(Received for publication, July 28, 1995; and in revised form, January 22, 1996)
From the
To elucidate the role of protein kinase C in vascular smooth
muscle cell proliferation, we examined the effects of phorbol
12-myristate 13-acetate (PMA) on G events in human arterial
cells. About 15 h after G
cells were stimulated with fetal
bovine serum and basic fibroblast growth factor,
[
H]thymidine incorporation started. PMA (10
nM) inhibited the incorporation over 90% when added earlier
than 3 h after stimulation, but had no effect when added 12 h or later.
PMA inhibited the phosphorylation of the retinoblastoma protein (pRb),
which normally began at about 9 h. PMA did not inhibit the gene
expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of
which began at 0-3 h. However, PMA reduced the expression of
cyclins E and A, which usually began at 3-9 h and about 15 h,
respectively. PMA inhibited the histone H1 kinase activity of Cdk2,
which increased from about 9 h, whereas PMA did not inhibit the pRb
kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable
from 0-3 h. These results suggested that the PMA-induced
inhibition of pRb phosphorylation is not mediated by suppressing cyclin
D-associated kinase(s) including Cdk4, but involves the suppression of
Cdk2 activity that results from the reduced expression of cyclins E and
A.
Hyperplasia of vascular smooth muscle cells (VSMCs) ()plays a central role in the formation of atherosclerotic
lesions and intimal thickening after angioplasty. It is also one of the
pathological changes that occurs during the development of
hypertension. Therefore, it is essential for understanding the etiology
of these disorders to elucidate the mechanism regulating VSMC
proliferation(1, 2, 3) .
VSMC
proliferation is promoted by several biological substances, such as
growth factors, cytokines, vasoactive peptides, catecholamines, and
arachidonate metabolites(1, 2) . Most, if not all, of
these substances activate protein kinase C (PKC), since upon
stimulation of their receptors, phospholipase C is activated, which
stimulates phosphoinositide turnover producing
1,2-sn-diacylglycerol (DAG), which then activates
PKC(4, 5) . PKC activity is sustained by DAG produced
from phosphatidylcholine by subsequently activated phospholipases C and
D(6) . Lysophosphatidylcholine and fatty acids produced by
phospholipase A may also contribute to PKC
activation(7) . Moreover, phosphatidylinositol 3,4-bisphosphate
and phosphatidylinositol 3,4,5-trisphosphate, the products of
phosphatidylinositol 3-kinase, reportedly activate some isoforms of the
PKC family(8, 9) .
The role of the PKC pathway in mitogenesis remains undetermined. PKC activators such as phorbol esters and membrane permeable DAGs mimic competence factors (10, 11) and induce the expression of immediate early genes such as c-fos and c-myc(12) . PKC directly phosphorylates and activates Raf-1(13, 14) , which would lead to the activation of MAP kinase. Therefore, PKC may be involved in the early phases of mitogenesis as a positive mediator.
Nevertheless, phorbol ester inhibits the transition from G to S phase in a variety of cell species including
VSMCs(15, 16, 17, 18) . Since this
effect is mimicked by repeated doses of membrane-permeable DAG and is
prevented by the down-regulation of
- and
-isoforms of PKC,
we suggested that PKC mediates the G
/S inhibition induced
by phorbol ester(18) . Moreover, PKC-mediated inhibition seems
to operate as a physiological mechanism, since DNA synthesis is
accelerated when PKC is down-regulated(18) . However, the
mechanism by which PKC inhibits the G
/S progression remains
to be investigated.
In the present study, we examined the effects of
phorbol ester on cellular events during the G and S phases,
including the phosphorylation of the retinoblastoma protein (pRb) and
the activation of cyclin-dependent kinases (Cdks), using VSMCs from
human umbilical arteries.
To measure the
activities of cyclin D-associated kinase and Cdk4, glutathione S-transferase-fused murine pRb C-terminal (amino acid residues
754-921) (GST-Rb) prepared as described (21) from pGEX-3X
containing the cDNA fragment provided by Hitoshi Matsushime, was used
as the substrate. The immunoprecipitates obtained using the antibodies
to cyclin D and Cdk4 were suspended in the buffer (40 µl)
containing 50 mM Hepes/NaOH (pH 7.5), 10 mM MgCl, 1 mM dithiothreitol, 20 µg/ml
GST-Rb, and 50 µM [
-
P]ATP
(14.8 GBq/mmol), then incubated for 40 min at 30 °C. The following
procedures were the same as those described for the Cdk2 assay.
Figure 1:
The
effect of PMA on the cell cycle progression. VSMCs seeded at 8
10
/dish (177 cm
) were cultured in growth medium
for 48 h, after which they were incubated in DMEM containing 0.1% BSA
for 48 h to be synchronized to the G
phase. Cells were then
stimulated with growth medium in the presence of 0.1% Me
SO
(control) or 10 nM PMA. At the indicated times after
stimulation, cells were trypsinized and stained with propidium iodide.
The DNA content in about 1
10
cells was measured by
flow cytometry. The x and y axes represent the
intensity of fluorescence and cell number,
respectively.
However, as shown in Fig. 1, some of the control cells
did not enter S phase even after mitogenic stimulation. Since the cell
viability was over 95% according to trypan blue exclusion, it was
unlikely that these non-responding cells were injured or dead.
Therefore, we considered that these cells were those remaining in
G phase due to contact inhibition, which cannot be avoided
when using normal diploid cells. Nevertheless, we used these cells,
because we felt that contamination with G
cells would not
interfere with the interpretation of our experimental results.
The
incorporation of [H]TdR started to increase about
15 h after mitogenic stimulation and reached a plateau at about 33 h (Fig. 2a), which corresponded well with the results of
the flow cytometry (Fig. 1). There was little incorporation when
cells were not stimulated, suggesting that entry into S phase was
strictly dependent on mitogenic stimulation (Fig. 2a).
To determine when PMA inhibits the cell cycle, PMA (10 nM) was
applied at several time points during the G
and S phases (Fig. 2b). PMA added earlier than 3 h after stimulation
inhibited [
H]TdR incorporation over 90%. However,
the effect was attenuated when PMA was added 6 h or later, and there
was no effect when added 12 h or later, suggesting that PMA inhibited
the G
, but not S phase progression. The inhibition was
maximal at a PMA concentration of 10 nM and partially reversed
above this concentration (Fig. 2c).
Figure 2:
The effect of PMA on DNA synthesis. a, cells seeded at 1 10
/well (1.8
cm
) were synchronized in the G
phase by serum
starvation, after which they were incubated in growth medium (
) or
DMEM containing 0.1% BSA (
), in the presence of
[
H]TdR (37 kBq/well). The amount of incorporated
radioactivity was determined at the indicated times. Data represent the
means ± S.D. (n = 3). b, G
cells were labeled with [
H]TdR in growth
medium for 33 h, during which PMA (10 nM) or vehicle (0.1%
Me
SO) was added at the indicated times. Data are the means
± S.D. (n = 3) of the PMA-induced inhibition (%)
against the values obtained with vehicle. *, p < 0.01 versus vehicle-treated cells. c, G
cells
were labeled with [
H]TdR in DMEM containing 0.1%
BSA (lane 1) or in growth medium (lanes 2-8) in
the presence of various concentrations of PMA added at time 0. The
amount of incorporated radioactivity was determined at 33 h. Lane
1, no stimulation (0.1% BSA); lane 2, growth medium; lane 3, growth medium + 0.1% Me
SO (vehicle); lanes 4-8, growth medium + PMA (0.1, 1, 10, 100,
and 1000 nM, respectively). Data represent the means ±
S.D. (n = 3). *, p < 0.01 versus lane
3.
Figure 3:
The
effect of PMA on pRb phosphorylation. a, G cells
were incubated in growth medium in the presence of PMA (10 nM)
or vehicle (0.1% Me
SO) for various times as indicated
below, after which they were collected and lysed. The proteins
precipitated by the anti-pRb antibody (XZ104) were separated by 7.5%
SDS-PAGE and immunoblotted against another anti-pRb antibody
(PMG3-245). Lanes 1 and 2, not stimulated with growth
medium; lanes 3-18, incubated in growth medium for 3 h (lanes 3 and 4), 6 h (lanes 5 and 6), 9 h (lanes 7 and 8), 12 h (lanes 9 and 10), 15 h (lanes 11 and 12), 18 h (lanes 13 and 14), 21 h (lanes 15 and 16), and 24 h (lanes 17 and 18),
respectively. Odd-numbered lanes, vehicle; even-numbered
lanes, PMA. b, G
cells were incubated in DMEM
containing 0.1% BSA (no stimulation) or in growth medium containing
various concentrations of PMA as indicated. *, 0.1% Me
SO
(vehicle) was added. Cells were lysed at 24 h and immunoblotted. c, after G
cells were stimulated with growth
medium, PMA (10 nM) or vehicle (0.1% Me
SO) was
added at 18 h. Cells were lysed at the indicated times and
immunoblotted. To indicate the position of hypophosphorylated pRb, the
immunoprecipitate from the G
cell lysate was also blotted
(shown as time 0).
Figure 4:
The effects of PMA on Cdk gene expression.
G cells were incubated in growth medium in the presence of
PMA (10 nM) or vehicle (0.1% Me
SO). Total cellular
RNA was extracted at indicated times. Equal amounts of RNA (20
µg/lane) were resolved by electrophoresis and hybridized with
P-labeled cDNA fragments of Cdks. The sizes of the
transcripts (in kilobase pairs) were: Cdc2, 1.6; Cdk2, 2.5; Cdk3, 2.7;
Cdk4, 1.7; Cdk5, 1.4.
We then examined the effects of PMA on the gene
expression of cyclins, since association with a cyclin is required to
activate Cdks. Cyclins G, C, D1, D2, and D3 were all induced from early
G (Fig. 5a). Cyclin G was expressed even in
quiescent cells without significant changes in its mRNA levels
throughout the G
and S phases. Cyclin C was also expressed
in quiescent cells, but this increased after mitogenic stimulation. The
expression of cyclins D1, D2, and D3 was low in quiescent cells, but
all were induced from the early G
phase. PMA (10
nM) did not reduce the expression of any of these cyclins at
least for the first 12 h, but rather tended to enhance that of cyclins
G, C, D1, and D2 in the early G
phase. PMA attenuated the
expression of cyclin D2 after 12 h.
Figure 5:
The effects of PMA on the gene expression
of cyclins. Northern blotting was performed as described in the legend
to Fig. 4. a, cyclins expressed from the early G phase. b, cyclins expressed later than early G
phase. The sizes of the transcripts (in kilobase pairs) were:
cyclin G, 2.7; cyclin C, 2.4; cyclin D1, 4.5; cyclin D2, 6.5; cyclin
D3, 2.2; cyclin E, 1.9; cyclin A, 2.9; cyclin B, 1.9;
-actin,
2.1.
Fig. 5b demonstrates the mRNA expression of cyclins induced later than
those shown in Fig. 5a. It was difficult to clearly
determine when the expression of cyclin E began, since small amounts of
the mRNA were detected 3-9 h after stimulation and before the
expression was markedly enhanced. Cyclin A was expressed from about 15
h, which corresponded to around the G/S border, and cyclin
B was expressed a few hours later than cyclin A, probably after entry
into S phase. In the presence of PMA (10 nM), however, the
expression of all these mRNAs was suppressed.
Figure 6:
The
effect of PMA on the activity of Cdk2. a, G cells
were incubated in growth medium in the presence of 0.1%
Me
SO (control) or 10 nM PMA. Cell lysates prepared
at the indicated times were precipitated with anti-Cdk2 antibody for
histone H1 kinase assay. After the enzyme reaction, assay mixtures were
resolved by 12.5% SDS-PAGE, electroblotted, and autoradiographed. b, G
cells were incubated in growth medium for 24
h and analyzed as described in a. PMA (10 nM) was
added at the indicated times after the release from G
. NS and S, not stimulated and stimulated with growth
medium in the absence of PMA, respectively. c, G
cells were incubated in growth medium for 24 h in the presence of
various concentrations of PMA or vehicle, and analyzed as described in a. NS, not stimulated with growth
medium.
Since Cdk2 phosphorylates pRb in
vitro(23) , the PMA-induced inhibition of Cdk2 activity
seemed to explain the inhibition of pRb phosphorylation. However, it
remained to be determined whether PMA inhibits the cellular events that
arise before Cdk2 activation. Cyclins of the D class and their major
partner Cdk4 may play crucial roles in the G/S transition
by phosphorylating pRb(30, 31) . As shown in Fig. 4and Fig. 5, D-type cyclins and Cdk4 were expressed
from the early G
phase, probably preceding the expression
of cyclins E and A. We therefore tested whether PMA influences the
activity of cyclin D-associated kinase(s), including Cdk4.
To
confirm that PMA does not inhibit the expression of Cdk4 and D-type
cyclins during the G phase, the protein levels of Cdk4 and
cyclin D were analyzed by Western blotting (Fig. 7). Consistent
with the result of Northern blotting, PMA did not reduce their protein
levels. Moreover, PMA did not reduce the amount of Cdk4 that
co-precipitated with cyclin D, indicating that PMA did not inhibit the
association of cyclin D with Cdk4.
Figure 7:
The
effect of PMA on the association between cyclin D and Cdk4. G cells were incubated in growth medium in the presence of PMA (10
nM) or vehicle and lysed 7.5 h after the release from
G
. Lysates were immunoprecipitated (IP) with
antibodies to Cdk4 or cyclin D as indicated. Precipitates were
separated by 10% SDS-PAGE, and immunoblotted with antibodies to Cdk4 or
cyclin D as indicated. The position of the IgG heavy chain (about 50
kDa) is also shown.
The activities of cyclin
D-associated kinase and Cdk4 were measured by the in vitro phosphorylation of GST-Rb, since pRb may be the physiological
substrate for these enzymes and the histone H1 kinase activity of Cdk4
was very low (not shown). The activity that co-precipitated with cyclin
D, of which the level was low in quiescent cells, increased during the
G phase to peak at 7.5 h (Fig. 8a). After
it dropped once at 9 h, the activity again increased from 12 until 24
h, when the activity was maximal during the observation period. In the
presence of PMA (10 nM), the elevation in the G
phase was enhanced and lasted for a much longer period, although
there was no second increase. The activity of Cdk4 was relatively high
even in quiescent cells and not markedly changed during the G
phase, although it significantly increased at 12-24 h (Fig. 8b). PMA also enhanced Cdk4 activity for the
first 9 h.
Figure 8:
The effects of PMA on the activities of
cyclin D-associated kinase and Cdk4. a, G cells
were incubated in growth medium in the presence of PMA (10 nM)
or vehicle for the indicated periods. Cell lysates were then prepared
and precipitated with anti-cyclin D antibody. After the kinase
reaction, assay mixtures were resolved by 10% SDS-PAGE. Proteins were
transferred to membranes and autoradiographed. The arrows indicate the positions of GST-Rb (46 kDa). b, the
procedure was the same as described in a, except that
anti-Cdk4 antibody was used for
immunoprecipitation.
The cellular event mediating the cell cycle arrest induced by
PMA should take place during G, but not S phase, since PMA
added after the advance into S phase failed to inhibit DNA replication.
This was consistent with flow cytometry studies on melanoma and U937
cells, which showed the inability of phorbol ester to block S phase
progression(32, 33) . Therefore, we studied the events
that arise prior to the G
/S transition.
Several afferent
signals converge onto pRb, including positive as well as negative
signals that inhibit the G/S transition. In turn pRb
transmits the integrated signal to a number of downstream effectors,
such as E2F transcription factors(31) . The pRb function is
regulated by phosphorylation, which occurs in the middle of the G
phase, close to and perhaps simultaneously with the R
(restriction) point(34) . Hypophosphorylated pRb actively
represses the advance into S phase, whereas the hyperphosphorylated
form loses this ability(31) . Transforming growth factor-
(TGF-
), cAMP, and cell-cell contact inhibit pRb
phosphorylation(31, 35) . PMA also inhibits pRb
phosphorylation in leukemia and lymphoma cell lines, and vascular
endothelial cells(35, 36, 37) . Although
mechanisms for the inhibition by the former three signals have been
proposed as described below, it remains to be determined how PMA
inhibits pRb phosphorylation.
In our VSMCs, PMA added simultaneously
with mitogens inhibited the in vivo phosphorylation of pRb in
a dose-dependent fashion similar to that of its effect on DNA
synthesis. However, once pRb was phosphorylated, PMA failed to reverse
it. Consistently, the inhibition of DNA synthesis was abolished, when
PMA was added later than 12 h after the release from G,
when pRb had already undergone phosphorylation. Therefore, PMA seemed
to arrest cells at the G
phase by inhibiting an event that
occurs before pRb phosphorylation.
Cdk2 phosphorylates pRb in
vitro and it may be a mediator of G/S
transition(23, 38) . Cdk2 activity increased from
about 9 h after the release from G
. Therefore, the onset of
the activation appeared to correspond to that of pRb phosphorylation.
The mobility of pRb appeared to have shifted by 15-18 h, whereas
Cdk2 activity continued to increase until 24 h. The progressive
elevation of Cdk2 activity after completion of the mobility shift could
contribute to the maintenance of phosphorylation, or induce the
phosphorylation of other sites on pRb that is undetectable by the
mobility shift. PMA inhibited Cdk2 activity in a dose-dependent manner
again similar to that for the inhibition of DNA replication. Therefore,
suppressed Cdk2 activity may explain the PMA-induced inhibition of pRb
phosphorylation. Once phosphorylated, pRb may not be dephosphorylated
even when Cdk2 activity is suppressed, because PMA added in the S phase
inhibited Cdk2 activity but did not reverse the mobility shift of pRb.
Coupling with either cyclins E or A is prerequisite for Cdk2
activation(26, 27, 28, 29) . The
time course of cyclin E expression appeared to correspond well with
that of Cdk2 activation, whereas cyclin A expression apparently
occurred later than cyclin E, and therefore, than pRb phosphorylation.
PMA inhibited the expression of these two cyclins, but not Cdk2.
Therefore, it was likely that the inhibition of Cdk2 activity resulted
from the reduced gene expression of cyclin E and, possibly, cyclin A.
In vascular endothelial cells, cyclin A appeared to be expressed
from the late G; therefore, we suggested that PMA caused
G
/S arrest by reducing cyclin A expression(39) . In
VSMCs, however, there was little expression of cyclin A until the cells
entered the S phase. This difference may have resulted from the
difficulty in synchronizing endothelial cells in the G
phase. There was little incorporation of
[
H]TdR until 15 h after growth stimulation in
VSMCs, whereas the incorporation gradually increased from 4 h in
endothelial cells. We consider that the endothelial cells assumed to be
synchronized in the G
phase contained a substantial number
of G
cells, which expressed cyclin A earlier than truly
G
-synchronized cells.
The onset of Cdk2 activation
appeared to be simultaneous with that of pRb phosphorylation, implying
that these two events were closely linked. However, Cdk2 activation did
not clearly precede pRb phosphorylation. It was conceivable, therefore,
that the initiation of pRb phosphorylation is mediated by other
kinases, and that PMA inhibits their activities. Cdks associated with
D-type cyclins, such as Cdk4 and Cdk6, also phosphorylate pRb in
vitro(21, 24, 25) . They may be the
kinases most prominently involved in the phosphorylation of pRb (31) . In contrast to Cdk2, the cyclin D-associated kinase
activity increased in the early to middle G phase forming a
peak 7.5 h after the release from G
, thus clearly preceding
the initiation of pRb phosphorylation. PMA did not suppress this
activity, but rather enhanced it in the early to middle G
phase. PMA did not inhibit the activity of Cdk4, either.
Consistently, PMA did not inhibit the expression of cyclins D1, D2, and
D3, and Cdk4 at least for 12 h after mitogenic stimulation. Moreover,
PMA did not impair the association of cyclin D with Cdk4. Therefore, it
was unlikely that the PMA-induced inhibition of pRb phosphorylation
resulted from the inhibition of cyclin D-dependent kinases. It remains
to be investigated why Cdk4 activity was relatively high even in
quiescent cells and its dependence on growth stimulation appeared lower
than the activity that co-precipitated with cyclin D.
However, if
cyclin D-dependent kinases such as Cdk4 and Cdk6 are responsible for
pRb phosphorylation, our results are contradictory, in that PMA
inhibited the in vivo phosphorylation of pRb regardless of its
inability to suppress the in vitro phosphorylation of GST-Rb
induced by cyclin D-associated kinase. Although we could not resolve
this discrepancy, there are some explanations. Collaboration between
cyclin D and cyclin E might be required for pRb phosphorylation. This
has been suggested by a report on pRb phosphorylation in Saccharomyces cerevisiae(40) . This phosphorylation
required specific combinations of yeast G cyclins, Cln3 and
either Cln1 or Cln2. The functions of Cln3 and Cln2 in pRb
phosphorylation were complemented by human cyclin D1 and cyclin E,
respectively. Therefore, it is possible that in our cells, reduced
cyclin E restrains pRb phosphorylation, even in the presence of active
cyclin D-Cdk complexes.
Alternatively, PMA could activate pRb
phosphatase. Although studies on the mechanism of pRb dephosphorylation
are so far limited, evidence suggests that one of the pRb phosphatases
is a type 1 protein phosphatase (PP1)(41) . The pRb directly
binds to the catalytic subunit of PP1, and the complex is detected in
G/G
, mid-G
, and M phase cells,
suggesting the importance of PP1 for the maintenance of
hypophosphorylated pRb from late M to mid-G
phase(42) . Therefore, if PMA can stimulate pRb
phosphatase, our apparently contradictory results would be coincident,
since pRb may be able to interact with pRb phosphatase in
vivo, but not in our in vitro pRb kinase assay system,
which lacks pRb phosphatase.
Negative growth signals generated by
TGF-, cAMP, and cell-cell contact may be mediated by inhibitor
proteins of Cdks(43, 44, 45) . Several Cdk
inhibitors have been found in mammalian cells, including
p21
, p27
, p16
,
and p15
(46) . Although there are only a few
reports on their mechanisms of action, p27 may inhibit Cdk4 and Cdk2 by
preventing the phosphorylation with Cdk-activating
kinase(44, 47) . p16 interrupts the association of
cyclin D1 with either Cdk4 or Cdk6(48, 49) . However,
PMA inhibited neither the activities of cyclin D-associated kinase and
Cdk4 nor the binding of cyclin D to Cdk4, suggesting that the effect of
PMA is not mediated by these inhibitors.
Alternative mechanisms have
been suggested for TGF- and cAMP. TGF-
reduces the levels of
Cdk2 and Cdk4 in human keratinocytes (50) and of Cdk4 in mink
lung epithelial cells(51) , and cAMP inhibits the expression of
cyclin D in murine macrophages(52) . However, in contrast, PMA
did not inhibit, but sometimes enhanced the cellular events arising in
the G
to early G
phase, that included the
expression of cyclins G, C, D, Cdk2, Cdk3, Cdk4, and Cdk5. This
corresponded to other reports that phorbol ester stimulates the
G
/G
transition by mimicking competence
factors(10) . Significantly, these contrasts between PMA and
other growth inhibitory signals indicate that PMA is a unique
inhibitory agent that suppresses pRb phosphorylation in a different
manner from others. PMA inhibits the proliferation of a lymphoma cell
line by inducing TGF-
(53) . However, this may not be so in
our cells due to the above reason, and moreover, because neutralizing
antibodies to TGF-
did not attenuate the PMA-induced inhibition of
G
/S transition. (
)
We showed that PKC mediates
the phorbol ester-induced inhibition of G/S transition,
since the inhibition was mimicked by repeated doses of
membrane-permeable DAG and abolished by down-regulation of PKC by a
preincubation with phorbol ester(18, 39) . The PMA
dose-effect relationship also suggested the involvement of PKC, because
the maximal inhibitory effects on DNA synthesis, pRb phosphorylation,
and Cdk2 activity were always obtained around 10 nM. This was
in agreement with the reported concentrations for PKC activation. This
inhibition was always partially reversed at concentrations over 100
nM, which we assumed to result from the down-regulation of
PKC.
It seems that the PKC pathway inhibits the cell cycle of VSMCs
at the mid-to-late G, but not the early G
phase. Cells remain competent for proliferation while PKC is
being activated. We thus speculated that PKC is a physiological
regulator of the G
phase, hindering DNA synthesis just
before the R point by preventing pRb phosphorylation until the
circumstances are favorable for advance into the remainder of the cell
cycle. Alternatively, PKC could contribute to cell differentiation,
since several lines of evidence have suggested that the prevention of
pRb phosphorylation and the resulting cell cycle arrest are necessary
for subsequent cell differentiation(31) . To test these
possibilities, it is essential to understand how PKC activity is
controlled during the cell cycle and cell differentiation.
In
addition, we found that vascular endothelial cells are arrested in the
G, as well as the G
phase by PMA and DAG (54) . If this is also a phenomenon universal among various
cell species, it would suggest that PKC operates as a regulator of the
cell cycle clock at two points, namely in the G
and G
phases. There are multiple phosphorylation sites on pRb (55) , and phosphorylation progresses by multiple steps, not
only in the G
, but also in the G
phase(56) . Hence, the G
inhibition could
also involve the inhibition of pRb phosphorylation. It is essential to
characterize each phosphorylation site in terms of the regulation of
pRb function.