mitosis and meiosis.
On the other hand, several cell cycle regulatory proteins (eg,
retinoblastoma [Rb]) are expressed in meiotic cells as well as in
cells going through the cell cycle of somatic cells.1 Cyclin A1 has
been demonstrated to be essential for spermatogenesis,2 and
high-level expression in the healthy organism is restricted to
pachytene spermatocytes undergoing meiosis.3 We reported previously that human cyclin A1 is also expressed in acute myeloid
leukemia (AML) and in cell lines derived from leukemic blasts.4,5
In addition, cyclin A1 directly interacts with E2F-1 and the Rb
family of proteins.6 However, the functions of cyclin A1 in the cell
cycle of somatic cells and especially its role in the pathogenesis of
AML have remained elusive.
B-myb plays an important role in the cell cycle of somatic cells,
and its expression sharply increases at the G1/S transition.7 It is
ubiquitously expressed in proliferating somatic cells and is thought
to be essential for G1/S progression. B-myb expression correlates
with proliferation in human hematopoietic cells,8 and antisense
oligonucleotides to B-myb inhibit proliferation of hematopoietic
cell lines.9 B-myb was found to be strongly expressed in non-small
cell lung cancer.10 In addition, high levels of B-myb are associated
with increased bcl-2 expression and resistance to apoptosis.11
B-myb expression has been demonstrated in male germ cells.12,13
The exact stage of spermatogenesis where B-myb expression can
be found is unclear. Whereas one group12 reported B-myb expression in spermatogonia, Sitzmann and colleagues13 provided evidence for B-myb expression during the first meiotic division.
The B-myb–mediated transactivation of mammalian promoters
is cell-type specific14 and might depend on the B-myb phosphorylation status.15 The transactivation capacity of B-myb in transient
transfections can be markedly enhanced by cotransfection of cyclin
A2 (also known as cyclin A) and cyclin E.16 B-myb is phosphorylated in a cell cycle–dependent manner, and the cyclin A2/cdk2 and
cyclin E/cdk2 complexes phosphorylate B-myb at several sites.17-19
Phosphorylation of B-myb is restricted to some cyclin/cdk complexes; for example, cyclin D1/cdk4 cannot phosphorylate Bmyb.18 In addition, a B-myb/cyclin D1 complex strongly inhibits
B-myb activity.20 Although cyclin A2/cdk2 complexes phosphorylate B-myb, a stable interaction between B-myb and cyclin A2 has
From the Department of Medicine, Hematology, and Oncology, University of
Münster, Germany; the Division of Hematology/Oncology, Cedars-Sinai
Research Institute/UCLA School of Medicine, Los Angeles, CA; the Division
of Biology, California Institute of Technology, Pasadena, CA; and the Ludwig
Institute for Cancer Research, Imperial College School of Medicine,
London, England.
Foundation and the Innovative Medizinische Forschung (IMF) Program at the
University of Münster to C.M.; and by project grant SP2358/0101 from The
Cancer Research Campaign to M.S. H.P.K. holds the Mark Goodson Chair in
Oncology Research and is a member of the Jonsson Cancer Center.
Submitted May 11, 2000; accepted November 21, 2000.
Supported by grants from the National Institutes of Health, Bethesda, MD; the
U.S. Department of Defense, the Parker Hughes and C. and H. Koeffler Funds,
the Lymphoma Foundation of America, the Horn Foundation to H.P.K.; and by
grants from the Deutsche Forschungsgemeinschaft (Mu-1328/2-1), the
Deutsche Krebshilfe (10-1539-Mü1), the Deutsche José-Carreras Leukemia
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
Reprints: Carsten Müller-Tidow, Department of Medicine, Hematology and
Oncology, University of Münster, Domagkstr 3, Münster, Germany; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
2091
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2092
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
MÜLLER-TIDOW et al
not been detected. The regulatory role of cyclin A1 for B-myb is
currently unknown.
Here we present data suggesting that cyclin A1 is an important
partner of B-myb. Cyclin A1 and B-myb co-immunoprecipitate
from leukemic cells in vivo, and cyclin A1 is able to interact
directly with B-myb. Baculovirus-expressed human cyclin A1/
cdk2 complexes can phosphorylate murine as well as human
B-myb in vitro at several sites that have been shown to be
functionally relevant. Phosphorylation is likely to be the mechanism by which cyclin A1 can activate B-myb to transactivate the
human cyclin A1 promoter. Our findings suggest that cyclin A1
might be a tissue-specific regulator of B-myb function and that
cyclin A1 activates B-myb function in leukemic blasts.
Materials and methods
Plasmids
The human cytomegalovirus (CMV)–B-myb expression plasmid, a gift of
Dr Sala, has been described previously.16 The myb-TK (thymidine kinase)
plasmid, gift of Dr Klempnauer, consists of a minimal thymidine kinase
promoter and several myb consensus binding sites driving expression of a
luciferase reporter gene. The human glutathione S-transferase (GST)–Bmyb plasmid was constructed by PFU–polymerase chain reaction of the
CMV–B-myb plasmid using gene-specific primers encompassing restriction sites for BamHI and NotI. The PCR product was then cloned in frame
into pGEX-2T. The murine GST–B-myb fusion plasmid and the generation
of point mutations in predicted cdk2 phosphorylation sites have been
described previously.18 GST fusion proteins encompassing the C-terminal
portion of B-myb were constructed by either restriction digestion of the
full-length plasmid (PvuII and XbaI) and religation or PFU-PCR amplification of the desired part of B-myb (base pair [bp] 819-2640) and subsequent
cloning into pGEX-2T. The plasmid encoding the N-terminal portion of
B-myb was constructed by restriction digestion of the full-length GST–Bmyb plasmid with SalI and EcoRI, Klenow fill-in, and subsequent ligation.
GST fusion proteins were expressed in BL21 cells and purified using glutathione
sepharose beads as described.18 The cyclin A1 promoter (⫺190 to ⫹145)
luciferase reporter plasmid, the pCDNA3-cyclin A1 expression plasmid, and the
baculovirus expression plasmids have been described previously.5,18,21
Co-immunoprecipitation and GST precipitation
Co-immunoprecipitation was carried out as described.6 The antibody
against B-myb (Santa Cruz Biotechnology, Santa Cruz, CA) was purchased.
GST fusion proteins were prepared as described above. The amount of
either GST fusion proteins or GST alone was standardized on a protein gel
stained with Coomassie blue prior to the precipitation. Lysates of U937
cells were prepared in RadioImmuno Precipitation Assay (RIPA) buffer;
these lysates were incubated with GST fusion proteins for 2 hours at 4°C.
Beads were washed 4 times in RIPA buffer, denatured in sodium dodecyl
sulfate (SDS) sample buffer, and run on a 4% to 15% gradient gel. Western
blot analysis was performed using antibodies against cyclin A1, cyclin A2,
and cdk2 as described.6
In vitro kinase assays and tryptic phosphopeptide mapping
Cyclin A1 and cdk2 were baculovirus-expressed in Sf9 insect cells, and in
vitro kinase assays were performed as described.6 Tryptic phosphopeptide
mapping was performed essentially as described22 using the pH 1.9
electrophoresis buffer and the phosphochromatography buffer for ascending chromatography.
Cell lines and transfection
U937 and NB4 cells were grown in Roswell Park Memorial Institute
(RPMI) medium plus 10% fetal calf serum (FCS) as described.23 CV-1 cells
were cultured in Dulbecco modified essential medium (DMEM) supple-
mented with 10% FCS containing 100 U/mL penicillin and 100 ␮g/mL
streptomycin. CV-1 cells were transfected in 60-mm plates using Superfect
(Qiagen, Hilden, Germany) following the protocol of the manufacturer. A
total of 6.5 ␮g DNA, which consisted of 0.5 ␮g luciferase reporter plasmid,
1 ␮g CMV–␤-gal expression vector (or 20 ng pRL-SV40 expression
vector), and where appropriate, 1 ␮g CMV–B-myb and pcDNA3/cyclin A1,
was transfected. Empty expression vector was used to equalize the amount
of DNA. Transfections were performed in duplicate, and ␤-galactosidase
activity or renilla luciferase activity was used to standardize for transfection
efficiency. Values present the mean ⫾ SE of 3 independent experiments.
Expression of B-myb in male germ cells
The generation of transgenic mice expressing Enhanced Green Fluorescence Protein under control of the cyclin A1 promoter is described
elsewhere.24 Frozen tissue sections from male testis were analyzed using a
Zeiss confocal laser scanning microscope (Zeiss, Jena, Germany). The flow
cytometric cell sorting of testis cells has been described previously.24
EGFP-expressing and EGFP-nonexpressing cell populations were sorted,
and equal amounts of proteins (20 ␮g) were loaded on a 4% to 15% gradient
gel. The anti–B-myb antibody mentioned above was used to analyze B-myb
expression in both cell populations. To analyze whether the 2 strong bands
detected by the anti–B-myb antibody represented differentially phosphorylated forms of B-myb, RIPA lysates from mouse testis were incubated with
Protein Phosphatase 1 (New England Biolab, Beverly, MA) for 2 hours at
room temperature before addition of the SDS buffer and loading of the gel.
Results
Cyclin A1/cdk2 complexes phosphorylate B-myb
of human and murine origin in vitro
Cyclin E and cyclin A2 have previously been shown to activate
B-myb in transient cotransfection assays and to direct cdk2 to
phosphorylate B-myb. Other cyclins, such as cyclin B1 or D1,
failed to activate or even inhibit B-myb.16,20 To study potential
phosphorylation of B-myb by cyclin A1/cdk2, we expressed cyclin
A1 and cdk2 in Sf9 insect cells using a baculovirus system. Strong
phosphorylation in an in vitro kinase assay using phosphorous-32–
adenosine 5⬘-triphosphate (32P-ATP) occurred only with lysates
expressing cyclin A1 and cdk2 together, but not with either one
expressed alone (Figure 1). Both human and murine GST–B-myb
became phosphorylated. These findings indicate that cyclin A1 can
direct cdk2 to phosphorylate B-myb.
Tryptic phosphopeptide mapping
Several sites in the C-terminal portion of murine B-myb have
previously been identified as targets for phosphorylation by cyclin
A2/cdk2 complexes in vitro and in vivo.18,19 To analyze cyclin
A1/cdk2 phosphorylation sites in the C-terminal portion of B-myb,
we incubated the murine GST–B-myb fusion protein (amino acid
[aa] 225-705) with 32P-ATP and cyclin A1/cdk2 expressed in insect
cell lysates. The 32P-phosphorylated B-myb protein was run on a
protein gel and excised after drying and autoradiography. Subsequent to purification and solubilizing, the protein was digested to
completion using trypsin. The phosphopeptides were resolved in 2
dimensions on TLC plates and exposed to film for 2-6 days. More
than 20 spots indicating phosphorylated peptides were observed on
the film (Figure 2). B-myb contains more than 20 sites that possess
the minimum specificity for cdk phosphorylation serine/threonineproline (S/T-P). To identify specific sites of phosphorylation, we
used GST–B-myb fusion proteins that harbored point mutations at
potential target sites. Wild-type and mutant GST–B-myb fusion
proteins were then phosphorylated in vitro by cyclin A1/cdk2,
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
Figure 1. In vitro kinase assay for human GST–B-myb (full length) and murine
GST–B-myb (aa 225-705). GST fusion proteins were phosphorylated in vitro by
cyclin A1 and cdk2 expressed in Sf9 lysates using 32P-ATP. Neither cyclin A1 nor cdk2
alone phosphorylated B-myb, but the combination of both led to strong phosphorylation of the murine as well as the human B-myb.
CYCLIN A1 INTERACTS WITH B-MYB
2093
presence or absence of cyclin A1. Initially we used a construct
containing a series of myb-consensus sites upstream of a minimal
TK promoter. These experiments confirmed that neither B-myb nor
cyclin A1 alone activated the promoter. However, co-expression of
both genes led to more than a 60-fold induction of luciferase
activity. These findings provide clear evidence that cyclin A1 can
activate B-myb (Figure 3). Previously we demonstrated that C-myb
transactivates the human cyclin A1 promoter.23 Because cell cycle
regulatory genes are often autoregulated (eg, the E2F gene), we
analyzed whether B-myb either alone or activated by cyclin A1
could transactivate the human cyclin A1 promoter. A CMV–B-myb
expression plasmid was cotransfected into CV-1 cells with a
luciferase reporter containing ⫺190 to ⫹145 bp of the promoter of
human cyclin A1. An empty expression vector was cotransfected
with the luciferase construct as a control (Figure 3). The forced
expression of B-myb alone did not transactivate the cyclin A1
promoter in CV-1 cells, but it reduced promoter activity by 20%.
Also, cyclin A1 alone did not have any effect on promoter activity.
However, B-myb and cyclin A1 together significantly activated the
cyclin A1 promoter, leading to a 3-fold increase in promoter
activity (Figure 3).
B-myb interacts with cyclin A1 in vivo
digested with trypsin, and analyzed by 2-dimensional phosphopeptide mapping. The mutations of threonine to alanine in aa 447
(447T⬎A), 490T⬎A, and 497T⬎A resulted in the loss of single
spots on the TLC plate (Figures 2B-D). The mutation of 581S⬎A
led to the loss of 3 spots. These results are similar to those obtained
for cyclin A2/cdk2 complexes.18 These phosphorylation sites have
also been identified as targets for cdk2 phosphorylation in vivo, and
their mutation resulted in a loss of B-myb transcriptional activity.18
These findings indicate that cyclin A1/cdk2 can phosphorylate
B-myb at functionally relevant amino acid residues.
To analyze for an interaction between cyclin A1 and B-myb in vivo,
RIPA buffer lysates from highly cyclin A1-expressing U937
leukemic cells were incubated with polyclonal anticyclin A1
antibody or with rabbit immunoglobulin G (IgG) as a control.
Immunoprecipitation products were separated on a 4% to 15%
gradient gel, and the blot was probed with anti–B-myb polyclonal
antibody (Figure 4). The anticyclin A1 antibody specifically
co-immunoprecipitated B-myb, which appeared as 2 bands of
approximately 95-105 kd. The 2 bands and their sizes were similar
to those described by Sala and colleagues.16 Two non-specific
bands were precipitated by control IgG as well as by the anticyclin
A1 antibody.
Cyclin A1 activates B-myb to transactivate myb
site-containing promoters
The C-terminus of B-myb interacts with cyclin A1
but not with cyclin A2
We investigated the functional relevance of the cyclin A1/B-myb
interaction by analyzing the transcriptional activity of B-myb in the
To confirm the interaction between cyclin A1 and B-myb, we used
GST fusion proteins of B-myb to precipitate cyclin A1 from RIPA
Figure 2. Identification of cyclin A1/cdk2 phosphorylation sites in the C-terminal region of murine B-myb.
Wild-type or point-mutated B-myb fused to GST were
phosphorylated in vitro by cyclin A1/cdk2 and subjected to
2-dimensional tryptic phosphopeptide mapping. (A) More
than 20 different spots were identified when wild-type
GST–B-myb was phosphorylated. Point mutations at potential phosphorylation sites were used to determine the
identity of several of these spots. (B-D) Point mutations
that changed the indicated threonine to an alanine (an
amino acid that cannot be phosphorylated) led to the
disappearance of single spots indicated by the arrow. (E)
The mutation of S581 (serine to alanine) erased 3 radioactive spots. The reason for the absence of several spots is
probably that S581 is located in a region of B-myb
containing multiple basic amino acids, and trypsin cleavage after either arginine or lysine residues might lead to
differently sized peptides containing the same S581.
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2094
MÜLLER-TIDOW et al
BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
Figure 3. Cyclin A1 activates B-myb to transactivate myb-site containing
promoters. A luciferase construct containing 3 myb-consensus binding sites upstream of a minimal thymidine kinase promoter was transiently transfected into Cos
cells (left side). Whereas no significant activation of this construct was detected by
either cyclin A1 or B-myb transfection alone, a more than 60-fold increase in
luciferase activity occurred upon cotransfection of cyclin A1 and B-myb expression
vectors. In the right side of the figure, the human cyclin A1 promoter (bp ⫺190 to
⫹145) was analyzed for transactivation by B-myb alone or by the combination of
cyclin A1 and B-myb. Cotransfection of B-myb alone decreased cyclin A1 promoter
activity by an average of 20%. Cyclin A1 alone did not have a significant effect on
cyclin A1 promoter activity. However, when cyclin A1 and B-myb were transfected
together, cyclin A1 promoter activity was induced 3-fold.
lysates of U937 cells (Figure 5). Two different fusion proteins were
employed: one containing the murine C-terminal part (aa 225-705)
of B-myb that encloses the region where cyclin A2/cdk2 complexes
have been shown to phosphorylate B-myb and the other
containing the N-terminal region (aa 1-184) of B-myb. GST
coupled to glutathione sepharose beads was used as a negative
control. The C-terminal fusion protein precipitated cyclin A1, as
Figure 5. The C-terminal portion of B-myb interacts with cyclin A1 in a GST
precipitation assay. Different portions of B-myb were fused in frame to GST
(“Materials and methods”) and bound to glutathione beads. These were incubated
with U937 lysates for 2 hours. SDS sample buffer was added to the extensively
washed beads. Proteins were electrophoresed, blotted on nitrocellulose, and subjected to Western blotting with the indicated antibodies. Lysate (20 ␮L total) and
cyclin A1 expressed in Sf9 insect cells (upper gel) were run in parallel. Overexposed
films are shown for cyclin A2 and cdk2 to demonstrate that even prolonged exposure
of the blots did not lead to the appearance of specific bands in any lane except for the
lysate. The band in the aa 1-183 lane of the cdk2 Western blot runs slower than the
band seen for cdk2.
shown by a specific band (approximately 64 kd) of the same size
as cyclin A1 from U937 lysate and recombinant protein from
Sf9 insect cells (Figure 5). No bands were seen for the
N-terminal portion of B-myb or the GST control. Interestingly,
cyclin A2 was not pulled down with GST–B-myb (Figure 5).
The antibody detected cyclin A2 in the U937 lysate, but there
was no band detected in the lane of either one of the GST–Bmyb fusion proteins, thereby providing evidence that cyclin A2
was not able to interact with B-myb in this assay. In addition,
cdk2 was not precipitated by the C-terminus of GST–B-myb,
indicating that the interaction between cyclin A1 and the
C-terminus of B-myb might be independent of cdk2 (Figure 5).
Binding of cyclin A1 to B-myb occurs independent of cdk2
Figure 4. B-myb co-immunoprecipitates with cyclin A1 in vivo. U937 leukemic
cells were lysed in RIPA buffer, and cyclin A1 was immunoprecipitated with rabbit
polyclonal anticyclin A1 antibody. Polyclonal mouse IgG antibody served as a control.
The Western blot was probed with anti–B-myb antibody. Besides the IgG band and 2
non-specific bands of similar strength in both lanes, 2 specific bands were seen that
represent B-myb in its phosphorylated and non-phosphorylated forms (compare to
Figure 7B).16
To analyze whether cdk2 was required for the binding between
B-myb and cyclin A1, we produced cyclin A1 and cdk2 as
recombinant proteins in a baculovirus expression system in Sf9
cells. Approximately equal expression of cyclin A1 in both lysates
and strong expression of cdk2 in the cyclin A1/cdk2 lysate was
confirmed by Western blotting (data not shown). The GST–B-myb
(aa 225-705) or GST alone was incubated with the baculovirus
extracts. After extensive washing, proteins were separated on a gel,
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
CYCLIN A1 INTERACTS WITH B-MYB
2095
and Western blotting for cyclin A1 was performed. Cyclin A1
bound to GST–B-myb in the presence as well as in the absence of
cdk2 (Figure 6). To confirm that cyclin A2 did not bind to B-myb
even in the presence of high levels of cyclin A2 and cdk2,
baculovirus-expressed cyclin A2, either alone or in combination
with cdk2, was incubated with GST–B-myb (aa 225-705) (Figure
6). No binding could be detected, confirming the specificity of the
cyclin A1 and B-myb binding.
B-myb is expressed in cyclin A1 expressing male germ cells
The spatial organization of spermatogenesis leads to concentric
layers of cells representing successive stages of male germ cell
differentiation. These specific stages of male germ cell differentiation parallel the location of the cells within the seminiferous tubuli.
Using cyclin A1 promoter-driven expression of EGFP in transgenic
mice in vivo, we found that the human cyclin A1 promoter is
strongly induced in spermatocytes undergoing their first meiotic
division (Figure 7A). These EGFP-expressing cells also express
endogenous murine cyclin A1, indicating the specificity of the
transgenic promoter.24 EGFP-expressing male germ cells from
transgenic mice were sorted by flow cytometry, and protein lysates
were prepared. Western blot analysis of 20 ␮g protein from these
lysates revealed that B-myb expression was exclusively found in
the EGFP-expressing cells (Figure 7A). Similar to the human
B-myb detected in U937 leukemic cells, B-myb protein presented
as 2 distinct bands of sizes between 95 and 105 kd. To confirm that
the 2 bands represented differentially phosphorylated forms of
B-myb, protein lysates from murine testis were incubated with
protein phosphatase before Western blotting. Phosphatase treatment led to a signal increase of the faster migrating band (Figure
7B). The increase was highly reproducible in different experiments
and also occurred in U937 lysates (data not shown). These
experiments show evidence that B-myb is phosphorylated during
spermatogenesis.
Figure 7. Cyclin A1 and B-myb are localized in similar stages of spermatogenesis in meiotic male germ cells. (A) Cyclin A1 expression was visualized as EGFP
expression in the testes of mice that were transgenic for a cyclin A1 promoter-EGFP
construct. The confocal laser microscopy image (top) shows EGFP expression in
green and non-specific fluorescence in Leydig cells in red. The EGFP expression
accurately reflects expression of cyclin A1 as determined previously by Western
blotting of cells sorted by flow cytometry.23 To analyze B-myb expression in testis cells
with and without cyclin A1 expression, EGFP-expressing testis cells were sorted and
analyzed for B-myb expression using Western blot analysis (bottom). There was no
B-myb expression found in cells not expressing EGFP, whereas expression could be
found in cyclin A1 (EGFP)–expressing cells. (B) The 2 B-myb bands in Figure 7A
were similar to the bands seen in human leukemic cells. To analyze whether the 2
bands represented differentially phosphorylated forms of B-myb, protein lysates from
mouse testis were treated with Protein Phosphatase 1 before Western blotting.
Phosphatase treatment led to an increase in the intensity of the faster migrating band,
which is consistent with protein dephosphorylation.
Discussion
Figure 6. Cyclin A1 and B-myb interaction is independent of cdk2. Baculovirusexpressed cyclin A1 was incubated with GST or GST–B-myb (aa 225-704) in the
presence or absence of baculovirus-expressed cdk2. The Western blot membrane
was probed with anticyclin A1 antibody. The second blot shows that cyclin A2 and
B-myb did not interact even when high levels of cyclin A2 and B-myb were present.
Baculovirus-expressed cyclin A2 was incubated with GST–B-myb or GST alone. The
Western blot was probed with anticyclin A2 antibody.
Most cyclins regulating S-phase progression and mitosis are
ubiquitously expressed in proliferating cells. In contrast, expression of cyclin A1 is restricted to very few cell types.4,25 Cyclin A1
does interact with E2F-1 and the Rb family of proteins,6 but the role
of cyclin A1 in the cell cycle of somatic cells remained unknown.
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BLOOD, 1 APRIL 2001 䡠 VOLUME 97, NUMBER 7
MÜLLER-TIDOW et al
In this study we demonstrate that cyclin A1 and B-myb interact
with each other in vivo and in vitro. This interaction involves the
C-terminal region of B-myb and is specific for cyclin A1 because
cyclin A2 did not bind to B-myb. Also, cyclin A1/cdk2 complexes
phosphorylate murine B-myb at functionally relevant sites in the
C-terminal region of B-myb. In addition, these proteins are
expressed in the same cell types during spermatogenesis, and
cyclin A1 can activate B-myb to transactivate myb site-containing
genes including the human cyclin A1 promoter. The latter finding
suggests a possible autoregulatory feedback loop.
B-myb is a member of the myb family of transcription factors. It is
ubiquitously expressed and is thought to play an essential role in G1/S
progression.7 Controversy existed over whether B-myb was actually an
activator of transcription or a transcriptional repressor.14,26 Most of these
issues have been resolved because studies have shown that transactivation by B-myb can be greatly enhanced by several cyclin family
members.16 The activity of myb proteins is controlled by their Cterminal domain. For example, c-myb activity can be enhanced by
C-terminal truncation.27 In contrast to the negative regulatory function
of the C-terminus in c-myb, the C-terminal portion of B-myb functions
as an activator of transcription.28 So far, cyclin A2 and cyclin E have
been the only cyclins shown to direct cdk2 to phosphorylate B-myb at
several sites in its C-terminus.19 These cyclins are known to be essential
for the cell cycle of somatic cells. In contrast, it is still unclear whether
the tissue-specific cyclin A1 can play a role in cell cycle progression in
mammalian cells. Our current study demonstrates that cyclin A1/cdk2
complexes are able to phosphorylate the C-terminus of B-myb at T447,
T490, T497, and S581. These sites have been found to be phosphorylated in vivo, and mutations at these sites greatly reduced the transactivation potential of B-myb.18,19 Also, S581 appears to be involved in DNA
binding of B-myb.19 In addition to the sites that we examined in our
studies, 6 other sites have been described that can be phosphorylated by
cyclin A2/cdk2 complexes.19 The functional relevance of these other
sites is unclear because mutation of the 4 phosphorylation sites (T447,
T490, T497, and S581) alone greatly reduced the activating properties of
cyclin A2.18
Cyclin A2 did not bind to B-myb in our present study, which
confirms previous results.18 In contrast, cyclin A1 binds to the
C-terminal region of B-myb, but not to its DNA binding domain.
This physical interaction does not require cdk2 because cdk2 was
not pulled down along with cyclin A1 from U937 lysates. In
addition, there was no observed enhancement of cyclin A1 binding
to GST–B-myb when cdk2 was co-expressed in the baculovirus
system. In contrast, phosphorylation of B-myb required coexpression of cyclin A1 and cdk2 (Figure 1). The finding that cyclin
A1, but not cyclin A2, interacts with B-myb indicates that cyclin
A1 might be an important partner for B-myb in cyclin A1–
expressing tissues or that functional regulation of B-myb differs
between cyclin A1 and cyclin A2. Cyclin A1 and B-myb are both
expressed during spermatogenesis and hematopoiesis as well as in
AML. In the current study we demonstrate the interaction of these
proteins in leukemic cells in vivo as well as the expression in
similar cell types during murine spermatogenesis. These data
suggest that cyclin A1 might function by tissue-specifically altering
B-myb function.
The appropriate timing of expression of cell cycle regulatory
genes is crucial for the successful passage of the cell cycle. The
BMYB gene is strongly induced at the G1/S boundary by an
E2F-mediated mechanism.29 Subsequently, the B-myb protein is
phosphorylated and further activated. Expression of the cyclin A1
gene also rises after the cell enters S-phase. In the current study we
found that B-myb, when activated by cyclin A1, transactivates the
cyclin A1 promoter. This interesting finding suggests an autoregulatory feedback loop in which cyclin A1, through activation of
B-myb activity, further increases transcription of its own gene. The
cis-reacting sites that are relevant for the transactivation of B-myb
are currently unknown, but 2 mechanisms are likely to play a role.
First, a myb site is located close to the transcriptional start site of
the cyclin A1 promoter, and c-myb binds to this site as determined
by electophoretic mobility shift assay.23 Second, several Sp1
binding sites are located upstream of the cyclin A1 promoter.
B-myb has been shown to transactivate its own promoter presumably through Sp1 sites.30 We have previously shown that the Sp1
sites of the cyclin A1 promoter are essential for cell cycle–
regulated transcription of the cyclin A1 gene.22 An appealing
hypothesis is that cell cycle regulation of cyclin A1 transcription might be influenced by B-myb, which alters Sp1 sitemediated activity.
Our data provide evidence that cyclin A1 interacts with B-myb
in a functionally important way. These findings suggest that cyclin
A1 does have a role in the cell cycle of somatic cells which express
this cyclin. B-myb is important for the proliferation of normal
hematopoietic cells and leukemic blasts, and cyclin A1 may be one
of the co-activators that enable the transactivating ability of B-myb
in these cell types. Cyclin A1 is essential for spermatogenesis, and
murine males that have a homozygously deleted cyclin A1 gene are
infertile.2 Currently, the target molecules of cyclin A1/cdk2, which
are critical during spermatogenesis, are unknown. BMYB closely
interacts with cyclin A1 and might be a potential target gene.
Taken together, our data establish that cyclin A1 interacts with
B-myb in vivo and in vitro. Cyclin A1 is likely to be an important
regulator of B-myb function in the cell cycle of normal hematopoietic cells and in leukemic blasts. Tissue-restricted expression as
seen for cyclin A1 is rare for cell cycle regulators and suggests that
cyclin A1 might be an interesting target gene for new therapeutic
approaches in AML.
Acknowledgments
We are grateful to Dr A. Sala, Department of Molecular Pharmacology and Pathology, Consorzio Mario Negri Sud, Chieti, Italy;
and Dr K. H. Klempnauer, Institute of Biochemistry, University of
Münster, Germany, for providing expression vectors and reporter
plasmids, respectively.
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2001 97: 2091-2097
doi:10.1182/blood.V97.7.2091
Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate
B-myb at functionally important serine and threonine residues: tissue-specific
regulation of B-myb function
Carsten Müller-Tidow, Wenbing Wang, Gregory E. Idos, Sven Diederichs, Rong Yang, Carol Readhead,
Wolfgang E. Berdel, Hubert Serve, Mark Saville, Roger Watson and H. Phillip Koeffler
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