Molecular Medicine |
From the Molecular Cardiology Unit (S.A., S.Z., M.A., M.D.S.), Departments of Medicine (S.A., S.Z., M.A., M.D.S.), Cell Biology (M.D.S), and Molecular Physiology & Biophysics (M.D.S), and the Graduate Program in Cardiovascular Sciences (S.Z., M.D.S.), Baylor College of Medicine, Houston, Tex.
Correspondence to Dr Michael D. Schneider, Molecular Cardiology Unit, Baylor College of Medicine, One Baylor Plaza, Room 506C, Houston, TX 77030. E-mail michaels{at}bcm.tmc.edu
| Abstract |
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Key Words: adenovirus cell cycle cyclin-dependent kinase E1A p21
| Introduction |
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To identify and override cell cycle constraints in cardiac muscle cells, we and others26 27 28 29 30 have used adenoviral gene transfer to dissect growth control pathways in primary cell culture, without the immortalizing events, clonal variance, and fortuitous mutations that may confound the use of permanent cell lines. Moreover, no permanent cardiac cell line has had adequate fidelity to differentiated ventricular myocytes themselves,31 although cardiac myocytes differentiated from clonal bone marrow stromal cells are promising in this regard.32 By using point mutations of the viral cell cycle regulator E1A, cardiac myocytes, like other cell types, were shown to possess dual growth inhibitory pathways, one dependent on E1A's binding to pocket proteins and one dependent on binding the transcriptional coactivator, p300, and its homologue, CREB-binding protein (CBP).26 29 30 Forced expression of E2F-1, the prototype for E2F proteins that are the best understood endogenous occupants of the "pocket," was sufficient by itself to reproduce the effect of pocket protein binding by E1A on G1 exit in cardiac muscle,26 27 a finding also seen in other cell backgrounds.13 33 34 Thus, G1 exit could be provoked by overexpression of a mammalian protein that is native to the cells, not merely by the viral protein E1A. However, multiple forms of E2F exist, which may be distinct functionally,35 36 and pocket proteins also associate physically with numerous factors apart from E2F.37 For these reasons, despite apparent similarities, it is ambiguous if G1 exit provoked by E2F-1 can replicate all aspects of G1 exit provoked by Rb-binding E1A proteins.
To test the hypothesis that pocket protein binding by E1A might engage mechanisms beyond those used by E2F-1, we expressed E2F-1 versus E1A proteins in cardiac myocytes in the absence or presence of 3 complementary cell cycle inhibitors: p16 (INK4a), p21 (Cip1/Waf1/Sdi1), or dominant-negative Cdk2. Unlike E2F-1, E1A was found to trigger G1 exit that is both refractory to p21 and independent of inducing Cdk2 function. This mechanism specifically requires the ability of E1A to bind pocket proteins but not its ability to bind p300/CBP.
| Materials and Methods |
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U2OS osteosarcoma cells (American Type Culture Collection, Manassas, Va) were propagated in DMEM containing 10% FBS (Hyclone Laboratories) and were growth-arrested using serum reduced to 0.5% for 48 hours before viral gene transfer.
AdE2F-1, AdE2F-1-VP16, and AdcycE, driven by the cytomegalovirus
immediate-early promoter, were obtained from J. Nevins (Duke
University Medical Center, Durham, NC),13 39 Adp21 from G.
Nabel (University of Michigan, Ann Arbor),40 and Adp16
from V. Sandig (Max-Delbrück-Zentrum, Berlin-Buch,
Germany).41 AdE2F-1-VP16 contains E2F-1 residues 1 to 368
fused to the activation domain of herpes simplex virus VP16. AdE2F-1
E138 was constructed by cloning E2F-1 E13842 into
p
E1sp1ACMV,43 which then was cotransfected with pJM17
into 293 cells. Adenovirus encoding dominant-negative Cdk2 was
constructed analogously, by first cloning Cdk2-D145N44
into p
E1sp1ACMV. AdHCMVsp1LacZ, encoding Escherichia coli
ß-galactosidase, was provided by F. Graham (McMaster University,
Hamilton, Ontario).43 Recombinant adenoviruses in the
Ad5dL309 background, encoding wild-type and mutant 12S E1A (lacking
conserved region 3), driven by the native E1A promoter, and
coexpressing E1B, were provided by E. Moran (Temple University,
Philadelphia, Pa).45
Epifluorescence Microscopy
Cells were cultured on 18-mm glass coverslips, fixed with 100%
methanol, and incubated with antibodies against E2F1 (2 µg/mL; C-20,
Santa Cruz Biotechnology) and sarcomeric myosin heavy chains (1
µg/mL; MF20, University of Iowa Hybridoma Bank) for 1 hour at 37°C,
followed by FITC-conjugated antibody against rabbit IgG and Texas
Redconjugated antibody against mouse IgG (4 µg/mL; Molecular
Probes) for 1 hour at 37°C. To detect E1A and p21, mouse monoclonal
antibody to E1A (M73, 2 µg/mL; Santa Cruz Biotechnology) and rabbit
polyclonal antibody to p21 (C-19, 2 µg/mL; Santa Cruz Biotechnology)
were used, followed by rhodamine-conjugated anti-mouse IgG (10 µg/mL;
Boehringer Mannheim) and FITC-conjugated anti-rabbit IgG
(10 µg/mL; Boehringer Mannheim); the primary and secondary
antibody incubations were for 60 minutes each. Nuclei were stained with
2.5 µg/mL DAPI (Figure 1
) or 0.2
µg/mL Hoechst dye 33258 (Figure 6
). Cells were photographed
using a Zeiss Axioplan 2 microscope.
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Flow Cytometry
Cardiac myocytes were seeded at 106 cells
per well in Falcon Primaria 6-well plates (Becton Dickinson) and
infected with recombinant viruses as above. MF20 antibody was purified
by affinity chromatography (MAbTrapGII, Pharmacia),
concentrated by centrifugation using Microcon-50
filters (Amicon), and directly conjugated to FITC (200 µg/reaction)
using the FluoReporter FITC protein labeling kit (Molecular Probes).
Cardiac myocytes were harvested 36 to 48 hours after infection by
trypsinization, fixed in 70% ethanol at -20°C, washed in PBS
containing 1% BSA, labeled with 2 µg/mL FITC-MF20 at 4°C, and then
labeled with 50 µg/mL propidium iodide (PI) in the presence of 50
µg/mL RNAse A. FITC and PI fluorescence were analyzed
by two-color flow cytometry (Coulter): total populations were gated to
remove doublets and debris, and >5000 MF20-positive cells were
assessed for each DNA histogram, using MODFIT 2.0 software. U2OS cells
were labeled in 500 µL of PBS and 1% BSA, containing 10 µg/mL of
PI and 250 µg/mL of RNAse A, for 30 minutes at 37°C.
Western Blot Analysis and Immune Complex Kinase
Assays
Cells were harvested at the indicated times after gene transfer.
Lysates (50 µg/lane) were resolved by SDS-PAGE and transferred to
nitrocellulose membranes. Filters were incubated with primary
antibodies (0.2 µg/mL, 1 hour) followed by HRP-conjugated donkey or
goat antibody against rabbit or mouse IgG (Amersham, 1:2000, 30
minutes). Bound secondary antibody was detected as chemiluminescence
using ECL reagents (Amersham). Murine monoclonal antibody to E1A (M73)
and rabbit antibodies against E2F-1 (C-20), p21 (C-19), p16 (C-20),
cyclin D3 (C-16), cyclin E (M-20), cyclin A (C-19), cdk2 (M2), and cdk4
(C-22) were from Santa Cruz Biotechnology. Rabbit antibody to NPAT
(nuclear protein mapped to the
ataxia telangiectasia locus) was provided by
E. Harlow (Massachusetts General Hospital,
Charlestown).46 The identity of all proteins illustrated
in the Western blot montages was corroborated using appropriate
molecular size markers (not shown).
Immune complex kinase assays for Cdk4 and Cdk2 were performed 24 hours
after infection, using anti-Cdk4 (C-22AC) and anti-Cdk2 (M2AC)
antibody-agarose conjugates (Santa Cruz Biotechnology) for the
immunoprecipitations (30 µL; 2 hours at 4°C) and 500 or 200 µg of
cell lysate, respectively, as previously detailed.13 For
Cdk2 activity, immune complexes were washed twice in 20 mmol/L
Tris-HCl (pH 8.0), 250 mmol/L NaCl, 1 mmol/L EDTA, and 0.5%
NP-40, twice in the same buffer containing 100 mmol/L NaCl, and
once in kinase buffer (50 mmol/L Tris-HCl [pH 7.5], 10
mmol/L MgCl2, and 1 mmol/L DTT). Kinase
activity was assayed for 30 minutes at 30°C, using 5 µg of histone
H1 (Boehringer Mannheim) as substrate, 1 µmol/L ATP, and
5 µCi of [
-32P]ATP (3000 Ci/mmol, Amersham
Pharmacia Biotech), in a final volume of 50 µL. For Cdk4 activity,
immune complexes were washed four times with 50 mmol/L HEPES (pH
8.0), 150 mmol/L NaCl, 1 mmol/L EDTA, 2 mmol/L EGTA,
1 mmol/L DTT, 0.1% Tween-20, 10% glycerol, 0.1 mmol/L PMSF,
1 mmol/L NaF, and 0.1 mmol/L
Na3VO4 and once with Rb
kinase buffer (50 mmol/L HEPES [pH 8.0], 10 mmol/L
MgCl2, and 1 mmol/L DTT). Kinase activity
was assayed for 30 minutes at 30°C, using 2 µg of GST-Rb(769)
(Santa Cruz Biotechnology) as substrate, 2.5 mmol/L EGTA, 10
mmol/L ß-glycerophosphate, 0.1 mmol/L
Na3VO4, 1 mmol/L NaF,
20 µmol/L ATP, and 5 µCi of
[
-32P]ATP in a final volume of 30 µL.
Statistics
Results, shown as mean±SE, were compared by ANOVA and
Dunnett's one-tailed t test, with a significance level
of P<0.05.
| Results |
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To monitor the cell cycle in authenticated myocytes after gene transfer
with virus for E2F-1 proteins, in the absence or presence of p21, we
used two-color flow cytometry. PI was used to define DNA content, in
concert with FITC-conjugated MF20 antibody to sarcomeric myosin heavy
chains as a marker of cell type. A representative
two-color plot is shown in Figure 2A
; MF20-positive cells (quadrants 1 and
2) are selected as the region of interest for constructing the DNA
histograms (Figure 2B
). Numbers presented adjacent to
each histogram in the figure correspond to the single histograms shown;
numbers presented in the text below correspond to the mean
values of replicate studies (Figure 2C
). In uninfected cells or
LacZ-infected cells, the predominant myocyte population comprised a
single peak, with DNA content corresponding to G0/G1, the percentage of
myocytes with greater DNA content (S phase plus G2/M) was 16.4±0.53%
for uninfected cells and 17.4±0.8% for LacZ-infected cells. As seen
previously with the more limited approach of
immunostaining for BrdU27 a measure of
DNA synthesis not contentefficient G1 exit was provoked in
ventricular myocytes by wild-type E2F-1 (S+G2/M:
37.1±3.7%). E2F-1induced G1 exit was potentiated by E1B (not
shown), a viral homologue of Bcl-2, which inhibits cell loss from
apoptosis in this setting.27 However, E2F-1 did
not elicit the hypodiploid subG1 peak associated with
apoptosis from other causes.47 The lack of a
hypodiploid population at this early a time point is consistent
with published results for E2F-1 in other cell types, where a subG1
peak required 4 to 5 days,35 perhaps because this mode of
apoptosis and the associated DNA fragmentation occur
preferentially in the S+G2/M cells.
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To exclude the possibility that an E1A-like mechanism (competition for
the pocket, at high levels of expression) might explain the activity of
E2F-1 in cardiac muscle cells, we made use of an E2F-1/VP16 chimera: in
this hybrid protein, the Rb-binding and transactivation domains were
replaced with an activation domain that does not bind
Rb.13 Notably, the chimeric E2F-1 increased the prevalence
of ventricular myocytes in S phase plus G2/M to a similar
extent as did wild-type E2F-1 (S+G2/M: 27.6±1.3%; Figure 2B
).
Conversely, G1 exit was not elicited by E2F-1 E138, containing a point
mutation that prevents DNA binding42 (S+G2/M:
12.3±1.2%). Thus, as in other systems, Rb binding was neither
sufficient nor necessary for E2F-1 to drive the G1/S transition,
whereas an intact DNA-binding site was obligatory.48
Together, these findings therefore suggest a requirement for the
induction of one or more E2F-dependent genes.
As anticipated by this conclusion, forced expression of E2F-1 resulted
in the induction of cyclins E and A, demonstrated by Western blot
analysis (Figure 3A
). To test
whether the induction of cyclins E and A was accompanied by an increase
in the activity of Cdk2, the target of these cyclins, in vitro immune
complex kinase assays were performed (Figure 3B
). E2F-1 was
sufficient to evoke a marked increase in Cdk2 activity; by contrast,
Cdk4 function was not increased. (The upper and lower panels were
exposed for 15 minutes and 12 hours, respectively, for the Cdk4 kinase
activity to be visualized.)
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Cdk2 Kinase Activity Was Necessary, but Not Sufficient, for
E2F-1Induced G1 Exit
To ascertain whether this increase in Cdk2 function was merely an
epiphenomenon or instead a prerequisite for G1 exit provoked by E2F-1,
cardiac myocytes were coinfected with viruses encoding either of two
Cdk inhibitorsp16, which is selective for Cdk4/6, versus
p21, which also inhibits Cdk2. Coinfection with p21 had no adverse
effect on E2F-1 expression (Figure 1B
) but prevented G1 exit
completely, whether wild-type E2F-1 was used (S+G2/M: 12.1±0.5%;
Figure 2B
and 2C
) or the E2F-1/VP16 chimera (S+G2/M:
11.1±0.6%). p21 was highly effective, even when E1B was present
(not shown). Despite the arrest of cells in G0/G1, p21 did not prevent
upregulation of the E2F-inducible proteins cyclins E and A (Figure 3A
). By contrast, despite the induction of these
endogenous cyclins, p21 did completely block activation of
their target, Cdk2 (Figure 3B
). Although these results do not
exclude the possibility that diminished induction of the cyclins
contributed, in part, to the effect of p21, the all-or-none effect on
Cdk2 function is more readily reconciled with the known effect of p21
on Cdk2 itself.
As one criterion to test whether this block to G1 exit by p21 was
contingent on the inhibition of Cdk2 activity, parallel studies were
performed with p16; Western blots, to corroborate expression, and
two-color flow cytometry are shown in Figure 4A
. Consistent with the
expected specificity of p16, like other INK4 proteins, for Cdk4/6,
adenoviral delivery of p16 failed to suppress the observed increase in
Cdk2 activity induced by E2F-1 (Figure 3B
) and had no effect on
G1 exit induced by E2F-1 (E2F-1, S+G2/M: 49.9±9.2%; E2F-1+p16,
49.6±8.8%; Figure 4B
).
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Taken together, these results for E2F-1 suggest, as in other
preparations, that p21 can block E2F-dependent G1 exit, whereas p16
cannot. This ability of Cip/Kip Cdk inhibitors to override
E2F-1 has been observed by some authors34 49 50 but not
others,13 33 possibly because of levels of expression or
activation of endogenous cell cycle proteins differing
between experimental systems. Wherever this block has been shown to
exist, it has been ascribed to the ability of p21 or p27 to inhibit
Cdk2, not merely Cdk4/6, as do p16 and other INK4
proteins.34 49 50 Consequently, as a second criterion to
support this inference in the present study, we coinfected cardiac
myocytes with E2F-1 plus dominant-negative Cdk2 itself (Figure 5
). As expected, the dominant-interfering
form of Cdk2 largely inhibited G1 exit (S+G2/M: 32.8±2.4% versus
E2F-1 alone: 42.8±5.1%). Conversely, an increase in Cdk2 activity,
comparable to that seen with E2F-1, could be induced by overexpression
of exogenous cyclin E but was not accompanied by G1 exit (S+G2/M:
25.2±2.5%; Figure 5
). Thus, in cardiac myocytes, an increase
in Cdk2 function was required for G1 exit provoked by E2F-1, but it was
not sufficient for this response, unlike several other cell
backgrounds.8 51 52
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The Pocket Protein Binding Form of E1A Provokes G1 Exit That Is
Refractory to Inhibition by p21
To isolate the pocket protein versus p300dependent actions of
E1A, we used 2 well-characterized point mutations, R2G (which disrupts
binding to p300/CBP) and Y47H, C124G (which disrupts pocket protein
binding).26 45 Thus, these mutations elicit the pocket
protein- and p300/CBP-dependent effects of E1A, respectively. Each of
these three E1A viruses, driven by the native E1A promoter, also
expressed E1B to avert apoptosis. By indirect
immunofluorescence, all three E1A proteins were
widely expressed in cardiac myocytes and were appropriately targeted to
the nucleus, even on coinfection with virus encoding p21 (Figure 6A
). All three also were comparably
expressed, by Western blot analysis (Figure 6B
), and
evoked comparably efficient G1 exit, by two-color flow cytometry
(Figure 7
). A 3-fold-reduction of
myocytes in G0/G1, with a preponderant accumulation of cells in G2/M,
was seen (S+G2/M: wild-type 12S E1A, 77.6±6.0%; R2G, 81.1±2.8%; and
Y47H, C124G, 71.1±7.8%; Figure 7
, bottom). Because the
relative contribution of p300/CBP versus pocket protein binding has
previously been questioned, as a trigger for G1 exit by E1A in cardiac
myocytes,29 30 the use of flow cytometry is technically
advantageous compared with immunostaining for BrdU or
other S-phase markers, given the very large number of cells in each
sample (>5000). In addition, this method confirms not only S-phase
entry but also its completion. Under these conditions, however, neither
E2F-1 nor E1A yields mitotic figures or increases cardiac muscle cell
number,26 27 suggesting the need for additional signals
for M-phase entry.
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To determine if either or both of the E1A-dependent pathways were
sensitive to disruption by Cdk inhibitors, as was forced
expression of E2F-1, coinfection was performed with the virus encoding
p21 (Figure 7
). G1 exit via the p300/CBP pathway (by Y47H, C124G
E1A) was blocked by p21 (S+G2/M: 20.1±4.6%), yet G1 exit caused by
wild-type 12S E1A or R2G E1A was not (S+G2/M: wild-type E1A,
78.2±7.2%; R2G, 77.2±8.2%). Thus, although the three E1A viruses
were comparable in their effect on the DNA content of the myocytes,
their sensitivity to block by this Cip/Kip Cdk inhibitor
was not.
E1A Does Not Override the p21 Block to Cdk2 Function
To compare the effect of the pocket protein and p300/CBP pathways
on endogenous cell cycle regulators, Western blot
analysis was performed, as shown earlier for E2F-1. Each form
of E1A was sufficient to induce both cyclins E and A (Figure 6B
). Each of the three E1A proteins also induced the expression
of NPAT, a recently described substrate for cyclin E-Cdk2, which
accumulates at the G1/S boundary and promotes S-phase
entry.46 p21 had little or no effect on cyclin A and NPAT
induction by E1A via pocket proteins and did not prevent cyclin E
induction by any form of E1A. However, p21 did prevent the induction of
both cyclin A and NPAT by E1A via the p300/CBP pathway, responses that
may contribute to the observed growth arrest. Whether this interference
is mediated here by transcriptional mechanisms,
posttranscriptional mechanisms, or both has not been established.
However, illustrated by results presented earlier (Figure 3A
), E2F-1 can upregulate cyclin A in the presence of p21,
despite a complete block to G1 exit. Thus, growth arrest per se cannot
explain the failure of cyclin A to be induced by E1A via p300/CBP, when
delivered together with p21. One mechanism consistent, in
theory, with these results is a block to the
phosphorylation of p300/CBP by Cdk2, affecting its
histone acetyltransferase activity.53 54 By contrast,
induction of cyclin A by E1A via the pocket protein pathway was
refractory to inhibition by p21 and presumably independent of Cdk2
activity.
In the presence of exogenous p21, both E2F-1 and pocket
proteinbinding forms of E1A can cause the accumulation of cyclins A
and E (Figures 3A
and 6B
), despite their markedly
differing sensitivity to growth arrest by this Cdk
inhibitor. Hence, an alternative explanation is required,
such as differential sensitivity of Cdk2 to p21 after E2F-1 versus E1A,
respectively. To test this candidate mechanism directly, Cdk2 activity
was compared after infection of cardiac myocytes with viruses encoding
E1A or E2F-1 versus coinfection of these in turn with virus encoding
p21. Surprisingly, given the marked differences that occurred in its
effect on G1 exit, p21 blocked induction of Cdk2 activity to a
comparable extent after delivery either of R2G E1A, Y47H, C124G E1A, or
E2F-1: whereas each of these genes increased Cdk2 activity at least
12-fold in the absence of exogenous p21, little or no increase was
detected in the presence of exogenous p21 (Figure 8
). Thus, G1 exit caused by the pocket
proteinbinding form of E1A was independent of increasing Cdk2
function beyond that seen in the quiescent cells, distinct from G1 exit
provoked by E2F-1 or the p300/CBP pathway.
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To test the generality of our observations, another cell type was used,
U2OS osteosarcoma cells, with similar results for both flow cytometry
and Cdk2 activity (Figure 9
): G1 exit
produced by E1A via the p300/CBP pathway was sensitive to inhibition by
p21 (%S+G2/M: 35.6 versus 55.7), whereas G1 exit mediated by pocket
protein binding was refractory to inhibition by p21 (%S+G2/M: 72.4
versus 74.0). In U2OS cells as in cardiac myocytes, the inhibition of
Cdk2 activity by p21 was comparable for both forms of E1A.
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One remaining concern was that levels of Y47H, C124G E1A were
consistently diminished in the presence of exogenous p21
(Figure 6B
), raising the theoretical possibility that
differences between this virus and R2G E1A might merely be dose
related. That is, in principle at least, downregulation of Y47H, C124G
E1A might itself contribute to the p21-induced block of G1 exit.
However, downregulation of E1A is a foreseeable consequence of cell
cycle arrest, mediated by relative transcription of the native E1A
promoter: in transgenic mice, the E1A promoter is active in
proliferative tissue but is poorly expressed in postmitotic tissue,
including the heart.55 Therefore, to exclude this
possibility, we compared cardiac myocytes that were subjected to 3
different doses of the R2G and Y47H, C124G viruses in the absence or
presence of exogenous p21 (Figure 10
, top and middle). The level of E1A accumulation in myocytes infected
with Y47H, C124G+p21 was inherently sufficient for G1 exit, but this
form of E1A could not override the p21 block, eg, compare results of
p21+Y47H, C124G, with MOI of 10, with results of p21+R2G, with MOI of
5.
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| Discussion |
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A seemingly essential role for activation of Cdk2 in the G1/S transition has been inferred from dominant-negative Cdks,44 mutagenesis of p21,56 DNA replication in vitro,57 and microinjection studies.58 On the basis of these findings collectively, it has been inferred that Cdk2 supports a unique function that cannot be complemented by other Cdks. Despite these several lines of evidence that Cdk2 itself is necessary, E2F-1 itself was reported to override the requirement for Cdk2 in other circumstances, when E2F-1 was used in the presence of mitogenic serum.13 This counter example does not provide a plausible mechanism for the findings in the present study, however, given that E1A, but not exogenous E2F-1, could overcome the p21-dependent growth arrest. Our findings do not exclude the possibility that basal levels of Cdk2 function play an obligatory, permissive effect, in concert with other events, that must be activated by E1A. Because p21 was equally effective at inhibiting Cdk2 activity induced by either the pocket protein or p300-binding form of E1A, the suggested binding of E1A to p2159 is unlikely to explain the differential sensitivity of these two E1A-dependent pathways to p21-induced growth arrest.
What E1A-induced (or E1A-modulated) target proteins might account for escape from p21 and for the independence from an increase in Cdk2 function? One possibility is that alternative cell cycledependent kinases could be operative, such as Cdk314 44 or more distantly related G1/S kinases,60 presumably resistant to p21. Alternatively, H1 kinase activity might misrepresent the physiological state of Cdk2, for which other substrates now are known.46 61 62 Ultimately, any explanation for G1 exit without increasing Cdk2 function must address satisfactorily the functional disparity we have shown between bypassing and overriding Rb.
Highlighted by the model shown in Figure 11
, one attractive theoretical
possibility is that E1A might, directly or indirectly, substitute for
Cdk2 and activate a Cdk2 substrate54 (pathway 1).
Two possibilities exist to explain the ability of p21 to inhibit Y47H,
C124G but not R2G E1A. More simply, the activation of this substrate
might be impaired by the Y47H, C124G mutation. Alternatively, this
activation might not suffice for G1 exit by itself. Suggesting the
likelihood of this latter possibility, Cdk2 activity was necessary but
not sufficient for G1 exit induced by E2F-1, implying that other
effectors of E2F-1 must also function, in concert (pathway 2). In an
effort to define a biochemical basis for this novel action of E1A,
Rb-binding proteins other than E2F-1 are especially attractive as a
focus for future effort (pathway 3), especially those that modulate or
complement E2F-1, such as Myc and brahma-like transcription factors, in
addition to the functionally distinct E2F isoform
E2F-3.36 37 50 However, although the pocket
proteinbinding site was essential to override p21, a role for
alternative or additional E1A targets, acting in concert with this
site, is not excluded (pathway 4).
|
Since the submission of our manuscript to this journal, other investigators have reported the analogous ability of E1A to override p27 and the requirement for increased Cdk2 activity in various cell backgroundsRat1 fibroblastic cells, NIH 3T3 cells, and rat embryo fibroblastsand have suggested that a requirement exists for a second E1A domain, amino acids 26 to 35, concurrent with the Rb-binding domain.63 This region of E1A is required for interaction with p400,64 65 postulated to be a homologue of p300 on the basis of peptide mapping,66 and perhaps for interaction with other cellular proteins.67 68 69 70 Consistent with the interpretation that a second region of E1A may be required in addition, it is noteworthy that pocket protein binding by SV40 T antigen did not induce S phase in the presence of dominant-negative Cdk2.14 Given that p21 is highly abundant and Cdk2 is poorly expressed in the adult ventricle23 24 25 two developmentally regulated events that are thought to underlie the postmitotic phenotypethis p21-resistant pathway may possibly offer inherent advantages for the manipulation of cardiac growth.
| Acknowledgments |
|---|
Received April 30, 1999; accepted June 11, 1999.
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