(Received for publication, April 5, 1995; and in revised form, May 5, 1995)
From the
Phosphorylation of cyclin-dependent kinases (CDKs) by the
CDK-activating kinase is required for the activation of CDK enzymes.
Members of two families of CDK inhibitors, p16/p18 and p21/p27, become
physically associated with and inhibit the activity of CDKs in response
to a variety of growth-modulating signals. Here, we show that the
representative members of both families of CDK inhibitors,
p21
The primary control of the eukaryotic cell cycle is provided by
a family of serine/threonine protein kinases known as cyclin-dependent
kinases (CDKs). ( Direct physical interaction between CDK inhibitors and CDK enzymes
is necessary for CDK inhibition, but the precise mechanism remains to
be elucidated. We asked whether CDK inhibitors would affect CDK
phosphorylation by CAK. Here, we show that CAK phosphorylation of CDK2
is prevented by the CDK inhibitors, p21 and p27, and that the
inhibition of CAK activity by p21 or p27 does not appear to involve a
direct interaction between CAK and p21 or p27. In addition, we find
that the CDK6-specific inhibitor p18 belonging to the p16 family of CDK
inhibitors also blocks CAK phosphorylation of CDK6.
Direct phosphorylation of
GST-CDK6 by CAK was performed by incubation of immunoprecipitated CAK
(100 µg of total protein) with 0.5 µg of GST-CDK6, 0.5 µg
of GST-cyclin D1, and 10 µCi of [
Figure 1:
p21 and p27 do not directly inhibit CAK
activity. Extracts of NHF cells were incubated with indicated
quantities of p21 or p27 and immunoprecipitated with anti-CDK2 (A) or anti-CDK7 (B) antibodies. The kinase activity
of immunoprecipitated CDK2 was measured by histone H1 phosphorylation (A), and the kinase activity of CAK was measured by GST-CDK2
phosphorylation (B). Lanes1 of both A and B show the immunoprecipitated CDK2 or CAK activities
from the lysates in the absence of CDK inhibitors. In lanes2 of both A and B, anti-CDK2 or
anti-CDK7 antibodies were blocked with correspondent antigenic peptides
before immunoprecipitation. Of note is the fact that none of the CDK
inhibitors block the immunoprecipitation of
CDK2.
Sequence similarity between CAK catalytic subunit CDK7 and
CDKs initially prompted us to ask whether CDK7 could also be regulated
by the p21 CDK inhibitor. To address this question, we raised
polyclonal antibodies against the bacterially expressed C-terminal
portion of human CDK7. This antibody was found not to cross-react with
different members of CDK family. The anti-CAK immunoprecipitates
possess kinase activity toward GST-CDK2.As reported
previously(5, 8, 9) , CDK2 alone without its
associated cyclin partner can be readily phosphorylated by CAK, but the
cyclin subunit is necessary for the activation of the CDK2 histone H1
kinase activity.Under our conditions the amount of immunoprecipitated
CAK was a limiting factor, therefore phosphorylation of histone H1 by
GST-CDK2 To test the
potential effect of p21 on CAK activity, we first assessed whether p21
interacts with the catalytic subunit of CAK, CDK7, as is the case for
other CDKs. Normal human fibroblasts (NHF) synchronized at different
phase of the cell cycle by serum starvation and re-stimulation were
metabolically labeled with [ Since it remained possible that the failure to detect the
presumptive p21-CAK association may have resulted from low levels of
CDK7 expression or from antibody disruption, we tested the potential
direct interaction of p21 and CAK by assaying the activity of CAK in
presence of high quantities of bacterially expressed p21. In these
experiments we also used a related CDK inhibitor p27. When added to NHF
cell lysates, purified recombinant p21 and p27 effectively inhibited
the histone H1 kinase activity of immunoprecipitated CDK2 (Fig. 1A). In contrast, no inhibition of CAK kinase
activity (as assessed by CDK2 phosphorylation) by p21 or p27 was
detected (Fig. 1B). Thus, from these data, we believe
that neither p21 nor p27 physically interact with CAK. Although p21
and p27 do not appear to interact with CAK directly, it is still
possible that the binding of CDK inhibitors with a CDK may render the
threonine at position 160 (in CDK2, or 161 in CDC2) inaccessible to the
CAK enzyme, thereby preventing the phosphorylation and activation of
the CDK. To test this possibility, we assembled the CDK2-cyclin A
complex in vitro using bacterially expressed and purified
GST-CDK2 and GST-cyclin A and used this complex to determine possible
effect of p21 and p27 on CDK2 phosphorylation by CAK. Intriguingly, the
addition of the p21 or p27 proteins to the CDK2-cyclin A complex
dramatically decreased CDK2 phosphorylation by CAK (Fig. 2, A and B). To further address the specificity of p21
and p27 inhibition of CDK2 phosphorylation by CAK, and to exclude the
possibility that this inhibition may be caused by a trace amount of Escherichia coli proteins copurifying with the p21 or p27
proteins, we tested the effect of another newly identified CDK
inhibitor, p18, on CDK2 phosphorylation by CAK. p18 is a homolog of p16
that strongly inhibits the activity of CDK6 and weakly inhibits that of
CDK4, but exhibits no detectable interaction with four other CDKs
including CDK2(21) . When we applied a recombinant p18 protein
that was prepared in a manner similar to that for p21 and p27, no
inhibition of CDK2 phosphorylation by CAK was detected (Fig. 2C). The same result was obtained when we added
bovine serum albumin instead of CDK inhibitors in the reaction (Fig. 2D). These results indicate that inhibition of
CDK2 phosphorylation by CAK is dependent on p21/p27 and not on p18.
Figure 2:
p21
and p27 proteins inhibit CDK2 phosphorylation by CAK. Complexes of
GST-CDK2, GST-cyclin A, and different amounts of p21 (A) or
p27 (B) were used as a substrates for CAK
immunoprecipitated from NHF cells. p18 (C) or BSA (D)
were mixed with GST-CDK2
The differences between two families of CDK inhibitors raise an
important question of whether the p16 family of CDK inhibitors can also
block the CAK phosphorylation on CDK. In order to analyze the effect of
the p16/p18 family of inhibitors on CDK phosphorylation by CAK, we
first determined whether the bacterially expressed GST-CDK6 protein
could be phosphorylated and activated by CAK/CDK7. Unlike CDK2
activation that has been well studied, no data about phosphorylation
and activation of CDK6 is available. Fig. 3(toppanel) shows that in the absence of immunoprecipitated
CAK, no phosphorylation of CDK6 takes place. Some phosphorylation of
GST-CDK6 can be detected when the recombinant CDK6 is coincubated with
CAK in the absence of cyclin. However, the addition of GST-cyclin D1
enhances CAK phosphorylation of CDK6 and renders the CDK6 enzymatically
active in the phosphorylation of the Rb protein (Fig. 3, bottompanel). To prove that CDK6 phosphorylation by
CAK occurs at the unique site at threonine at position 177
that is homologous to Thr-160 in CDK2 and Thr-161 in CDC2, we used
site-directed mutagenesis to replace Thr-177 by Ala. When GST-CDK6
T177A was treated with immunoprecipitated CAK, no phosphorylation of
the mutant CDK6 protein (Fig. 3, toppanel) or
Rb kinase activity (Fig. 3, bottompanel) was
detected. These data show that CDK6 is activated through
phosphorylation by CAK, and that this phosphorylation is enhanced by
cyclin D1.
Figure 3:
Immunoprecipitated CAK phosphorylates
GST-CDK6 and activates its Rb kinase activity. Top panel,
immunopreciptated CAK was incubated with
[
We then tested the CDK6-specific inhibitor p18, a homolog
of p16(21) , for its effect on CDK6 phosphorylation by CAK. In
our experiment, the p18 protein and GST-cyclin D1 were first mixed,
then added to GST-CDK6. After 30 min of incubation, the resultant
complex was mixed with immunoprecipitated CAK and assessed for
CAK-dependent phosphorylation. Fig. 4(toppanel) shows that p18 efficiently blocked the CDK6
phosphorylation by CAK. When recombinant Rb protein was added to the
CDK6/p18/cyclin D mixture after CAK activation, no Rb phosphorylation
was found (Fig. 4, bottompanel). These data
show that, like CDK2, CDK6 activation by CAK is also regulated by p18
CDK inhibitor. Thus, we demonstrate that both families of CDK
inhibitors block CDK phosphorylation and activation by CAK and that
this block is most likely through the same mechanism: by rendering the
CDKs inaccessible to CAK.
Figure 4:
p18 inhibits GST-CDK6 phosphorylation by
CAK. In the toppanel, increasing amounts of p18 were
pre-mixed with GST-cyclin D1 and then added to GST-CDK6. This complex
was then incubated with immunoprecipitated CAK showing inhibition of
CDK6 phosphorylation by p18. In the bottompanel, Rb
phosphorylation in presence of p18 was
tested.
During the preparation of this manuscript,
Kato et al.(7) reported that in mouse macrophages
addition of cyclic AMP (cAMP) resulted in growth arrest in mid-G
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, p27
, and p18,
can prevent the phosphorylation of their CDK partners, CDK2 and CDK6,
by CDK-activating kinase. No direct interaction between CDK-activating
kinase and the CDK inhibitors could be detected, suggesting that
binding of these CDK inhibitors to CDK subunits renders CDK
inaccessible to the CDK-activating kinase phosphorylation. These
findings suggest that a general mechanism of CDK inhibitor function is
to block the phosphorylation of CDK enzymes by CDK-activating kinase.
)The enzymatic activity of a CDK is
regulated at three different levels: cyclin binding and activation,
subunit phosphorylation, and inhibition by a group of heterologous
small proteins. Phosphorylation of Thr-161 (or Thr-160 in CDK2) is
required for p34
/p33
activation(1, 2) . The enzyme that causes
the phosphorylation of p34
,
p33
, and perhaps other members of CDK family on
Thr-161, termed CDK-activating kinase
(CAK)(3, 4, 5, 6, 7) . In
mammalian cells, the catalytic subunit of CAK, CDK7, is associated with
and activated by a regulatory subunit, cyclin
H(8, 9) . CDK activity is also regulated by a number
of small proteins that physically associate with cyclins, CDKs, or
their complexes. There exist at least two distinct families of CDK
inhibitors in mammalian cells: the p21/p27 family and the p16/p18
family. p21 and p27 proteins share considerable sequence homology and
have been shown to be a potent inhibitors of almost all cyclin-CDK
enzymes(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ,
In contrast, the p16 family of CDK inhibitors specifically interacts
with the D-type cyclin-dependent kinases 4 and 6 (CDK4 and CDK6). The
p16 family currently includes four isolated genes that share sequence
similarity and were evolved from a common ancestor,
p16
(19) ,
p14/p15
(20, 21) , p18(21) , and p20.
The prototype
member of this family, p16, is homozygously deleted at a high
frequency in a wide variety of human tumor-derived cell lines (23, 24) and is also mutated or deleted in several
specific types of primary
tumors(25, 26, 27, 28, 29) .
Cell Lines and Cell Culture
Synchronization
Primary human foreskin fibroblasts (NHF)
cultivation, serum starvation, and serum stimulation were performed as
described in (30) .Antibodies to Human CDK7
Polyclonal antibodies
were raised in rabbits against C-terminal 131 amino acids (see (31) ). Corresponding fragment was PCR-amplified and subcloned
into a pQE31 vector (Qiagen, Chatsworth, CA) as a BamHI-KpnI fragment. Antigenic polypeptide was
expressed in bacteria as a hexahistidine-tagged recombinant protein and
purified according to Qiagen instruction. Antisera was
affinity-purified using bacterially expressed antigenic polypeptide
bound to Ni-NTA-agarose (Qiagen). 1 ml of the
antibodies was loaded into the column, the column was extensively
washed with phosphate-buffered saline, and antibodies were eluted with
1 ml of ActiSep media (Sterogene, Arcadia, CA) and desalted according
to manufacturer's instructions.
Construction of Mutagenic Recombinant CDK6
To
replace threonine 177 in GST-CDK6 for alanine, the fragment containing
two unique sites (SphI and DraIII) was PCR-amplified
using forward primer with SphI site:
5`-CGAGGAGGGCATGCCGCTCT-3` and reverse mutagenic oligonucleotide with DraIII restriction site:
5`-GTACCACAGCGTGACGACCACTGAGGCTAGAG-3`. The PCR fragment was digested
with SphI and DraIII and inserted into SphI/DraIII-cut GST-CDK6. The presence of mutation
was confirmed by sequencing reaction.Purification of Recombinant Proteins from
Bacteria
GST-cyclins A and D were purified according to Solomon et al.(1) . GST-CDK2 and GST-CDK6 were purified
according to Poon et al.(5) . Purification of
bacterially expressed CDK inhibitors will be described elsewhere. ()
Kinase Assay
Cell lysates (100 µg
of total protein) prepared in Nonidet P-40 lysis buffer (10) were used for immunoprecipitation with anti-CDK7
antibodies. CAK activation of GST-CDK2 and direct phosphorylation of
GST-CDK2 were performed as in (5) . To test an effect of CDK
inhibitors, 1 µg of GST-CDK2 and 1 µg of GST-cyclin A were
mixed with p21 or p27 in amounts indicated on the top of the pictures
in a total volume of 20 µl in kinase buffer for 30 min on ice. Then
5 µCi of [-
P]ATP was added, and the
reaction mixture was transferred to the tube with immunoprecipitated
CAK for 10 min at 23 °C, followed by SDS-polyacrylamide gel
electrophoresis and autoradiography.
-
P]ATP
for 40-60 min at 30 °C in the kinase buffer. To assay for the
activation of CDK6 by CAK, the first step was performed with 1 mM cold ATP as above. Then GST fusion protein with the 137 C-terminal
amino acids of Rb (21) was added together with 10 µCi of
[
-
P]ATP for another 20 min at 30 °C.
Direct Effect of p21 and p27 on CDK2 and CAK
Activity
To analyze direct effect of CDK inhibitors on CDK2 or
CAK (Fig. 1), 100 µg of cell lysates were incubated with
indicated amounts of p21 or p27 for 1 h at 4 °C. Then CAK or CDK2
were immunoprecipitated, washed two times with lysis buffer and two
times with kinase buffer, and activities were measured using GST-CDK2
as a substrate for the anti-CDK7 immunoprecipitates or histone H1 as a
substrate for the anti-CDK2 immunoprecipitates.
GST-cyclin A complex always correlated with
phosphorylation of GST-CDK2 by CAK (data not shown).
S]methionine and
immunoprecipitated with antibody specific to CDK7. This experiment did
not reveal the presence of a 21-kDa protein in anti-CDK7
immunoprecipitates. Furthermore, when anti-p21 immunoprecipitates were
probed with antibodies against CDK7 we found no detectable CDK7 protein
in asynchronously growing fibroblasts. Similarly, when anti-CDK7
immunoprecipates were probed with antibodies against p21, we found no
p21 protein in fibroblasts synchronized by serum starvation and
restimulation. The same results were also obtained in human lymphocytes
stimulated to proliferate by PHA and IL-2 treatment (data not shown).
GST-cyclin A instead of p21/p27, showing
no effect on CDK2 phosphorylation.
-
P]ATP and GST-CDK6, in the absence (lane3) or presence (lane4) of
GST-cyclin D1 and analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography. Bottom panel, immunoprecipitated CAK was
incubated in the presence of cold ATP and GST-CDK6, with or without
cyclin D1. The reaction was then assayed for kinase activity using a
purified GST-Rb protein as substrate. In lanes5 of
both panels, a GST-CDK6 protein mutated at threonine 177 (threonine
alanine, GST-CDK6 T177A) was used.
phase, where CDK4 exerts its effect on Rb phosphorylation. The
study of events that take place after cAMP treatment revealed that
induction of the p27 CDK inhibitor blocked the CDK4 activation. Similar
to our results, the authors did not find direct interaction of p27 with
CAK; thus the inhibition of CDK activation by p27 bound to CDK4-cyclin
D1 complex was inferred. Taken together, these data suggest that, in
addition to the block of CDK phosphorylation of downstream substrates
such as Rb or histone H1, another general mechanism by which CDK
inhibitors regulate the cell cycle is by preventing CDK phosphorylation
and activation by CAK.
We are grateful to Rolf Craven for critical reading of
the manuscript and to Aalok Kuthiala and Edward Baptist for help with
experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.