From the Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, September 13, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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The E2A gene products, E12 and E47, are
multifunctional transcription factors that as homodimers regulate B
cell development, growth, and survival. In this report, the E2A gene
products are shown to be targets for regulation by the
G1 cyclin-dependent kinases. Two novel
G1 cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47. One site displays homology to a
preferential D-type cyclin-dependent kinase site (serine 780) on the retinoblastoma susceptibility gene product (pRB) and, consistent with this homology, is more efficiently phosphorylated by
cyclin D1-CDK4 than by the other cyclin-dependent kinases
(CDK) that were tested. The second kinase site is phosphorylated by both cyclin D1-CDK4 and cyclin A/E-CDK2 complexes. Mutation studies indicated that phosphorylation of the cyclin D1-CDK4 site, or more
potently, of both the cyclin D1-CDK4 and cyclin A/E-CDK2 sites,
negatively regulates the growth suppressor function associated with the
N-terminal domain of E12/E47. Transient expression studies showed that
ectopic expression of cyclin D1 or E negatively regulates sequence-specific activation of gene transcription by E12/E47. Analysis
of site mutants, however, indicated that inhibition of E12/E47
transcriptional activity did not require the N-terminal G1
cyclin-dependent kinase sites. Together, the results
suggest that the growth suppressor and transcriptional activator
functions of E12/E47 are targets for regulation by G1
cyclin-dependent kinases but that the mechanisms of
regulation for each function are distinct.
The products of the e2a gene are basic
helix-loop-helix (bHLH)1
DNA-binding proteins that control gene expression as homodimers in B
cells and as heterodimers in other tissues (1, 2). Expression of the
e2a gene produces two splice products, E12 and E47 (3, 4).
One of these two proteins, E47, was originally identified by its
ability to bind specific sites (E boxes) in the enhancer regions of the
immunoglobulin genes (3, 5, 6). The E2A gene products later were found
to be part of a larger, widely expressed family of bHLH regulators (the
E proteins) that function primarily as heterodimers with
tissue-specific bHLH proteins (1). Functional heterodimers between the
E proteins and other tissue-specific bHLH regulators have been observed
in differentiating neuronal, myogenic, and pancreatic cells (7-11). The E2A gene products function as homodimers in B cells (10, 12-14),
and mature B cells are absent from e2a gene null mutant mice
(15, 16). On the other hand, development of other tissues, including
muscle and nerves, appears normal in these mice. This result suggests
that homodimers of the E2A gene products are essential for B cell
development but that their role in forming active heterodimers with
tissue-specific bHLH proteins may be played by other members of the E
protein family (1, 3, 5, 17).
Homodimers of E2A gene products bind and activate transcription from a
minimal reporter construct containing four tandem repeats of µE5,
µE2, and µE3 sites ([5,2,3]x4-TATA-CAT). In
the absence of ectopically expressed E2A gene products, expression of
this reporter is restricted to B cells (18). The reporter is activated
in NIH3T3 cells when ITF-1 (E47/E2-5) is expressed ectopically (18).
The function of E2A gene products as transcriptional activators is
critical for the completion of immunoglobulin gene rearrangement and
normal B cell development. In e2a gene null mice, B cell
development is severed between the pre-B and pro-B cell stages, and
rearrangement of the immunoglobulin genes is interrupted (15, 16). The
importance of the E2A gene products for initiation of immunoglobulin
gene rearrangement is underscored by experiments showing that ectopic
expression in T cells induces immunoglobulin gene rearrangement (6).
Since transcriptionally active E2A gene products are found in B cells
during most stages of their development, it is likely that they also
perform critical functions in cells that are more mature than pro-B
cells, although the nature and importance of these functions has not
been well explored.
The DNA-binding and dimer assembly domain (the bHLH) of E12/E47 maps to
the extreme C-terminal region of the proteins, leaving a large
N-terminal domain. The transcriptional activation domains of the E2A
gene products have been mapped to this N-terminal domain and are
conserved among members of the E protein family (19, 20). One
activation domain has been structurally characterized as a loop
followed by an Recent studies have indicated that the E2A gene products activate
transcription of the cyclin-dependent kinase inhibitor
p21CIP/WAF/SD11 (22), suggesting a direct mechanism through
which E12/E47 can regulate cell growth. In addition, other functions
for E12/E47 in the regulation of cell growth that are not directly
associated with the activation of gene transcription have been
described. These functions have been structurally mapped to the
N-terminal segment of E12/E47 and include regulation of apoptosis
(23-28) and mitogenic growth (29). Recent studies have shown that
e2a gene null mice develop T cell lymphomas and that ectopic
expression of E12/E47 in these lymphoma cells induces their death (27). A role for E47 in growth suppression was first observed in NIH3T3 cells
(29). In colony-forming assays, deletion mutants of E47 that contained
the N-terminal region suppressed growth of NIH3T3 fibroblasts as well
as wild-type E47 (29). The growth suppressor activity of E47 contrasts
that of MyoD (30, 31) and other bHLH proteins, as E47 does not require
the bHLH domain for this function. Growth suppression by E47 is induced
during entry into or progression through the G1 phase of
the cell cycle (29), suggesting that the G1
cyclin-dependent kinases (CDKs) may negatively regulate
this function under appropriate growth conditions.
In this report, we identify the E2A gene products as novel targets for
regulation by G1 cyclin-dependent kinases. Two
novel G1 cyclin-dependent kinase sites are
identified on the N-terminal domain of E12/E47, and evidence is
presented that phosphorylation of these sites negatively regulates the
growth suppressor activity of E47. One of the sites displays a strong
preference for phosphorylation by cyclin D1-dependent
kinase, a characteristic previously associated only with certain kinase
sites of another class of growth suppressors, the retinoblastoma
susceptibility gene (RB) family. In addition, ectopic expression of
cyclin D1 or E is shown to regulate negatively site-specific
transcriptional activation by E12/E47 but through a mechanism that does
not require the N-terminal cyclin-dependent kinase sites.
Together, the results suggest that the products of the e2a
gene are targets through which the G1
cyclin-dependent kinases may regulate the proliferation and
gene expression of certain cell types.
Cell Culture
C3H10T1/2 mouse embryonic fibroblasts (given by H. Weintraub,
Fred Hutchinson Cancer Research Center) and CV-1 monkey kidney fibroblasts (provided by P. Palese, Mt. Sinai School of Medicine) were
grown in Dulbecco's minimum essential medium (DMEM, Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (FBS, HyClone). NIH3T3
mouse fibroblasts (given by S. Aaronson, Mt. Sinai School of Medicine)
were maintained in DMEM supplemented with 10% newborn calf serum (Life
Technologies, Inc.). Mantle lymphoma cell line MO2058 (given by T. Meeker, University of Kentucky) was grown in suspension in DMEM
supplemented with 15% FBS. Sf9 insect cells (PharMingen) were
maintained as monolayers in supplemented Grace's Medium (Life
Technologies, Inc.) supplemented with 10% FBS.
Eukaryotic Expression Plasmids
Transcriptional activities of E47 and site mutants were measured
with an E protein-specific reporter, [µE5 + µE2 + µE3]x4-TATA-CAT (18) generously provided by T. Kadesch (University of Pennsylvania). Parallel transient expression
assays were performed with a control reporter, pRSV-CAT (32) generously
provided by E. Johnson (Mt. Sinai School of Medicine). The cDNAs
coding for human cyclin A (33), cyclin E (34, 35), and cyclin D1 (36)
were generously provided as pRc/CMV (Invitrogen) expression constructs
by P. Hinds and R. Weinberg (Whitehead Institute). The vector, pECE
(37), and pECE expression construct coding for the human
E47·(ITF-1/E2-5 (5)) cDNA with a C-terminal SV40 tag were given
by B. Wold (California Institute of Technology). E47 deletion mutants
were generated that code residues 1-402 (with additional amino acids at the C terminus, GRQRDQAGGEGGRGEHVSG; pECE-E47Not) and residues 1-45
and 415-559 (pECE-E47bHLH). The E47 site mutants E47-S48A, E47-S154A,
and E47-S48A/S154A were prepared as described below and were ligated
into the BglII and XbaI sites of pECE. The
Baculovirus Expression Constructs
The cDNAs of cyclin A, cyclin E, and cyclin D1 were
blunt-inserted into the transfer vectors, pVL1392 or pVL1393
(PharMingen). The cDNA for mouse CDK4 (38) was given by C. Sherr
(St. Jude Children's Research Hospital) and subcloned into the
EcoRI site of the transfer vector, pAcGHLT-A (PharMingen).
Bacterial Expression Constructs
The cDNA for human p21 (39-43) was given by D. Beach (Cold
Spring Harbor Laboratory) and inserted into the NcoI and
EcoRI sites of pTAT-HA (44), generously provided by S. Dowdy
(Washington University School of Medicine). The
His6-tagged expression constructs were prepared by
inserting PCR fragments containing coding sequence into the
BamHI and HindIII sites of pQE30 (Qiagen).
Wild-type and mutant E47 His6-tagged expression constructs
were prepared by using sequences coding for residues 9-559. Sequence
coding for residues 1-912 of mouse CDK4 was used to prepare the
His6-tagged CDK4 expression construct.
Site-directed Mutagenesis
A method was devised to generate site mutants based on two
separate PCRs. The gene was divided into two fragments flanking the
mutation site and extending to vector sequences at the 5' and 3' ends
of the gene. To generate the 5' fragment, a forward (sense) primer
containing a vector restriction site 5' to the gene (e.g.
BglII) and a backward (antisense) primer starting with the
mutant nucleotide and extending into the gene were used in one PCR. To
generate the 3' fragment, a forward (sense) primer starting 1 nucleotide down from the mutant and a backward (antisense) primer
starting from a vector restriction site 3' to gene (e.g. XbaI) were used in a second PCR. A protocol modified from
that described by Liang and Pardee (45) was used to perform the PCRs. Reactions were carried out by a Techne Progene thermal cycler programmed to perform 35 cycles of a three-stage process and a final
elongation step. The 1st stage was set at 95 °C for 40 s, 2nd
stage set at 59 °C for 2 min, and 3rd stage set at 72 °C for 1 min. Reactions were extracted with phenol/chloroform to remove proteins
and dNTPs. The 5' and 3' PCR fragments were digested by
BglII and XbaI, respectively, whereas the vector
was digested by both BglII and XbaI and then
further digested with CIP (New England Biolabs). The two fragments and
vector were combined and incubated with ligase, which resulted in a
blunt-end ligation at the site of the mutation and sticky-end ligations
at the BglII and XbaI sites. Vent DNA polymerase
(New England Biolabs), a thermophilic DNA polymerase that generates
blunt ends instead of overhangs, was used for the PCRs. Competent XL-1
blue cells were transformed with the ligation reactions as described
elsewhere (46). Plasmid DNA was isolated from randomly selected clones
with the Wizard SV Mini-prep DNA Isolation Kit (Promega) and digested
with both BglII and XbaI to check for inserts.
Positive clones were then sequenced to confirm mutations and correct junctions.
DNA Sequencing
Plasmid DNA was denatured under alkaline conditions and used as
template for sequencing by the Sanger method (Sequenase version 2.0 DNA
Sequencing Kit supplied by Amersham Pharmacia Biotech/U. S.
Biotechnology Corp.). Sequence reactions were then run on a 6%
polyacrylamide/urea gel in TBE buffer (46). The gel was fixed, dried
under vacuum, and then exposed to x-ray film. Sequences were read
directly from the autoradiographs.
Transient Expression Assays
Cells were transfected at 80% confluency with a standard
calcium phosphate method (55) and incubated for 18 h. After
transfection, cells were grown in low mitogen medium (DMEM supplemented
with 3% horse serum) for another 48 h. Cells were then
harvested for CAT activity. CAT assays were performed as described
elsewhere (47). CAT activities were then quantified from TLC plates by PhosphorImager analysis. To control for nonspecific effects on [µE5 + µE2 + µE3]x4-TATA-CAT reporter activity,
parallel experiments were performed with a constitutively active
reporter, pRSV-CAT. Relative CAT activities were then determined by
normalizing CAT values from [µE5 + µE2 + µE3]x4-TATA-CAT reporter with respective values
obtained with the control reporter (pRSV-CAT).
Generation of Recombinant Proteins
Proteins were prepared by protocol modified from that described
elsewhere (Qiagen His6 Protein Expression Manual). TB-1
competent cells expressing pREP4 were transformed with expression
constructs by a standard protocol, and small scale protein preparations
were performed to isolate high protein-expressing clones. Clones were grown overnight in LB medium containing both ampicillin and kanamycin and were then used for large scale protein preparations. For large scale protein preparations, bacteria were grown for 1 h in LB medium at 37 °C and then induced to express protein with 0.5 mM isopropyl-1-thio- Preparation of Active Cyclin-CDK Complexes
The four steps used in the preparation of active cyclin-CDK
complexes included generation of baculoviruses, titration of
baculoviruses, coinfection with cyclin and CDK baculoviruses, and
purification of active cyclin-CDK complexes. The baculovirus expressing
HA-tagged CDK2 (34, 48-50) was generously provided by C. Prives
(Columbia University). Baculoviruses expressing human MO15/CDK7
(51-54) and His6-tagged cyclin H (52) were gifts from D. Morgan (University of California, San Francisco).
Generation of Baculoviruses--
The baculoviruses
expressing cyclin A, cyclin E, cyclin D1, and CDK4 were generated with
the Baculovirus Protein Expression Kit from PharMingen. Monolayers of
Sf9 insect cells (2 × 106) passaged into T25
flasks were cotransfected with transfer vectors (driven by the
polyhedrin promoter) that contain the gene of interest and
BaculoGoldTM DNA (linearized viral DNA) by a calcium
phosphate method for 4 h. The use of BaculoGoldTM DNA
results in the generation of baculoviruses that express only the
protein of interest, thus eliminating the need to select positive expressing clones. After transfection, cells were grown in fresh medium
and incubated for 5 days. The medium was collected, and 1 ml was used
to infect 2 × 107 Sf9 cells seeded on a 15-cm
tissue culture plate. After 3 days of infection, the medium was
collected, and the titer of the virus was initially measured by end
point dilution assay. Multiple rounds of amplification were performed
until viral stocks reached titers greater than or equal to 1 × 108 plaque-forming units/cell.
Titration of Baculoviruses--
Since cells are infected with
two viruses simultaneously, infection efficiencies may differ from one
virus to another. Infection efficiencies of cyclin and CDK viruses were
normalized by comparing relative protein expression levels at different
virus concentrations. Sf9 cells were passaged into 24-well
plates containing 2 × 105 cells/well. Amplified virus
was added to the wells at dilutions values of 1:10, 1:20, 1:40, 1:100,
1:200, 1:400, and 1:1000. After 2 days of infection, cells were then
harvested, and lysates were analyzed by SDS-PAGE (55). Proteins were
visualized by staining gels with Coomassie Blue R-250 solution
(55).
Coinfection with Cyclin and CDK Viruses--
The dilution value
where maximum protein expression levels were observed with each virus
was used in coinfections to generate cyclin-CDK complexes. Cyclin and
CDK viruses were mixed together and then added to T75 flasks containing
1 × 107 Sf9 cells. After 48 h of
infection, cells were detached from flasks by pipetting up and down and
gently spun at 4 oC. Cell pellets were then washed with
ice-cold wash buffer (25 mM HEPES, pH 7.5, 150 mM NaCl), spun again at 4 C, and then resuspended with 2 volumes of Sonication Buffer (25 mM HEPES, pH 7.5, 10 mM NaCl, 40 µg/ml aprotinin, and 40 µg/ml leupeptin).
Lysates were sonicated and then flash-frozen.
Purification of Cyclin-CDK Complexes--
Cyclin A/E-CDK2
complexes were affinity-purified with 12CA5-protein A-agarose beads.
Briefly, 12CA5-protein A-agarose beads were generated by coupling 12CA5
monoclonal antibody (provided by T. Moran, Mt. Sinai School of
Medicine) to protein A-agarose beads (Roche Molecular Biochemicals)
with dimethyl pimelimidate (Sigma). Frozen lysates were thawed,
clarified, and incubated with 12CA5-protein A-agarose beads. Purified
cyclin A/E-CDK2 complexes were then eluted from the beads with HA
peptide (Roche Molecular Biochemicals) containing Arg-insulin (Sigma)
as carrier.
Production of Purified p21 Protein
BL-21 (DE3)-competent cells were transformed with pTAT-HA-p21,
and small scale protein preparations were performed to isolate high
expressing clones. High expressing clones were grown overnight in LB
medium with ampicillin and then used for large scale protein preparations. Bacteria were grown in Superbroth at 37 oC
with shaking and induced to express protein with 0.8 mM
isopropyl-1-thio- Preparation of Active Cyclin D1-CDK Complexes
Sonicated cyclin D1-CDK4 baculovirus lysate (prepared as
described above) was thawed slowly on ice, and purified p21-TAT protein was added (molar ratio of 1:50). The lysate was incubated at 4 °C
for 15 min and then clarified by spinning at 13,000 rpm for 15 min at
4 °C. Glutathione-agarose beads pre-washed with wash buffer (25 mM HEPES, pH 8.0, 50 mM NaCl) were added to the
collected supernatant and incubated overnight with shaking at 4 °C.
Beads were washed 3 times with wash buffer and then resuspended in 25 mM HEPES, pH 8.0, with 20 mM MgCl2,
500 µM cold ATP, and clarified CAK baculovirus
lysate prepared from coinfection of Sf9 cells with cyclin H and
CDK7 baculoviruses. The kinase reaction was incubated at 37 °C for
15 min and then incubated at ambient temperature for an additional 45 min with shaking. The beads were washed 3 times with wash buffer and
then stored in wash buffer at 4 °C.
In Vitro Kinase Assays
Protein substrates were phosphorylated by purified cyclin-CDK
complexes in a reaction mixture containing 50 mM HEPES, pH
7.5, 20 mM MgCl2, 50 µM cold ATP,
and 10 µCi of [ Peptide Phosphorylation Assays
A list of the E47 peptides (generated by Research Genetics,
Inc.) used in kinase assays with cyclin-CDK complexes is shown in Table
I. Stock solutions were prepared by dissolving lyophilized peptides in
sterile water at a final concentration of 2 mg/ml. Peptides were
phosphorylated in a reaction volume of 20 µl containing 4 µg of
peptide, 25 mM HEPES, pH 7.5, 20 mM
MgCl2, active kinase (cyclin A/E-CDK2 complexes, or cyclin
D1-CDK4 bound to glutathione-agarose beads), 50 µM cold
ATP, and 10 µCi of [ Radioisotope Labeling and Immunoprecipitations
Suspension cultures of MO2058 lymphocytes grown in T75 flasks
were lipofected with LipofectAMINE reagent (Life Technologies, Inc.)
and expression constructs containing wild-type or mutant E47 as
described by the manufacturer (Life Technologies, Inc., LipofectAMINE
Lipofection Kit). Cells were incubated with lipofection mixture for
1.5 h at 37 °C in Opti-MEM low serum medium (Life Technologies,
Inc.), and then incubated at 37 °C in supplemented DMEM with 15%
FBS. After 18-24 h, cells were washed with phosphate-free medium.
Cells were labeled with 3 mCi/5 ml of medium of inorganic 32P (PerkinElmer Life Sciences) in phosphate-free medium
for 2 h. Alternatively, cells were washed with
methionine/cysteine-free medium and labeled with 1 mCi/5 ml medium
[35S]methionine and [35S]cysteine. After
labeling, cells were washed two times with PBS, and lysed with IP
buffer (25 mM HEPES, pH 7.5, 50 mM NaCl, 0.5% Triton X-100, 25 mM EDTA, pH 8.0, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 10 µg/ml bacitracin, 100 µg/ml benzamide, 1 mM NaF, 1 mM Na3VO4, 10 mM glycerophosphate, and 500 µg/ml acetylated bovine
serum albumin). Lysates were clarified by centrifugation in a
microcentrifuge at 13,000 rpm for 10 min (4 °C). Protein G-agarose (Roche Molecular Biochemicals) beads pre-washed with wash
buffer (25 mM HEPES, pH 7.5, 50 mM NaCl, 0.5%
Triton X-100) were added to the collected supernatants and incubated at
4 °C for 1 h with gentle shaking. Samples were centrifuged in a
microcentrifuge at 13,000 rpm for 2 min, and the supernatants were
collected. Antibody to the SV40 tag (KT-3, Babco) was added, and the
samples were incubated overnight at 4 °C. Samples were then
incubated with washed protein G-agarose beads for 1 h at 4 °C
with gentle shaking. The beads were washed five times with wash buffer
and then resuspended in sample buffer (100 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.025% phenol red, and 5%
Western Blot Analysis
After SDS-PAGE, proteins were electrophoretically transferred to
nitrocellulose filters. Filters were fixed with 25% isopropyl alcohol, 10% acetic acid, washed with distilled water, and then washed
three times with Tris-buffered saline (TBS, 25 mM Tris-Cl, pH 7.4, 100 mM NaCl). The filters were then incubated
overnight (4 °C) in Blocking Solution (5% nonfat dry milk in TBS).
Primary antibody was added to Blotting Solution (25 mM
Tris-Cl, pH 7.4, 500 mM NaCl, 0.5% Tween 20, 5% nonfat
dry milk, 5% horse serum) and incubated overnight with the filters
(4 °C). The blots were washed three times with TBST (25 mM Tris-Cl, pH 7.4, 500 mM NaCl, 0.5% Tween
20) and then incubated with secondary antibody at 1:10,000 in TBST for
1 h at room temperature. Three washes with TBST and one wash with
TBS were performed on the blots after incubation with secondary
antibody. The blots were laid on plastic wrap and incubated with
chemiluminescent reagent (Amersham Pharmacia Biotech ECL Western blot
Protein Detection Kit). After 1 min, the blots were drained of excess
reagent and wrapped in new plastic wrap and then exposed to x-ray film.
Primary antibodies used for detection of E47, cyclins, and p21 proteins
were obtained from Santa Cruz Biotechnology.
Colony-forming Assays
Cells passaged at 80% confluency in T25 flasks were
cotransfected with a plasmid conferring G418 resistance (pRc/CMV or
pSV2-Neo) and an expression construct of interest by a standard calcium phosphate method described elsewhere (55). After 24 h of
transfection, cells were trypsinized and passaged into 15-cm plates.
G418 (Life Technologies, Inc.) was added to the medium at an effective
concentration of 600 µg/ml the following day. Medium was replaced,
and G418 was replenished every 3 days until macroscopic colonies were
visible. Cells were washed 3 times with PBS and fixed with EFA (70%
ethanol, 10% formaldehyde, 5% acetic acid) for 10 min at
Preparation of Samples for Flow Cytometry
Cells were trypsinized, diluted with medium in a 50-ml Falcon
conical tube, and spun at 1,000 rpm for 5 min. Cells were washed once
with PBS and then resuspended with 10 ml of PBS. While vortexing gently, 30 ml of ice-cold ethanol was added dropwise to the resuspended cells. The cells were fixed at Identification of G1 Cyclin-dependent
Kinase Sites on E47--
By using the S/TPX consensus site, predicted
phosphorylation sites for cyclin A-CDK2, cyclin E-CDK2, or cyclin
D1-CDK4 were identified in the primary sequence of E47 (Fig.
1A). Peptides were synthesized
containing these predicted proline-directed kinase sites (Table
I) and assayed for phosphorylation with
purified cyclin A-CDK2, cyclin E-CDK2, or cyclin D1-CDK4. The
cyclin-dependent kinase preparations used for these
experiments initially were characterized using recombinant RB protein
and histone H1 as a substrates. Preparations of cyclin A-CDK2 and
cyclin E-CDK2 phosphorylated both histone H1 and RB protein, whereas
preparations of cyclin D1-CDK4 phosphorylated RB protein equivalently
but lacked histone H1 kinase activity (data not shown). Peptides were
incubated with kinases in the presence of [
We next confirmed that residues Ser-48 and Ser-154 are phosphorylated
in vitro on full-length recombinant E47 by purified cyclin
A-CDK2, cyclin E-CDK2, and/or cyclin D1-CDK4. Serine to alanine site
mutants of E47 were generated at Ser-48 (S48A), Ser-154 (S154A), and
both Ser-48 and Ser-154 (S48A/S154A). Recombinant wild-type E47,
E47-S48A, E47-S154A, and E47-S48A/S154A were used as substrates. An
unrelated E47 site mutant, E47-S250A was used as an additional control.
The results of phosphorylation experiments with recombinant E47 and E47
site mutants was consistent with the results obtained with the
synthetic peptides. Recombinant wild-type E47 was phosphorylated by
purified cyclin A-CDK2, cyclin E-CDK2, or cyclin D1-CDK4 (Fig.
2A). The E47-S154A mutant was phosphorylated nearly as efficiently as wild-type E47 by cyclin E-CDK2.
In contrast, cyclin E-CDK2 was strongly inhibited from phosphorylation
of the E47-S48A mutant (Fig. 2A). The data suggest that
cyclin E-CDK2 phosphorylates Ser-48 on E47 almost exclusively. Phosphorylation of the E47-S48A mutant by cyclin A-CDK2 also was significantly reduced from the level of phosphorylation observed with
wild-type E47, the E47-S250A mutant, or the E47-S154A mutant (Fig.
2A). A low level of phosphorylation by cyclin A-CDK2 was observed with the E47-S48A/S154A double mutant, suggesting that an
additional weak phosphorylation site may be present for this kinase
complex.
Cyclin D1-CDK4 phosphorylated wild-type E47, the E47-S250A mutant, and
the E47-S48A mutant (Fig. 2A). In contrast to the other cyclin-dependent kinases tested, cyclin D1-CDK4
phosphorylated the E47-S154A mutant much less efficiently than it did
wild-type E47. Virtually no phosphorylation of the E47-S48A/S154A
double mutant was observed using cyclin D1-CDK4 (Fig. 2A).
These data are consistent with the peptide phosphorylation studies,
which showed that cyclin D1-CDK4 phosphorylates Ser-154 more
efficiently than Ser-48. In summary, the data identifies two residues
on E47 that are phosphorylated by G1
cyclin-dependent kinases in a type-specific manner as
follows: Ser-48, which is preferentially phosphorylated by cyclin
A/CDK2 and cyclin E/CDK2, and Ser-154, which is preferentially phosphorylated by cyclin D1/CDK4.
We next asked whether Ser-48 and Ser-154 are phosphorylated in
vivo. A permanent cell line (MO2058; Refs. 56 and 57) derived from
a mantle B cell lymphoma was used for these experiments. This cell line
was chosen for two reasons as follows. 1) Because of a chromosomal
translocation characteristic of mantle cell lymphomas (t(11;14)(q13;q32)), these cells express high levels of cyclin D1-dependent kinase activity. 2) B cells have been shown to
hypophosphorylate other kinase sites on E12/E47 (21). As indicated by
Western blot, mutations of Ser-48 or Ser-154 do not adversely affect
accumulation of E47 protein in transiently transfected cells (Fig.
2B). This result also indicates that the viability of cells
is not altered by transient transfection of the E47 and E47 site mutant
expression plasmids, a conclusion that is supported by their equivalent
transcriptional activity in transient expression assays (described
below). MO2058 cells were transfected with expression constructs for
SV40-tagged wild-type E47 and E47 site mutants S48A, S154A, and
S48A/S154A. After 18 h, the transfected cultures were
metabolically labeled with inorganic 32P. Ectopically
expressed wild-type E47 and mutants were immunoprecipitated with an
SV40 tag monoclonal antibody (KT-3; Babco), and analyzed for
phosphorylation by SDS-PAGE and autoradiography. Incorporation of
32P was significantly reduced in the S154A and S48A/S154A
site mutants (Fig. 2C). The lowest level of incorporation
was observed in the S48A/S154A double mutant, and the S154A mutant
displayed a lower level of incorporation than the S48A mutant (Fig.
2C). These results indicate that both Ser-48 and Ser-154 may
be phosphorylated in proliferating mantle lymphoma cells.
Ectopic Expression of G1 Cyclins Inhibits Activation of
Transcription by E47, but Inhibition Is Not Mediated by Phosphorylation
of the G1 Cyclin-dependent Kinase
Sites--
Regulation of transcription by the E2A gene products has
been shown to be mediated by E box sequences in the promoter/enhancer region of the immunoglobulin heavy chain gene (3, 5, 10). Expression of
a synthetic reporter construct containing four tandem repeats of three
different E box sequences linked to the thymidine kinase TATA box
region [p4R(µE5 + µE2 + µE3)TATA-CAT] has been shown to be
activated by E47 in fibroblasts (18). We used this reporter construct
to study the effects of ectopic cyclin expression on transcriptional
activation by E47. NIH3T3 cells were transfected with an E47 expression
construct under control of the SV40 early region promoter (pECE-E47)
and a series of cyclin expression constructs under control of the CMV
promoter. These expression constructs have been shown in previous
studies to be expressed at high levels in fibroblasts (58). Ectopic
expression of cyclin D1 and cyclin E strongly inhibited activation of
transcription by E47 in NIH3T3 cells (Fig.
3A). Moderate inhibition
and/or stimulation of E47 transcriptional activity was associated with
ectopic expression of other cyclins, but the significance of those
results will not be explored in this report.
We next determined whether phosphorylation of serine 48 and/or serine
154 affects activation of transcription by E47. Activation of p4R(µE5 + µE2 + µE3)TATA-CAT transcription by E47, E47-S48A, E47-S154A, and
E47-S48A/S154A was compared in NIH3T3 cells. Base-line transcriptional
activity of the three mutants also did not vary significantly from that
of wild-type E47 in transient expression assays (Fig. 3B).
The effect of ectopic expression of cyclins on activation of p4R(µE5 + µE2 + µE3)TATA-CAT transcription by the E47 mutants was also
determined. In NIH3T3 cells, transcriptional activation by E47-S48A,
E47-S154A, and E47-S48A/S154A was inhibited by ectopic expression of
cyclin D1 and cyclin E equivalently to wild-type E47 (Fig.
3C). Together, these results indicate that phosphorylation
of serine 48 and/or serine 154 is not involved in the negative
regulation of E47 transcriptional activation by cyclins D1 and E.
G1 Cyclin-dependent Kinase Sites Negatively
Regulate Growth Suppression by E47--
Expression of the E2A gene
products has been shown to suppress the growth of fibroblasts in
culture (29). Consistent with previous observations, we have found that
ectopic expression of E47 in NIH3T3 cells reduces colony-forming
efficiency. Deletion mutants of E47 were generated that contained
either the N-terminal segment of the protein but lacked the bHLH motif
(residues 1-402; E47Not) or contained the bHLH motif but lacked most
of the N-terminal domain (residues 1-45, 415-559; E47bHLH). Growth
suppressor activity persisted, albeit at lower levels, in the mutant
containing the N-terminal segment but was completely absent from the
mutant lacking the N-terminal region (Table
III). The data are consistent with previous results (29) showing that inhibition of colony formation is
conferred by the N-terminal domain of E47.
We next asked whether phosphorylation of Ser-48 and/or Ser-154
regulates growth suppression by E47. Colony-forming assays were
performed with C3H10T1/2 cells. These cells express endogenous E
proteins and are more resistant to growth suppression by E47 than are
NIH3T3 cells. Expression plasmids for wild-type E47, E47-S48A,
E47-S154A, and E47-S48A/S154A were cotransfected with a plasmid
conferring G418 resistance into C3H10T1/2 cells. Colonies were selected
for G418 resistance, and the colony-forming efficiency for the
transfected cultures was compared. Consistent with the notion that
C3H10T1/2 cells are resistant to growth suppression by E47, ectopic
expression of wild-type E47 or E47-S48A in C3H10T1/2 cells did not
significantly reduce the efficiency of colony formation (Table
IV). In contrast, ectopic expression of
E47-S154A or E47-S48A/S154A severely reduced colony-forming efficiency
(Table IV). The double site mutant, E47-S48A/S154A, inhibited colony
formation more strongly than E47-S154A, even though the E47-S48A single
site mutant did not significantly inhibit colony formation. These
observations suggest that phosphorylation of Ser-154 blocks growth
inhibition by E47 and that phosphorylation of Ser-48 in conjunction
with phosphorylation of Ser-154 more potently blocks growth
inhibition.
Ectopic Expression of E47 Kinase Site Mutants Enhances Sensitivity
of C3H10T1/2 Cells to Growth Factor Depletion and Contact Inhibition of
Growth--
Cultures were cotransfected with expression plasmids for
either wild-type E47, E47-S48A, E47-S154A, or E47-S48A/S154A and an
expression plasmid conferring G418 resistance into C3H10T1/2 cells.
Colonies were selected for G418 resistance and then dissociated with
trypsin and grown as polyclonal cultures. Although colony-forming efficiency was reduced in cultures transfected with E47-S154A or
E47-S48A/S154A (see above), large scale experiments produced sufficient
clones from each to generate polyclonal cultures. Time course
experiments showed that C3H10T1/2 cells transfected with E47-S154A or
E47-S48A/S154A expression plasmids proliferated more slowly than cells
transfected with wild-type E47, E47-S48A, or vector control (Figs.
4A). In addition, the density
to which cultures transfected with E47-S154A and E47-S48A/S154A grew at
confluence was significantly reduced (Fig. 4, A and
B). The cultures were harvested during the logarithmic phase
of their growth curves, stained with propidium iodide, and processed
for fluorescence-activated cell sorter (FACS) analysis. The FACS
analysis revealed that during the logarithmic phase of their growth
(Fig. 4A, arrow), the distribution of cells between
G1, S, and G2 phases of the cell cycle varied only slightly between the cultures (Table
V). Growth suppression of cells
expressing ectopic E47-S154A and E47-S48A/S154A does not appear to
result from an increase in the time required for completion of a
particular phase of the cell cycle. Instead, the protracted growth
curves and reduced density at confluence suggest a greater sensitivity
of the cells to contact inhibition of growth and/or to depletion of
growth factors from the medium. Changes in these parameters may
manifest as delayed entry into or more frequent exit from the cell
cycle.
By using kinase assays with purified cyclin A-CDK2, cyclin E-CDK2,
cyclin D1-CDK4, and a series of synthetic peptides as substrates, we
identified two G1 cyclin-dependent kinase
phosphorylation sites on E47 as follows: one that is phosphorylated
more efficiently by cyclin A and cyclin E-dependent kinase
(Ser-48) and another that is phosphorylated more efficiently by cyclin
D1-dependent kinase (Ser-154). Further studies using as
substrates recombinant wild-type E47 or site mutants substituting
alanine for serine 48 (S48A), serine 154 (S154A), or both serine 48 and
154 (S48A/S154A) confirmed the results obtained with the synthetic
peptides. Finally, metabolic labeling studies in transfected MO2058
cells provided evidence that Ser-48 and Ser-154 are phosphorylated
in vivo. Transient expression assays in NIH3T3 cells using
the p4R(µE5 + µE2 + µE3)TATA-CAT reporter indicated that
wild-type E47 and the S48A, S154A, and S48A/S154A mutants sustained
equivalent transcriptional activity. In addition, none of the mutants
were resistant to inhibition by cyclin D1 or cyclin E, indicating that
phosphorylation of these sites was not required for inhibition of E47
transcriptional activity.
Ectopic expression of cyclin D1 has been shown in many studies to
inhibit activation of muscle gene transcription by the myogenic bHLH
factors (59, 60). We show here that ectopic expression of two
G1 cyclins, cyclin D1 and cyclin E, inhibits activation of
transcription by another member of the bHLH family, the E2A gene
products. So far, most results have indicated that direct phosphorylation of the myogenic bHLH factors by cyclin
D1-dependent kinase is not required for inhibition, and
this is consistent with our studies of inhibition of the E2A gene
products by cyclin D1 and cyclin E. The mechanism(s) by which
G1 cyclins inhibit either the myogenic bHLH factors or
products of the e2a gene family are not understood. The
ability of cyclin D1 to inhibit myogenic bHLH mutants that are
resistant to Id expression
(61)2 or to phosphorylation
by protein kinase C (59, 62) suggest alternative mechanisms of
inhibition. Studies from our laboratory have suggested that site
mutants of myogenin lacking the E protein-dependent kinase
sites (63) require somewhat greater doses of cyclin D1 for complete
inhibition,2 but it is not clear that these phosphorylation
sites are directly involved in the mechanism of inhibition.
Some studies have suggested that phosphorylation of the retinoblastoma
susceptibility protein (pRB) by D-type cyclin-dependent kinases may block its binding to the myogenic bHLH proteins and that
the interaction between these two proteins is essential to activate
transcription of muscle-specific genes (64-67). A problem posed by
this hypothesis is that pRB is efficiently phosphorylated by cyclin
D3-dependent kinases (68, 69), even though cyclin D3 does
not inhibit transcriptional activation by the myogenic bHLH factors
(59). A recent report has suggested that CDK4 directly binds and
inhibits the activity of MyoD (70) and that cyclin D1 is required for
translocation of CDK4 from the cytoplasm to the nucleus (71). The role
for cyclin D1 in this mechanism is to mediate nuclear translocation of
CDK4, thereby allowing it to bind and inhibit MyoD (70). This mechanism
has been shown so far to apply only to MyoD, although future studies
may reveal its role in the inhibition of other bHLH proteins by D-type cyclins.
Growth suppression of NIH3T3 cells by products of the e2a
gene was first observed by Philipson and coworkers (29). Cotransfection of E47 expression constructs with a plasmid construct conferring G418
resistance into NIH3T3 cells significantly reduced the efficiency of
colony formation under selective conditions. Consistent with these
results, we have observed that ectopic expression of E47 in NIH3T3
cells reduces colony-forming efficiency and that the bHLH domain of E47
is not required for growth suppressor activity. Since the bHLH domain
of E47 is required for dimer assembly, site-specific DNA binding
activity, and site-specific activation of gene transcription, these
functions apparently are not directly associated with growth suppression by E47. Previous studies have shown that binding to Id
proteins blocks growth suppression by E47, but when Id binding was
abrogated by removing the HLH domain, growth suppression by E47
remained intact (29). We show here that two G1
cyclin-dependent kinase sites (Ser-48 and Ser-154) in the
N-terminal domain of E47 negatively regulate growth suppression by E47.
Site-directed mutagenesis of Ser-154 strongly enhances growth
suppression of C3H10T1/2 cells. Site-directed mutagenesis of Ser-48
alone did not enhance growth suppression by E47 but instead as a double mutant augmented the increase in growth suppression associated with
mutation of Ser-154. As indicated above, Ser-154 is preferentially phosphorylated by cyclin D1-dependent kinases, whereas
Ser-48 is preferentially phosphorylated by cyclin A- or cyclin
E-dependent kinases. Together, these observations suggest
that phosphorylation of E47 first by cyclin D1-dependent
kinases at Ser-154 and then by cyclin A- or cyclin
E-dependent kinases at Ser-48 may negatively regulate
growth suppression as cells enter and/or progress through the cell cycle.
Enhanced cyclin D1 expression has been shown to be involved in the
oncogenesis of several malignancies, and the targets of cyclin D1 may
differ with each type. Expression of cyclin D1 is enhanced in a
significant percentage of human breast cancers (72-74). It has been
proposed that in breast cancer cyclin D1 does not function as a
regulatory subunit of a cyclin-dependent kinase complex but
rather as a transcriptional coactivator for the estrogen receptor (75,
76). Recent studies have not supported this hypothesis. Although cyclin
D1 null mutant mice display significant defects in mammary development
(77, 78), replacement of the coding region of the cyclin D1 gene with
the coding region of the cyclin E gene does not appear to affect murine
development (79). Since cyclin E is not a transcriptional cofactor for
the estrogen receptor, this result suggests that the proposed function of cyclin D1 as a transcriptional cofactor is not essential. It is
important to note that the mice with cyclin E substituted for the
cyclin D1 coding region still contained intact cyclin D2 and D3 genes,
and therefore their apparent normal phenotype does not prove that
cyclin E expression can substitute for or bypass all of the functions
of the D-type cyclin family. In addition, the results do not explain
the oncogenic potential of cyclin D1, which appears to be more
prominent than that of cyclin E.
Phosphorylation of RB family members by D-type
cyclin-dependent kinases regulates progression during the
G1 phase of the cell cycle (80). Perturbation of this
regulatory step through enhanced expression of cyclin D1, loss of p16
or other cyclin-dependent kinase inhibitors, or loss of RB
has been proposed as a common event leading to malignancy in many
tissue types (81, 82). Studies of neuronal precursor cell cycle
regulation have indicated that growth control by D-type cyclin-CDK4/6
complexes is targeted toward regulation of RB, whereas growth control
by CDK2 in these cells is regulated by additional targets (83).
Enhanced expression of cyclin D1 is achieved in mantle cell lymphoma (a
B cell malignancy) through rearrangement of the cyclin D1 gene with the
immunoglobulin heavy chain locus (84, 85). The role of enhanced cyclin
D1 expression in this malignancy has not been determined, although the
critical importance of the E2A gene products in B cell growth and
development has been well established (15, 16). Some investigations have provided strong evidence that pRB is not the oncogenic target of
cyclin D1-CDK4 in mantle lymphoma cells (86, 87). It is possible that
enhanced expression of cyclin D1 may block growth suppression of mantle
B cells by products of the e2a gene, resulting in a
malignant phenotype.
The similarity between regulation of growth suppression by the
retinoblastoma susceptibility gene product (pRB) and E47 is intriguing.
Both proteins contain G1 cyclin-dependent
kinase sites that negatively regulate their functions as growth
suppressors, including sites that are preferentially phosphorylated by
cyclin D1-dependent kinase (88). Although some gene
products have been identified that are phosphorylated by cyclin
D1-dependent as well as other cyclin-dependent
kinases, to our knowledge only RB family members and now E47 have been
shown to contain sites that are preferentially
phosphorylated by D-type cyclin-dependent kinases. The
sequence surrounding Ser-154 on E47 displays significant homology to
the major preferred cyclin D1-dependent kinase site found
on pRB (serine 780 (88); Fig. 5). Some
studies have suggested that the optimal consensus for preferential
phosphorylation by D-type cyclin-dependent kinases differs
significantly from the more generic consensus for all
cyclin-dependent kinases, and our results support this
conclusion. Virtually all of the cyclin-dependent kinases identified so far are proline-directed, phosphorylating a serine or
threonine immediately N-terminal to this residue. In general, inappropriate residues N-terminal to the serine/threonine residue can
abrogate phosphorylation, whereas variations in residues C-terminal to
the directing proline can confer preferential phosphorylation by
certain cyclin-dependent kinase types (88). Many
cyclin-dependent kinase sites are phosphorylated
efficiently by both D-type cyclin-dependent kinases and
cyclin A/E-dependent kinases. As shown here, sites preferentially phosphorylated by D-type cyclins may be distinguished by
alternating hydrophobic and proline residues C-terminal to the
determining proline.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amphipathic helix and is referred to as a loop-helix
motif (19). Within an acidic stretch of residues immediately N-terminal
to the bHLH are two sites that are phosphorylated by casein kinase II
and protein kinase A (21). In electrophoretic mobility shift studies,
the DNA binding ability of E47 homodimers was blocked by
phosphorylation of these residues, whereas the DNA binding ability of
MyoD/E47 heterodimers was unaffected (21). Consistent with this,
phosphorylation of E47 at these sites was observed only in non-B cells
(21), suggesting a unique mechanism for restricting the transcriptional
activity of E12/E47 homodimers to B cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression construct pCMV-
(Invitrogen) was used
in some transient expression assays as an internal control.
-D-galactopyranoside for
4-5 h. Cells were harvested, washed with ice-cold TES (10 mM Tris, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl), and lysed in sonication buffer (25 mM
HEPES, pH 8.0, 150 mM NaCl, and 8 M urea).
Lysates were sonicated, clarified, and then loaded onto
nickel-nitrilotriacetic acid-agarose (Qiagen) columns that were
prewashed with wash buffer (25 mM HEPES, pH 8.0, 150 mM NaCl, 8 M urea). Columns were washed several
times with wash buffer, and His6-tagged proteins were
eluted with 150 mM imidazole in wash buffer. Proteins were
then dialyzed twice against 10 mM HEPES, pH 8.0, 100 mM NaCl. Samples were aliquoted and flash-frozen.
-D-galactopyranoside. After 4-5 h of
growth, bacteria were harvested, washed with ice-cold TES, and lysed in
lysis buffer (8 M urea, 25 mM HEPES, pH 8.0, 100 mM NaCl). Lysate was then sonicated, clarified, and
loaded onto a nickel-nitrilotriacetic acid-agarose (Qiagen) column
pre-washed with wash buffer (8 M urea, 25 mM
HEPES, pH 8.0, 50 mM NaCl). The collected flow-through was
then loaded again onto the column for a second round. The column was
washed with several column volumes of wash buffer and then with 1 column volume of HBS (25 mM HEPES, pH 8.0, 50 mM NaCl). The p21-TAT protein was eluted with elution
buffer (25 mM HEPES, pH 8.0, 2% SDS) and dialyzed twice in
10 mM HEPES, pH 8.0, 50 mM NaCl, 0.5% Triton
X-100. After dialysis, p21-TAT protein samples were aliquoted and
frozen in liquid nitrogen.
-32P]ATP. The reactions were
incubated at 30 °C for 1 h and then stopped by the addition of
EDTA to a final concentration of 50 mM. The samples were
mixed with an equal volume of Sample buffer (100 mM Tris,
pH 6.8, 2% SDS, 10% glycerol, 0.025% phenol red, 5%
-mercaptoethanol), boiled at 100 °C for 5 min, and then analyzed by SDS-PAGE. Gels were fixed, rehydrated, dried under vacuum, and
phosphorylation was detected by autoradiography.
-32P]ATP. Reactions containing
cyclin A/E-CDK2 were incubated at room temperature for 1 h,
whereas those containing cyclin D1-CDK4 bound to beads were first
incubated at 37 °C for 15 min and then at room temperature for 45 min with shaking. Kinase reactions were stopped with the addition of
EDTA to a final concentration of 50 mM. Kinase reactions
were loaded onto cellulose/polyethyleneimine TLC flexible plates with
fluorescent indicator (J. T. Baker Co.) and allowed to air-dry
overnight at room temperature and kept away from light. TLC plates were
placed upright in distilled water until running front reached the line
of origin and dried at room temperature away from light. Plates were
then placed upright in TLC glass chambers saturated with a solvent
mixture containing n-butyl alcohol:ethanol:30%
ammonium hydroxide:chloroform (4:5:9:2). Plates were taken out after
the running front reached 1 inch below the top of the plates and
allowed to air-dry for 15 min. Plates were wrapped with plastic wrap
and exposed to x-ray film. Phosphorylated peptide bands were quantified
by PhosphorImager analysis. Total peptide bands were visualized by
ninhydrin staining of TLC plates.
mercaptoethanol). Samples were heated in a boiling water bath for
5 min and then analyzed by SDS-PAGE and autoradiography.
20 oC. After fixation, cells were rehydrated with water
and stained with 0.15% crystal violet solution for 5 min. Cells were
then washed three times with water to remove excess crystal violet stain. Colonies were counted and photographed.
20 °C for at least 2 h. After fixation, cells were spun at 1,000 rpm for 5 min and rehydrated with
PBS. Cells were then incubated with RNase A and incubated on ice for 10 min. Cells were spun down, washed once with Wash Buffer (PBS, 50 mM EDTA pH 8.0), and resuspended in PBS with propidium iodide at 25 µg/ml. Cells were incubated for at least 15 min before processing for flow cytometry. FACS analysis was performed with a
Becton Dickinson FACS Calibur cell sorter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP,
and phosphorylation was detected by thin layer chromatography (TLC) and
autoradiography (Fig. 1B). The relative incorporation of
32P into phosphorylated peptides was quantified by
PhosphorImager analysis (Table II).
Phosphorylation by cyclin A-CDK2 and cyclin E-CDK2 was observed on
peptides containing serine 48 (Ser-48) and serine 154 (Ser-154),
with a strong preference for phosphorylation of Ser-48 (Fig.
1B and Table II). Cyclin D1-CDK4 also phosphorylated Ser-48
and Ser-154; however, preferential phosphorylation by cyclin D1-CDK4
was observed at Ser-154 (Fig. 1B and Table II).
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Fig. 1.
Identification of G1
cyclin-dependent kinase sites on E47. A,
functional domains and predicted proline-directed kinase sites of E47.
The predicted primary sequence maps of three reported E47 cDNA
clones is shown. Residue numbers for the cyclin D1-CDK4 and cyclin
A/E-CDK2 sites for each of the three clones are shown. The cDNA
clone designated E47 (61) was used for the studies presented in this
report, and the relative positions of all potential proline-directed
kinase sites are marked (small bars). The cDNA clone
designated E2-5 was numbered from the first predicted amino acid (5).
The third cDNA clone (E2A; see Ref. 27) is thought to contain an
additional transcriptional activation domain on the N-terminal region
(TAD1). All three cDNA clones contain a central
transcriptional activation domain (TAD2) with a loop-helix
motif (LH) and a C-terminal bHLH. B,
phosphorylation of E47 peptides by cyclin A-CDK2, cyclin E-CDK2, and
cyclin D1-CDK4. Synthetic peptides representing the 11-residue
neighborhoods surrounding the potential proline-directed kinase sites
in E47 (Table I) were incubated with purified cyclin A-CDK2, cyclin
E-CDK2, and cyclin D1-CDK4 in the presence of
[ -32P]ATP and Mg2+. Phosphorylated
peptides were then resolved by thin layer chromatography
(TLC) and detected by autoradiography. Phosphorylation at
serines 48 and 154 was detected (arrows).
List of E47 synthetic peptides
Phosphorimage analysis of E47 peptide phosphorylation by
cyclin-dependent kinases
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Fig. 2.
Phosphorylation of E47 by G1
cyclin-dependent kinases. A,
phosphorylation of recombinant histidine-tagged E47 and E47 site
mutants by cyclin A-CDK2, cyclin E-CDK2, and cyclin D1-CDK4 in
vitro. Purified cyclin A-CDK2, cyclin E-CDK2, and cyclin D1-CDK4
were incubated with recombinant wild-type E47 and E47 site mutants in
the presence of [ -32P]ATP and Mg2+.
Phosphorylated proteins were detected by SDS-PAGE and autoradiography.
Relative phosphorylation, as quantified by PhosphorImager analysis, is
indicated under the autoradiography. ND indicates not
determined. Cyclin A-CDK2 and cyclin E-CDK2 were inhibited from
phosphorylating E47 S48A site mutants, indicating preferential
phosphorylation of serine 48. In contrast, cyclin D1-CDK4 was inhibited
from phosphorylating E47 S154A site mutants, indicating preferential
phosphorylation of serine 154. Lower phosphorylation of the E47
S48A/S154A double site mutant than the S154A site mutant suggests that
cyclin D1-CDK4 phosphorylates serine 48, albeit more weakly than serine
154. The phosphorylated recombinant E47 substrate migrated as a single
band near 54 kDa. B, site mutation of serine 48 or serine
154 does not affect accumulation of E47 protein in transiently
transfected cells. CV-1 fibroblasts were transfected with expression
constructs for wild-type E47 (pECE-E47), E47-S48A site mutant
(pECE-E47-S48A), E47-S154A site mutant (pECE-E47-S154A), E47-S48A/S170A
double site mutant (pECE-E47-S48A/S170A), and empty vector (pECE).
After 36 h, a whole cell lysate was generated by dissolving the
cells in SDS-PAGE sample buffer. The proteins in the lysates were
resolved by SDS-PAGE and analyzed by Western blot for expression of E47
and E47 site mutants. Ectopically expressed E47 was detected as a
doublet migrating at 52-54 kDa; this doublet was not detected in CV-1
fibroblasts transfected with empty vector. C,
phosphorylation of E47 in a lymphoblastoid cell line derived from a
mantle cell lymphoma (MO2058). MO2058 cells were transfected by
lipofection with expression constructs for SV40-tagged wild-type E47,
E47-S48A site mutant, E47-S154A site mutant, and E47-S48A/S154A double
site mutant. After 18 h, the cells were metabolically labeled with
inorganic 32P for 2 h. Detergent lysates were then
generated, and ectopically expressed E47 and E47 site mutants were
immunoprecipitated with an antibody to the SV40 tag (KT-3).
Immunoprecipitated proteins were resolved by SDS-PAGE, and
phosphorylated proteins were detected by autoradiography.
Phosphorylation of E47 was inhibited in the S154A site mutant,
indicating that serine 154 on E47 is phosphorylated in MO2058 cells.
Lower phosphorylation of the E47 S48A/S154A double site mutant than the
S154A site mutant suggested that serine 48 also is phosphorylated in
MO2058 cells, albeit more weakly than serine 154. High molecular mass
phosphoprotein(s) consistently coimmunoprecipitated with wild-type E47
and the S48A site mutant, suggesting an affinity for the serine
154-phosphorylated form of E47. The identification of these bands is in
progress. D, cultures were transfected in parallel as
described in C and after 18 h metabolically labeled
with [35S]methionine and [35S]cysteine for
4 h. Detergent lysates were then generated, and ectopically
expressed E47 and E47 site mutants were immunoprecipitated with an
antibody to the SV40 tag (KT-3). Immunoprecipitated proteins were
resolved by SDS-PAGE and detected by autofluorography.
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Fig. 3.
G1 cyclin-dependent
kinase sites are not involved in the regulation of E47 transcriptional
activity. A, ectopic expression of cyclin D1 and cyclin
E inhibits activation of transcription by E47. NIH3T3 cells were
transfected with p4R(µE5 + µE2 + µE3)TATA-CAT reporter, and
expression plasmids for E47 (pECE-E47) and cyclins A, B1, B2, C, D1,
D3, and E (pRcCMV-cyclin). After 24 h in high mitogen medium, the
cells were switched to low mitogen for 48 h and then assayed for
CAT activity. Duplicate experiments were performed, and mean values of
quantification of CAT activity by PhosphorImager analysis are shown
beside the autoradiographs. B, mutation of G1
cyclin-dependent kinase sites does not affect
transcriptional activation by E47. NIH3T3 cells were transfected with
p4R(µE5 + µE2 + µE3)TATA-CAT reporter, and expression plasmids
for wild-type E47 (pECE-E47), E47-S48A site mutant (pECE-E47-S48A),
E47-S154A site mutant (pECE-E47-S154A), E47-S48A/S170A double site
mutant (pECE-E47-S48A/S170A), and empty vector (pECE). After 24 h
in growth medium, the cells were switched to low mitogen medium for
48 h and then assayed for CAT activity. Duplicate experiments were
performed, and quantification of CAT activity was performed by
PhosphorImager analysis of the TLC plates. The background value for
p4R(µE5 + µE2 + µE3)TATA-CAT expression in the presence of empty
vector was subtracted from all points. Mean values and variance are
shown. C, mutation of G1
cyclin-dependent kinase sites does not block inhibition of
E47 by ectopic expression of G1 cyclins. NIH3T3 cells were
transfected with p4R(µE5 + µE2 + µE3)TATA-CAT reporter, and
expression plasmids for wild-type E47 (pECE-E47), E47-S48A site mutant
(pECE-E47-S48A), E47-S154A site mutant (pECE-E47-S154A), E47-S48A/S170A
double site mutant (pECE-E47-S48A/S170A), and empty vector (pECE).
Cultures were also cotransfected with expression plasmids for cyclin D1
(pRcCMV-cyclin D1) and cyclin E (pRcCMV-cyclin E). After 24 h in
growth medium, the cells were switched to low mitogen medium for
48 h and then assayed for CAT activity. Quantification of CAT
activity was performed by PhosphorImager analysis of the TLC plates.
The background value for p4R(µE5 + µE2 + µE3)TATA-CAT expression
in the presence of empty pECE expression vector was subtracted from all
points. Mean values and variance are shown.
E47 growth suppression is conferred by the N-terminal domain
Mutation of G1 cyclin-dependent kinase sites
enhances growth suppression by E47
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Fig. 4.
Analysis of growth suppression by E47 site
mutants. A, stable expression of S154A or S48A/S154A
site mutants of E47 in C3H10T1/2 cells enhances sensitivity to
depletion of growth factors and/or contact inhibition of growth.
Expression constructs for E47 (pECE-E47) and S48A (pECE-E47-S48A),
S154A (pECE-E47-S154A), and S48A/S154A (pECE-E47-S48A/S154A) site
mutants of E47 were cotransfected with pRc/CMV (confers G418
resistance) into C3H10T1/2 cells. Transfection of vector (pECE) with
pRc/CMV was used a control. After 2 weeks selection in G418, colonies
were removed by trypsin and grown as polyclonal cultures. Growth curves
were generated after seeding T25 flasks with 3 × 104
cells. Cells were counted at the indicated intervals. An
arrow indicates when the cells were harvested for FACS
analysis (Table V); another arrow indicates when the culture
medium was replaced with new medium (Refresh medium). At the end point
(14 days), cells had reached confluence, and no mitotic figures were
observed in the cultures. B, phase contrast microscopy of
C3H10T1/2 cells permanently transfected with E47 and E47 site mutants.
Permanently transfected polyclonal cultures described in Fig.
5A were grown to confluence (14 days) and photographed by
phase contrast microscopy. Magnification is the same in all fields (× 400); approximate nuclear counts for each field are indicated
below the photographs. C3H10T1/2 cells transfected with
either the S154A or S48A/S154A site-mutants of E47 displayed
significantly greater sensitivity to contact inhibition of growth, as
indicated by the lower density of these cultures at confluence.
FACS analysis of cell cycle distribution of growing C3H10T1/2 cells
permanently transfected with E47 and E47 site mutant expression
constructs
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 5.
Homology between sites displaying
preferential phosphorylation by cyclin D1-CDK4 in E47 and pRb.
Phosphorylation of serine 154 on E47 displays a marked preference for
cyclin D1-CDK4, similar to serine 780 on pRb. Homology between these
two sites is shown. Most striking is the presence of prolines and
hydrophobic residues C-terminal to the directing proline in both the
E47 serine 154 site and pRb serine 780 site. In contrast, the pRb
serine 795 site, which displays roughly equivalent phosphorylation by
both cyclin E-CDK2 and cyclin D1-CDK4, has a C-terminal basic residue
characteristic of generic cyclin-CDK phosphorylation sites. Also shown
is the consensus sequence for generic cyclin-CDK phosphorylation sites,
which in contrast to sites preferentially phosphorylated by cyclin
D1-CDK4 contains basic rather than hydrophobic residues C-terminal to
the directing proline.
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ACKNOWLEDGEMENTS |
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We thank S. Aaronson, D. Beach, A. Bergemann, D. Burstein, F. Cole, P. Hinds, E. Johnson, T. Kadesch, R. Krauss, A. Lassar, M. Lisanti, J. Manfredi, T. Meeker, P. Palese, C. Sherr, R. Weinberg, and B. Wold for generous reagent gifts and helpful discussions.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant CA-72775 from the NCI of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology
(1194), Mount Sinai School of Medicine, 1 Gustave Levy Place, New York,
NY 10029. Tel.: 212-241-9169; Fax: 212-534-7491; E-mail: stave.kohtz@mssm.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008371200
2 C. Chu and D. Stave Kohtz, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: bHLH, basic helix-loop-helix; pRB, retinoblastoma susceptibility gene product; RB, retinoblastoma; CDKs, cyclin-dependent kinases; FBS, fetal bovine serum; DMEM, Dulbecco's minimum essential medium; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; CMV, cytomegalovirus.
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