Download A Cyclin A-Protein Kinase Complex Possesses

Wesleyan University
WesScholar
Division III Faculty Publications
Natural Sciences and Mathematics
1-10-1992
A Cyclin A-Protein Kinase Complex Possesses
Sequence-Specific DNA Binding Activity: p33 is a
Component of the E2F Cyclin-A Complex
Stephen Devoto
Wesleyan University, [email protected]
Follow this and additional works at: http://wesscholar.wesleyan.edu/div3facpubs
Part of the Neuroscience and Neurobiology Commons
Recommended Citation
Devoto, Stephen, "A Cyclin A-Protein Kinase Complex Possesses Sequence-Specific DNA Binding Activity: p33 is a Component of
the E2F Cyclin-A Complex" (1992). Division III Faculty Publications. Paper 59.
http://wesscholar.wesleyan.edu/div3facpubs/59
This Article is brought to you for free and open access by the Natural Sciences and Mathematics at WesScholar. It has been accepted for inclusion in
Division III Faculty Publications by an authorized administrator of WesScholar. For more information, please contact [email protected],
[email protected].
Cell, Vol. 66, 167-176,
January
10, 1992, Copyright
0 1992 by Cell Press
A Cyclin A-Protein Kinase Complex Possesses
Sequence-Specific
DNA Binding Activity: ~33~~~ Is
a Component of the E2F-Cyclin A Complex
Stephen H. Devote;
Maria Mudryj,’ Jonathon
Tony Hunter,t and Joseph R. Nevins’
Howard Hughes Medical Institute
Section of Genetics
Duke University Medical Center
Durham, North Carolina 27710
tThe Salk Institute
La Jolla, California 92037
Pines,t
l
Summary
The E2F transcription
factor has been found in association with the cyclin A protein, and this complex accumulates during the S phase of the cell cycle, suggesting that E2F may play a role in cell cycle control.
In independent
studies, cyclin A has been shown to
be associated with two other proteins, the M-related
~107 protein and the cdcbrelated
~33 cdk2 protein
kinase. Through an analysis of the EPF-cyclin A complex, we now find that both the ~107 protein and the
cdc2-related
~33~~ kinase are components
of the previously described
complex.
Moreover, the complex
possesses Hl kinase activity. These results thus define a cyclin A-cdk2 kinase complex that possesses
sequence-specific
DNA binding activity. This suggests that the cdk2 kinase may phosphorylate
other
DNA-bound
substrates,
and that one role of the E2F
factor may be to localize this protein kinase to the DNA.
Introduction
Through a combined biochemical and genetic approach,
much has been learned about the proteins whose expression and activity regulate progression through the cell cycle (for reviews, see Murray and Kirschner, 1989; Nurse,
1990; Draetta, 1990; Pines and Hunter, 199Oa). The primary participants in this regulatory circuit are the cdc2
class of protein kinases and the cyclin proteins that regulate the activity of the kinases. The S. cerevisiae CDC28
gene and the S. pombe c&2 gene encode 34 kd protein
kinases that are highly conserved in sequence and functionally interchangeable
(Hartwell et al., 1974; Beach et
al., 1982; Reed et al., 1985; Nurse and Bisset, 1981). Moreover, it is now clear that structurally and functionally related proteins are produced in many other species, including humans (Lee and Nurse, 1987; Dunphy et al., 1988;
Arion et al., 1988; Labbe et al., 1989).
Genetic analyses have demonstrated that cdc2ICDC28
gene function is required for entry into S phase as well as
for entry into mitosis in yeast (Nurse and Bisset, 1981;
Piggot et al., 1982). However, although the kinase activity
is modulated through the cell cycle, the actual level of the
c&2 gene product does not vary (Durkacz et al., 1986;
Simanis and Nurse, 1988; Draetta and Beach, 1988). The
cyclins were originally discovered as proteins that accumulated to high levels prior to mitosis and then rapidly
disappeared
at the end of mitosis (Evans et al., 1983;
Swenson et al., 1986). The accumulation of the cyclins
and the subsequent complex formation with cdc2 lead to
activation of the kinase activity (Draetta and Beach, 1988;
Meijer et al., 1989; Gautier et al., 1990; Solomon et al.,
1990).
In addition to the mitotic cyclins, there is recent evidence
in yeast for a class of cyclin-like proteins that are required
during Gl (Cross, 1988; Nash et al., 1988; Hadwiger et al.,
1989; Richardson et al., 1989; Forsburg and Nurse, 1991).
Moreover, several mammalian genes, as well as a Drosophilagene, have been isolated that encode proteinsthat
bear homology to the cyclins and that can provide the Gl
functioninyeast(Matsushimeetal.,
1991; Lewetal., 1991;
Koff et al., 1991; Leopold and O’Farrell, 1991). By analogy
with the activation of cdc2 at mitosis by the mitotic cyclins,
it is presumed that the Gl cyclins participate in the activation of c&2 or a cdcPlike kinase to promote entry into S
phase. In addition to the multiplicity of cyclin proteins, it is
now clear that in vertebrate cells there are several different
cyclin-dependent
protein kinases that bear a strong homology to cdc2 (Pines and Hunter, 1990b; Elledge and
Spottswood, 1991; Paris et al., 1991; Tsai et al., 1991;
Meyerson et al., 1991).
There are some indications that cyclin A, originally identified as a mitotic cyclin, may function in S phase. In mammalian cells, the cyclin A protein and its associated kinase
activity appear during S phase, do not show as dramatic
a peak in mitosis as cyclin B, and disappear prior to the
disappearance of cyclin B (Pines and Hunter, 1990b). Furthermore, cyclin A has been implicated in the phosphorylation of RP-A, a cellular protein complex required for the in
vitro replication of SV40 DNA (A. Dutta and 6. Stillman,
unpublished data). In addition, cyclin A is one component
of an activity purified from asynchronous cells that, when
added to a Gl extract, makes it competent for T antigendependent SV40 DNA replication (D’Urso et al., 1990).
An alternative approach to the analysis of cell growth
regulation has been the study of the products of mammalian cell and viral oncogenes. In particular, two cellular
genes, the retinoblastoma gene (Rf37) and the P53 gene,
appear to play major roles in regulating the progression
of cells through Gl (see Marshall, 1991). In addition, the
analysis of proteins such as adenovirus ElA has provided
insights into the mechanism by which proteins such as
the retinoblastoma gene product function to control cell
proliferation. The RBI gene product (pRb) and the f53
gene product (~53) can be inactivated by mutation, but
also by the interaction with viral gene products (e.g., see
Scheffneret al., 1991). For instance, the ability of adenovirus El A, SV40 T antigen, and HPV E7 to function as oncogenes is dependent on sequences that are also essential
for these proteins to interact with pRb (Kalderon and
Smith, 1984; Moran et al., 1986; Lillie et al., 1987; Schneider et al., 1987; Cherington et al., 1988; DeCaprio et al.,
1988; Moran, 1988; Smith and Ziff, 1988; Whyte et al.,
1989; Munger et al., 1989).
Cdl
166
A.
9
E2F
/
$
4” $
f?
rr,f‘a..’
p
f?
$
f?
fc
*
p
OCT
N
f2
(6
.
$
f?
fc
.
I
(0
(0
‘-
a
Figure 1. Copurification
of a cd&
the EPF-Cyclin
A Complex
Kinase with
(A) E2F binding assays.
Extracts
were prepared from growing mouse L cells as previously described
(Sagchi et al., 1990; Mudryj
et al., 1991). E2F binding activity was purified
through a heparin-agarose
column and then
one E2F DNA affinity column. The active fractions from the affinity eluate were divided in
half and chromatographed
on parallel affinity
columns, one specific for E2F and the other for
the octamer element. Aliquots of the fractions
were assayed for E2F binding activity by gel
retardation.
FT, flow-through;
Wl, W2, wash
fractions;
E, El, E2, E3, eluate fractions.
(6) Western blot assays. Aliquots of the various
column fractions were analyzed by SDS-gel
electrophoresis
as described
in Experimental
Procedures.
The separated
proteins
were
transferred
to nitrocellulose,
and portions of the
filter corresponding
to the appropriate
molecular weight were excised and then probed separately with the cyclin A antiserum
or the
PSTAIRE antibody.
B.
[a Cyclin A]
Recent studies directed at the E2F transcription factor
have provided a direct link between the action of viral oncogenes and cell cycle control. E2F has been shown to interact with the cyclin A protein during the S phase of the cell
cycle. This complex is targeted by the adenovirus EiA
protein, resulting in the dissociation of the complex and
the release of free E2F; the uncomplexed
E2F appears
to be the transcriptionally
active molecule (Mudryj et al.,
1991). E2F is also found in a complex with the retinoblastoma gene product (pRb) (Chellappan et al., 1991; Mudryj
et al., 1991; Bandara and LaThangue, 1991; Chittenden
et al., 1991; Bandara et al., 1991) and this complex is also
dissociated by El A, again releasing free E2F. Both the
cyclin A protein and the Rb protein have been shown to be
in stable complexes with El A (Whyte et al., 1988, 1989;
Pines and Hunter, 1990b); as such, it is possible that this
stable interaction with ElA is a result of the dissociation
of the E2F complexes. Two other El A-associated proteins, the Rb-related ~107 protein and the ~33~” kinase,
have also been shown to interact with cyclin A (Pines and
Hunter, 1990b; Tsai et al., 1991; Faha et al, 1991; Ewen
et al., 1991a). Although it is possible that these proteins
exist in a variety of distinct but overlapping interactions, it
seemed equally possible that these observations might
in fact reflect different views of one complex containing
p33*@ kinase, the ~107 protein, and cyclin A, all associated with E2F. We now report that both the ~107 protein
and the ~33~~ kinase are components of the E2F-cyclin
A complex. Moreover, this complex possesses kinase
activity, thus defining a cyclin A-~33~~~ kinase with
sequence-specific
DNA binding activity.
Results
The S Phase-Regulated
ESF-Cyclin A Complex
Also Contains a cdc2-Related
Protein
Our previous experiments have demonstrated an interaction between the E2F transcription factor and the cyclin A
protein generating a complex that accumulates during the
S phase of the NIH 3T3 cell cycle and disappears sometime in G2 or M (Mudryj et al., 1991). These experiments
did not, however, exclude the possible involvement of
other proteins in this complex. Indeed, several observations have prompted us to examine the nature of this complex further. First, it was apparent that the E2F-cyclin A
complex was likely composed of additional proteins,
E2F-Protein
169
Kinase
Complex
based on the size of the complex. The EPF-cyclin A complex appeared larger than the E2F-Rb complex, both by
gel mobility shift assays and by glycerol gradient sedimentation, despite the fact that the Rb protein is nearly twice
as large as cyclin A. Second, other experiments
have
shown that cyclin A forms protein complexes with at least
two distinct kinases, ~34~~ and ~33”~~(Pines and Hunter,
1990b; Tsai et al., 1991; Giordano et al., 1991a; Faha et
al, 1991). Recent experiments have shown that the ~33”~~
kinase is also associated with the ElA protein, as is cyclin
A(Giordano et al., 1989,199la;
Pines and Hunter, 1990b;
Tsai et al., 1991). Third, another El A-associated protein,
~107, which is related in sequence to pRb (Ewen et al.,
1991 b), has now been shown to interact with cyclin A (Faha
et al, 1991; Ewen et al., 1991a). Since we have previously
argued that the interaction of cellular proteins with ElA
may be a consequence of dissociation of complexes such
as those involving E2F (Bagchi et al., 1990; Chellappan et
al., 1991; Mudryj et al., 1991) we have now investigated
the possibility that a cdc2-like kinase, as well as the Rb-related ~107 protein, may be a component of the E2F-cyclin
A complex.
Our approach has been to purify the EPF-cyclin A complex and then probe for the presence of the kinase as well
as the ~107 protein using specific antibodies. Initially, we
have taken advantage of the fact that extractsof asynchronously growing L cells contain predominantly
the E2Fcyclin A complex (Mudryj et al., 1991) perhaps because
the proportion of ceils in S phase is high. Using such extracts, we have biochemically purified the EPF-cyclin A
complex, using conventional chromatography
followed by
one pass over an EPF-specific DNA affinity column. Half of
this material was then reapplied to the E2F affinity column,
and half was applied to an unrelated DNA affinity column,
a column containing the octamer element. The column
eluates were assayed for E2F and then analyzed for cyclin
A and cdc2 by Western blotting. We were not able to probe
for the ~107 protein in these samples, since the available
antibody (XZ37; Hu et al., 1991) does not recognize the
mouse protein.
As seen in Figure 1 A, E2F activity was bound by the E2F
affinity column but not by the octamer affinity column. As
observed in our previous experiments, the cyclin A protein
could be detected in the E2F affinity column eluate but not
in the eluate of the control column (Figure 1 B). The same
fractions were then probed with a PSTAIRE monoclonal
antibody (MAb) in order to detect cdc2-related proteins
(Lee and Nurse, 1987; Pines and Hunter, 1991a). As
shown in Figure 1 B, the same fractions that contained the
cyclin A protein as well as the E2F binding activity also
contained a 33 kd PSTAIRE-reactive
polypeptide. In contrast, the PSTAIRE specific polypeptide was not bound by
the octamer column but rather was found in the flowthrough fraction of the column. We thus conclude that in
addition to the cyclin A polypeptide, a PSTAIRE-reactive
polypeptide of 33 kd and, thus, a likely member of the
family of cdcBrelated
kinases (Pines and Hunter, 1991 b;
Meyerson et al., 1991) is in association with the E2F transcription factor.
To define further the interaction of a cdc2 kinase as a
component of the E2F complex, we have isolated the
E2F-cyclin A complex from NIH3T3 cells that have been
synchronized by serum starvation and stimulation. Our
previous experiments have shown that 18 hr after serum
addition, the cells are in S phase and contain the E2Fcyclin A complex (Mudryj et al., 1991). Extracts were prepared from these cells and fractionated by heparin agarose chromatography
followed by one pass over an E2F
DNA affinity column. As before, the affinity column eluate
was then divided in half and applied in parallel to an E2F
column and an octamer column. Fractions were assayed
with the cyclin A antiserum and the PSTAIRE antibody. As
shown in Figure 2A, a 33 kd, PSTAIRE-reactive
polypeptide again cofractionates with E2F binding activity and
cyclin A.
The S Phase E2F-Cyclin
A Complex Possesses Hl
Kinase Activity
The assays presented thus far demonstrate that a cdcB
like protein copurifies with the E2F-cyclin A complex.
Clearly, however, the critical issue concerns whether this
complex also contains protein kinase activity. To address
this issue, we have assayed the fractions from the isolation
of the S phase NIH3T3 E2F-cyclin A complex for histone
Hl kinase activity. As shown in Figure 2C, Hl kinase
assays revealed that the same fractions that contained
the E2F-cyclin A complex and the PSTAIRE-reactive p33
polypeptide also contained Hl kinase activity. The Hl kinase activity was retained by the E2F DNA affinity column
but not by the octamer-specific affinity column. If the E2Fcyclin A complex was dissociated with deoxycholate before being applied to the E2F DNA affinity column, then
the free E2F bound to the column, but cyclin A and the Hl
kinase did not (data not shown). We therefore conclude
that in addition to the cyclin A protein, a cdcPrelated
kinase is a component of an E2F complex that we have
previously shown to accumulate during the S phase of the
cell cycle (Mudryj et al., 1991). Moreover, these results
demonstrate that the EPF-cyclin A-kinase complex possesses both DNA binding activity and kinase activity.
The ESF-Cyclin A Complex Also Contains the
Rb-Related ~107 Protein
The use of the two mouse cell extracts did not allow an
assay for the ~107 protein, since the MAb XZ37 did not
efficiently recognize the mouse protein. To address the
question of the ~107 protein and to provide further evidence for the association of a cdcPlike kinase with E2F,
we have analyzed the E2F-cyclin A complex in extracts of
human U937 cells. The EPF-cyclin A complex detected in
the human monocytic cell line U937 has identical characteristics to those in NIH3T3 cell S phase extracts and L
cells, and since the complex derives from a human cell
line, we were able to use the XZ37 antibody to analyze
~107. Since the XZ37 antibody detects both ~107 and pRb
(Hu et al., 1991) and since U937 cells contain both the
E2F-Rb complex and the E2F-cyclin A complex (Chellappan et al., 1991) we have used an additional purification
step, glycerol gradient sedimentation,
to separate the
E2F-cyclin A complex from the EPF-Rb complex. The
Cell
170
A.
Glycerol
4
Gradient
5
6
7
Fraction
6
9
10
E2F--
B.
2345676910
P107,
p105/
p60 -
[a Cyclln
A]
[a PSTARE]
p33-
C.
I-
E2F
OCT
Figure 3. The ~107 Protein
EPF-Cyclin
A Complex
Figure 2. The S Phase
Kinase Activity
CI
E2F-Cyclin
A Complex
Contains
cdc2 and Hl
A whole cell extract was prepared from cultures of NIH 3T3 cells that
had been serum starved and then stimulated
by serum addition for
18 hr. The EPF-cyclin
A complex was purified by heparin agarose
chromatography
and thenapplied
toan E2FaffinitycolumnTheeluate
was then reapplied to an E2F column or an octamer-specific
column.
(A) E2F binding assays. Column fractions were assayed for E2F binding activity by gel retardation.
In this particular assay, the uncomplexed
probe has run off the bottom of the gel.
(B) Western blot assays. Aliquots of the fractions
were analyzed by
SDS-gel electrophoresis
as described
in Experimental
Procedures
and as described
in Figure 1.
(C) Histone Hl kinase assays. Aliquots of the fractions were assayed
for histone Hi kinase activity as described
in Experimental
Procedures. The position of histone Hl , as identified by Coomassie
blue
staining of the gel, is indicated.
gradient sedimentation also provides an analytical step,
since we could determine if the various candidate proteins
did indeed cosediment with the appropriate E2F complex.
E2F activity was purified by heparin agarose chromatography, concentrated by ammonium sulfate precipitation,
and fractionated by glycerol gradient sedimentation. Gra-
and the cdc2 Kinase Cosediment
with the
A U937 cell whole cell extract was prepared, fractionated
by heparin
agarose chromatography,
and then fractionated
by glycerol gradient
sedimentation
as described
in Experimental
Procedures.
(A) E2F assays. Fractions of the gradient were assayed for E2F by gel
retardation. The positions of the EPF-cyclin
A complex and the E2FRb complex are indicated. The top of the gradient is at right. Fractions
l-3 of the gradient were not assayed for E2F activity.
(8) Western blot assays. Fractions of the gradient were analyzed by
SDS-gel electrophoresis
and proteins were then transferred
to nitrocellulose. Appropriate
regions of the blot were excised and probed
with the indicated antisera.
dient fractions were then analyzed for E2F by gel retardation assays. As shown in Figure 3A, the faster-sedimenting
EPF-cyclin A complex clearly separated from the EPF-Rb
complex. Aliquots of the fractions were analyzed in SDSpolyacrylamide gels by Western blotting to detect the presence of ~107, pflb, cyclin A, and cdc2-related proteins. As
can be seen in Figure 38, the Western analysis demonstrated that the ~107 protein and a 33 kd PSTAIRE reactive
protein cosedimented with the EPF-cyclin A complex. The
cyclin A protein was distributed throughout the gradient,
although there was clearly a peak at the position of the
EPF-cyclin A complex. In contrast, the Rb protein, which
is also detected with the X237 antibody, cosedimented
with the EPF-Rb complex rather than with the EPF-cyclin
A complex, indicating that the Rb protein and the Rbrelated ~107 protein are components of distinct E2Fcontaining complexes. The fact that the cyclin A protein
was detected in fractions of the gradient not containing the
EpF-Protein
171
Kinase
Complex
A.
protein, cyclin A, and a 33 kd, PSTAIRE-reactive
protein
were all detected in the eluate of the E2F-specific column
but not in the eluate of the octamer-specific
column. Finally, histone Hl kinase assays of the affinity column fractions demonstrated the presence of kinase activity coincident with E2F, cyclin A, and cdc2 (Figure 4C). These
results suggest that the previously identified E2F-cyclin A
complex also contains the ~107 protein and a cdcPlike
kinase. We sought next to identify the kinase and to investigate further its interaction with E2F.
pm-r,
--
p33-
-
-
.-
-
--
[a PSTAIRE]
C.
*
Hl+
Figure 4. The ~107 Protein and a cdc2 Kinase
Cyclin A Complex from Human U937 Cells
Copurify
with the E2F-
(A) E2F binding assays. The EPF-cyclin
A complex was isolated from
a U937 extract by glycerol gradient sedimentation
as described
in
Experimental
Procedures
and as shown in Figure 3. Pooled gradient
fractions were applied to an E2F DNA affinity column or an octamer
DNA affinity column. Aliquots of fractions were assayed for E2F DNA
binding activity as described
in Experimental
Procedures.
(6) Western blot assays. Aliquots of the column fractionswere
assayed
by Western blotting as described in Figure 2 and Experimental
Procedures.
(C) Histone Hl kinase assays. Assays were as described
in Figure 2
and Experimental
Procedures.
E2F-cyclin A complex would suggest that cyclin A is
a component of several protein complexes in addition
to E2F.
Further evidence in support of the association of ~107
and a cdcPlike kinase in the EPF-cyclin A complex was
provided by DNA affinity chromatography
of the U937 cell
E2F-cyclin A complex. Glycerol gradient fractions containing the ESF-cyclin A complex were pooled and applied
in parallel to an E2F-specific column as well as the
octamer-specific column. As shown in Figure 4B, the ~107
The EPF-Associated
cdc2 Kinase Is ~33~~~
Previous experiments have shown that cyclin A is associated with a 33 kd, cdcPrelated kinase as well as the p34
molecule that is a component of the MPF complex (Giordano et al., 1989, 1991a; Pines and Hunter, 1990b; Faha
et al., 1991). The p33 kinase, which has now been termed
cdk2, has also been shown to be a protein that is found in
association with the ElA protein (Tsai et al., 1991). In the
Western blot analyses shown above (Figure lB), the
PSTAIRE antibody recognized a doublet at 33-34 kd in
relatively crude extract, whereas only the 33 kd band was
detected in the affinity-purified EPF-cyclin A complex. Although this could be due to electrophoretic
variation in
the ~34~~~~molecule, an additional possibility was that the
kinase in association with E2F and cyclin A would be the
p33Cd@ kinase. We have addressed this issue using a
cdk2specific
antiserum (Tsai et al., 1991).
Our assay took advantage of the availability of an antiserum that was specific for ~33~~~~ (Tsai et al., 1991) and that
was able to detect the kinase directly as a component of
the E2F-cyclin A-DNA complex. As shown in Figure 5A,
addition of the cyclin A antiserum to the U937 extract eliminated the E2F-cyclin A complex but had no effect on the
E2F-Rb complex. There was some reduction in intensity
of the Rb complex, but this appeared to be a nonspecific
effect, as it was also observed with the normal mouse
serum (NMS). In contrast, the addition of the Rb antibody
eliminated the E2F-Rb complex but not the E2F-cyclin A
complex. Similar to the effect of the cyclin A antiserum,
addition of the cdk2 antiserum to the U937 extract also
selectively eliminated the E2F-cyclin Acomplex. This was
accompanied
by the appearance of a slower-migrating
band that we presume results from binding of the antibody
to the complex. We also isolated the two E2F containing
complexes by glycerol gradient sedimentation
and assayed each independently
with the various antisera. As
shown in Figure 5B, the cyclin A antiserum and the ~33”~~~
antiserum abolished the E2F-cyclin
A complex (left
panel). In contrast, the Rb antibody did not alter the isolated EPF-cyclin A complex but eliminated the EPF-Rb
complex (right panel). The cdk2 antiserum had no effect
on the E2F-Rb complex, indicating that this complex does
not contain cdk2.
Finally, the specificity of the cdk2 antiserum, with respect to recognition of cdk2 in the EPF-cyclin A complex,
was demonstrated by a blocking assay. Whereas addition
of the cdk2 antiserum alone abolished the E2F-cyclin A
complex, there was no effect if the antiserum was preincubated with the GST-cdk2 fusion protein (Figure 5C). In
Cell
172
A
E*&sn AE2FRb-
Figure
5. The EPF-Associated
cdc2 Kinase
is ~33~~
(A) An extract of human U937 cells was assayed for E2F binding activity directly or following incubation with the indicated antisera. The positions
of the EPF-cyclin
A complex and the EPF-Rb complex are indicated. The arrow indicates the position of the EPF-cyclin
A complex after being
supershifted
by the cdk2 antiserum.
In each case, 2 ug of extract were assayed together with the indicated antiserum.
The addition of antibodies
in this experiment
caused a general reduction in the intensity of the E2F complexes,
but the cyclin A and cdk2 antibodies had a specific effect on
the EPF-cyclin
A complex, while having no specific effect on the EPF-Rb complex (compared
to NM).
(6) The EPF-cyclin
A complex and the EPF-Rb complex were isolated from a U937 extract by glycerol gradient sedimentation.
Each complex was
then assayed independently
as described
in (A). The E2F-Rb fraction also contained free E2F as indicated.
(C) Specificity
of the cdk2 antiserum.
The isolated EPF-cyclin
A complex was assayed with 1 pl of the cdk2 antiserum
alone or following a
preincubation
with 1.2 ug of GSTcdkP
or 1.2 ug of GST-cdc2
fusion protein.
contrast, the antiserum continued to recognize the E2Fcyclin A complex after pre-incubation
with a GST-cdc2
fusion protein. Based on these results, we conclude that
the ~33~~ polypeptide is a component of the EPF-cyclin
A complex. Moreover, these analyses demonstrate that
the interaction is stoichiometric, since the antiserum eliminated most of the complex, indicating that the ~33~~ molecule is a component of most if not all of the ESF-cyclin A
complexes.
Discussion
The data presented here demonstrate that the previously
described EPF-cyclin A complex that forms during S
phase following the release of E2F from Gl-specific interactions also contains the Rb-related protein ~107 as well
as the ~33~~ kinase, a member of the family of cdc2related protein kinases(Figure SA). Adistinct E2Fcomplex
contains the Rb protein. Each of the proteins in association
with E2F in these complexes are also proteins previously
found to interact with the ElA protein. We believe it likely
that the interaction of this group of proteins with ElA is,
in part, a consequence of the activity of El A to dissociate
E2F from the complexes. However, we do not also believe
that dissociation of the E2F complexes is necessarily the
sole purpose of ElA in this regard. it is certainly possible
that the cellular proteins that associate with E2F play important roles in the regulation of other cellular pathways.
For instance, the Rb protein has been shown to interact
with several cellular proteins (Kaeiin et al., 1991; Huang
et al., 1991; DefeoJones et al., 1991) and may well have
other roles in addition to the interaction with E2F. Two
additional cellular proteins, ~130 and ~300, have been
shown to be in complexes with ElA (Yee and Branton,
1985; Harlow et al., 1986). The interaction of ~130 with
ElA is dependent on sequences that overlap with those
required for the E2F-associated proteins (Giordano et al.,
1991 b), whereas theinteractionof
p3OOwith ElAisdependent on distinct, N-terminal sequences (Egan et al., 1988;
Whyte et al., 1989; Stein et al., 1990). Certainly, it is possible that the ElA association with ~130 and with ~300 may
have quite different functions. Nevertheless, the observation that so many of the El A-associated proteins are also
associated with E2F raises the possibility that these additional proteins, particularly ~130, may also interact with
E2F or with other transcription factors. Unfortunately, reagents for these two proteins are not yet available.
Although the identification of additional components of
the EPF-cyclin A complex is an important step forward
in the understanding of the control of the factor and the
mechanism of ElA action, it is the identification of the cdk2
kinase as one of these components that we believe to be
the critical finding. The original observation that the cyclin
A protein was a component of an E2F complex that accumulated during S phase of the NIH3T3 cell cycle (Mudryj
et al., 1991) raised two possible views of the functional
relevance of this interaction. On the one hand, the interaction of cyclin A with a transcription factor could represent
EPF-Protein
173
Kinase
Complex
A
B
a novel role for a cyclin. The function of cyclins, particularly
the mitotic cyclin B, in regulating the activity of the cdc2
protein kinase is now well established. The activation of
the cdc2 kinase at the beginning of mitosis is dependent
on the interaction of cyclin B with the kinase (Solomon et
al., 1990). Moreover, the inactivation of kinase activity at
the end of mitosis, which is essential for cells to exit mitosis, is dependent on the destruction of the cyclin molecule
(Murray et al., 1989). Cyclin A is also able to activate cdc2
and cause chromosome condensation in vitro and to initiate meiosis I and II in Xenopus oocytes (Roy et al., 1991).
Thus, in light of this body of information, a role for cyclin
A in regulating the activity of a transcription factor was
surprising, suggesting an apparently new and novel role
for a cyclin protein.
The finding that the E2F-cyclin A complex also contains
the ~33~~~ kinase, as described in the present experiments, supports a second and distinct possibility. Rather
Figure 6. Formation of an E2F Complex
taining the cdk2 Kinase
Con-
(A) The E2F-cyclin
A complex is formed during
S phase (Mudryj et al., 1991). The data presented here now demonstrate
that the E2Fcyclin Acomplexalsocontainsthe
pl07protein
and the cdk2 kinase. The interactions
of the
proteins in the complex are presented only for
illustrative purpose; we do not mean to imply a
specific set of contacts.
(B) Potential functional implications
of the association of a cyclin A-cdk2
kinase complex
with the sequence-specific
DNA-binding
protein E2F. The top figure represents
a multicomponent complex interacting with a specific DNA
element adjacent to an EPF-binding site. Upon
interaction of the EPF-cyclin
A-cdk2 complex
with the E2F element, the cdk2 kinase is able
to phosphorylate
the adjacent factor.
than the cyclin A protein playing an unexpected role in
regulating a transcription factor, it would now appear that
the E2F-associated cyclin A might function in a traditional
role of regulating a cdcBlike kinase. That is, although this
complex contains the E2F transcription factor, these results demonstrate that the EPF-cyclin A complex also contains an active cdcPlike kinase. We could envision two
possible roles for the protein kinase in this context. First,
it is conceivable that E2F and/or ~107 is a substrate for
the cdk2 kinase and that this complex is a phosphorylation
intermediate.
~107 is phosphorylated
in vivo and is an
excellent substrate in vitro for a cell cycle-regulated
serine/threonine
kinase activity that is associated with ElA
(Herrmann et al., 1991). It is tempting to speculate that
these activities are due to p3Pk2, but at present the true
identities of the p107- and El A-associated kinases are
unresolved. Nevertheless, it is clear that both E2F and
~107 are potential substrates for ~33~“~.
Cell
174
Although we cannot determine the proportion of ~107
that is associated with E2F, it is clear that nearly all of the
E2F found during the S phase of the cell cycle is in this
complex (Mudryj et al., 1991; M. M., S. H. D., and J. R. N.,
unpublished data). Thus, if the presence of E2F in this
complex were solely for the purpose of phosphorylation of
E2F, it would be highly unusual for an intermediate to be
so stable and in apparent vast excess over the substrate
and the product. Similarly, the relative stability and stoichiometryof the E2F-cyclin A-p1 07-cdk2 complex is difficult
to explain if the complex is merely an intermediate of ~107
phosphorylation.
Of course, these considerations
do not
rule out the possibility that E2F and/or ~107 are substrates
for p33cdk2, but it appears unlikely that a simple enzyme/
substrate interaction will account for the appearance of
this complex.
An alternative possibility is that E2F is functioning as a
co-factor for the cyclin A-cdk2 kinase. It is intriguing to
consider the possibility that E2F might target this cdcBlike
kinase to a specific substrate. The EPF-p107-cyclin
Acdk2 complex does bind to DNA with sequence specificity
(the E2F recognition site), and the complex Clearly possesses kinase activity. As suggested by the schematic
diagram shown in Figure 66, perhaps the E2F-p107cyclin A-cdk2 complex is responsible for the phosphorylation of an adjacent DNA bound factor and, as a
consequence of this action, activates or represses the
transcription potential of the factor. That is, the adjacent
factor may be poised for action and become activated or
repressed by cdkBmediated
phosphorylation.
In fact,
RNA polymerase II could be considered a target in this
context, since the C-terminal domain of the largest subunit
has been shown to be a substrate for a cdc2 kinase in in
vitro assays (Cisek and Corden, 1989).
Given the fact that E2F is a DNA-binding protein, and
that the cyclin A complex appears during S phase, it is also
possible that the EPF-p107-cyclin
A-cdk2 complex could
participate in DNA replication events. Examples of transcription factors that can also play a role in DNA replication
are now numerous (see DePamphilis, 1988). Thus, it is not
unreasonable to suggest that the E2F transcription factor
might perform a role in a DNA replication event by localizing an active cyclin A-cdk2 kinase to the DNA. A role for
cyclin A in S phase has been suggested by the kinetics of
synthesis of cyclin A, which precede that of cyclin 6 (Pines
and Hunter, 1990a, 1990b). Depletion of cdk2 from Xenopus egg extracts has been shown to inhibit DNA replication
in vitro(Fang and Newport, 1991). Otherexperiments
have
shown that depletion of cyclin A abolishes the controls that
ensure complete DNA replication prior to mitosis in vitro
(Walker and Mailer, 1991). It is known that cyclin A can
interact with cdk2 as well as cdc2, and it is thus possible
that distinct cyclin A complexes perform different functions
during S phase. If the cyclin A-~33*“~ complex that is
associated with E2F does indeed participate in an S phase
event, it is unlikely that it would be an essential role in DNA
replication, since the complex is largely dissociated in cells
that express the ElA protein (Mudryj et al., 1991). That is,
a cell can clearly replicate its DNA and proceed through
an S phase without any detectable E2F-cyclin A-cdk2
complex. Thus, a regulatory function might be the more
likely role for the complex if indeed it was participating in
DNA replication. Of course, this is only speculation, and
given the present lack of understanding
of the nature of
cellular DNA replication, we do not know if E2F sites are
critical elements involved in DNA replication. Nevertheless, given the S phase accumulation of the E2F-p107cyclin A-cdk2 complex, we believe that this must be considered as a possibility.
Experimental Procedures
Cells, Extracts, and Antibodles
The growth and maintenance of NIH 3T3 ceils, L ceils, and the U937
cell line have been previously
described,
as have the procedures
for
the preparation of whole cell extracts (Mudryj et al., 1991; Cheiiappan
et al., 1991).
Several of the antibodies
have been described
previously.
The
mouse poiycional antiserum raised against GST-cdkP was generously
donated by Li-Huei Tsai and Ed Harlow (Tsai et al., 1991), as were the
GST-cdk2
and GST-cdc2
fusion proteins; the cyciin A antiserum
has
been described
(Pines and Hunter, 199la). The PSTAIRE antibody
was a gifi of Dr. M. Yamashitaand
recognizes
cdc2, cdk2, and presumably other PSTAIRE-containing
proteins (Pines and Hunter, IQQia).
E2F Assays
The procedures
and probes used for the assay of E2F have
described
(Bagchi et al., 1989, 1990; Mudryj et al., 1991).
been
Glycerol
Gradient
Sxllmentation
E2F activity isolated by heparin agarose chromatography
was further
purified and concentrated
by ammonium
sulfate precipitation
(40%
saturation).
The precipitated
material was dissolved in 0.2 ml and applied to a 4 ml, 5%-20%
glycerol gradient. Sedimentation
was at
45,000 rpm in a SW65 rotor at 4OC for 18 hr. Fractions were collected
and aiiquots were assayed for E2F binding activity.
Afflnlty
Column Isolation
of E2F
Whole cell extracts were fractionated
by heparin agarosechromatography as described (Yee et al., 1989). The E2F-containing
fractions were
pooled, dialyzed, and applied to either an E2F DNA affinity column or
an octamer element affinity column as described previously
(Chellappan et al., 1991).
Western
Blot Asseys
Aiiquots of gradient fractions or affinity column fractions were precipitated with 20% trichioroacetic
acid and dissolved
in sodium dodecyi
sulfate (SDS) sample buffer. The L cell and 3T3 cell S phase material
was analyzed in 10% acryiamide-SDS
gels, while material from the
U937 ceil extracts was analyzed in a 6%-14%
gradient gel in order lo
resolve ~107, pflb. cyciin A, and the PSTAIRE antigens on the same
gel. After transfer to nitrocelluiose,
the blots were probed with the
primary antibodies,
followed by a biotin-conjugated
secondary
antibody (Sigma) and then avidinconjugated
alkaline
phosphatase
(Sigma) or avidin-conjugated
horseradish
peroxidase
(ABC Kit, Vector
Laboratories).
The procedures
for SDS gel electrophoresis.
transfer to
nitroceiiuiose,
and assay with specific antisera have been described
(Chellappan
et al., 1991).
Hlstone Hl Kinaae Aseeys
Aliquots of column fractions were assayed for histone Hi kinase activity according
to the procedure
described
by Sooher et al. (1989). The
reaction products
were analyzed by SDS-PAGE
and visuaiized
by
autoradiography.
Acknowledgments
We would like to thank Li-Huei Tsai for generously
providing specific
antiserum to cdk2 as well as GST fusion proteins. We are also grateful
for numerous
helpful discussions
with Ed Harlow, Nick Dyson, and
Liang Cao. We also thank Laszlo Jakoi for expert technical assistance,
EPF-Protein
175
Kinase
Complex
Scott Kyles for help in growing 3T3 cells, and Kaye Culler for help in
the preparation
of the manuscript.
S. H. D. was supported by an NIH
postdoctoral
fellowship
and M. M. was supported
by the Howard
Hughes Medical Institute. This work was supported
by the Howard
Hughes Medical Institute (J. Ft. N.).
The costs of publication
of this article were defrayed
in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement”
in accordance
with 18 USC Section 1734
solely to indicate this fact.
Dunphy, W. G., Brizuela, L., Beach,
Xenopus cdc2 protein is a component
of mitosis. Cell 54, 423-431.
Received
Egan, C., Jelsma, T. N., Howe, J. A., Bayley, S. T., Ferguson, B., and
Branton, P. E. (1988). Mapping of cellular protein binding sites on the
products
of early region 1A of human adenovirus
type 5. Mol. Cell.
Biol. 8, 3955-3959.
November
18, 1991; revised
December
20, 1991
References
Arion, D., Meijer, L., Brizuela, L., and Beach, D. (1988). cdcl is a
component
of the M phase-specific
histone Hl kinase: evidence for
identity with MPF. Cell 55, 371-378.
Bagchi, S., Raychaudhuri,
P., and Nevins, J. R. (1989). Phosphorylation-dependent
activation of the adenovirus-inducible
E2F transcription factor in a cell-free system. Proc. Natl. Acad. Sci USA 86,
4352-4356.
Bagchi, S., Raychaudhuri.
P.‘, and Nevins, J. R. (1990). Adenovirus
ElA proteins can dissociate heteromeric
complexes
involving the E2F
transcription
factor: a novel mechanism
for ElA trans-activation.
Cell
62, 659689.
D., and Newport, J. (1988). The
of MPF, a cytoplasmic
regulator
Durkacz, B., Carr, A., and Nurse, P. (1986). Transcription
cycle control gene of the fission yeast Schimsaccharomyces
EMBO J. 5, 369-373.
D’llrso,
(1990).
human
of cdc2 cell
pombe.
G., Marraccino,
R. L., Marshak,
D. R., and Roberts, J. M.
Cell cycle control of DNA replication
by a homologue
from
cells of the ~34~‘~ protein kinase. Science 250, 788-791.
Elledge, S. J., and Spottswood,
M. R. (1991). The new human ~34
protein kinase, CDKP, identified by complementation
of a cdc28 mutation in Saccharomyces
cefevisiae,
is a homolog of Xenopus Egl.
EMBO J. 70, 2653-2659.
Evans, T., Rosenthal,
E. T., Youngblom,
J., Distel, D., and Hunt, T.
(1983). Cyclin: a protein specified by maternal mRNA in sea urchin
eggs that is destroyed
at each cleavage division. Cell 33, 389-398.
Ewen, M., Faha, B., Harlow, E., and Livingston,
D. (1991a). ~107 interacts with cyclin A independent of complex formation with SV40 T antigen and adenovirus
ElA. Science, in press.
Bandara, L. R., and LaThangue, N. B. (1991). Adenovirus
Elaprevents
the retinoblastoma
gene product from complexing
with a cellular transcription factor. Nature 351, 494-497.
Ewen, M.,
Molecular
cDNA for
86, 1155-I
Bandara, L. R., Adamczewski,
J. P., Hunt, T.. and LaThangue,
(1991). Cyclin A and the retinoblastoma
gene product complex
common transcription
factor. Nature 352, 249-251.
Faha, B., Ewen, M., Tsai, L.-H., Livingston, D., and Harlow, E. (1991).
Association
between human cyclin A and adenovirus
El A-associated
~107 protein. Science, in press.
N. B.
with a
Xing, Y., Lawrence, J. B., and Livingston,
D. M. (199lb).
cloning, chromosomal
mapping, and expression
of the
~107, a retinoblastoma
gene product-related
protein. Cell
164.
Beach, D., Durkacz,
B.. and Nurse, P. (1982). Functionally
homologous cell cycle control genes in budding and fission yeast. Nature 300,
706709.
Fang, F., and Newport, J. W. (1991). Evidence that the Gl-S and
G2-M transitions
are controlled
by different cdc2 proteins in higher
eukaryotes.
Cell 66, 731-742.
Booher, R. N., Alfa, C. E., Hyams, J. S., and Beach, D. H. (1989). The
fission yeast cdc2/cdcl3/sucl
protein kinase: regulation of catalytic
activity and nuclear localization.
Cell 58, 485-497.
Forsburg, S. L., and Nurse, P. (1991). Identification
of a Gl-type cyclin
pucl+ in the fission yeast Scbizosaccharomyces
pornbe. Nature 357,
245-248.
Chellappan,
S. P., Hiebert. S., Mudryj, M., Horowitz, J. M.. and Nevins,
J. R. (1991). The E2F transcription
factor is a cellular target for the RB
protein. Cell 65, 1053-1061.
Gautier. J., Minshull, J., Lohka,
J. L. (1990). Cyclin is acomponent
Xenopus. Cell 60, 487-494.
Cherington,
V., Brown, M., Paucha, E., St. Louis, J., Spiegelman,
B. M., and Roberts, T. M. (1988). Separation of simian virus40 Iarge-Tantigen-transforming
and origin-binding
functions
from the ability to
block differentiation.
Mol. Cell. Biol. 8, 1380-1384.
Giordano, A., Whyte, P., Harlow, E., Franza,
Draetta, G. (1989). A 60 kd cdc2-associated
with the ElA proteins in adenovirus-infected
Chittenden,
T., Livingston, D. M., and Kaelin, W. G., Jr. (1991). The TI
El A-binding domain of the retinoblastoma
product can interact selectively with a sequence-specific
DNA-binding
protein. Cell 65, 10731082.
M., Glotzer. M., Hunt, T., and Mailer,
of maturation-promoting
factor from
B. R., Jr., Beach, D., and
polypeptide
complexes
cells. Cell 58, 981-990.
Giordano, A., Lee, J. H., Scheppler,
J. A., Herrmann,
C., Harlow, E.,
Deuschle,
U., Beach, D.. and Franza, 8. R.. Jr. (1991a). Cell cycle
regulation of histone Hi kinase activity associated with the adenoviral
protein EIA. Science 253, 1271-1275.
Cisek, L. J., and Corden, J. L. (1989). Phosphorylation
of RNA polymerase by the murine homologue of the cell-cycle
control protein cdc2.
Nature 339, 679-684.
Giordano, A., McCall, C., Whyte, P., and Franza, B. R. (1991 b). Human
cyclin A and the retinoblastoma
protein interact with similar but distinguishable sequences
in the adenovirus
ElA gene product. Oncogene
6, 481-486.
Cross, F. R. (1988). DAFl, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccbaromyces
cerevisiae. Mol.
Cell. Biol. 8, 4675-4684.
Hadwiger, J. A., Wittenberg,
C., Richardson,
H. E., Lopes, M. D. B.,
and Reed, S. I. (1989). A family of cyclin homologs that control the Gl
phase in yeast. Proc. Natl. Acad. Sci. USA 86, 8255-6259.
DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C.-M.,
Lee, W.-H., Marsilio, E., Paucha, E., and Livingston,
D. M. (1988).
SV40 large tumor antigen forms a specific complex with the product
of the retinoblastoma
susceptibility
gene. Cell 54, 275-283.
Harlow, E., Whyte, P., Franza, R., and Schley, C. (1988). Association
of adenovirus
early region IA proteins with cellular polypeptides.
Mol.
Cell. Biol. 6, 1579-1589.
DefeoJones,
D., Huang, P. S., Jones, R. E., Haskell, K. M., Vuocolo,
G. A., Hanobik, M. G., Huber, H. E., and Oliff. A. (1991). Cloning of
cDNAs for cellular proteins that bind to the retinoblastoma
gene product. Nature 352, 251-254.
DePamphilis,
of eukaryotic
M. L. (1988). Transcriptional
origins of DNA replication.
elements as components
Cell 52. 635-838.
Draetta, G. (1990). Cell cycle control in eukaryotes:
molecular
nisms of cdc2 activation. Trends Biochem. Sci. 75, 376-363.
mecha-
Draetta. G., and Beach, D. (1988). Activation of cdc2 protein kinase
during mitosis in human cells: cell cycle-dependent
phosphorylation
and subunit rearrangement.
Cell 54, 17-26.
Hartwell, L. H., Culotti, J., Pringle, J. R., and Reid, B. J. (1974). Genetic
control of the cell division cycle in yeast. Science 783, 46-51.
Herrmann.
associated
5859.
C., Su, L.-K., and Harlow. E. (1991). Adenovirus
ElA is
with a serinenhreonine
protein kinase. J. Virol. 65, 5848-
Hu, Q., Bautista, C., Edwards, G. M., DefeoJones,
D., Jones, Ft. E.,
and Harlow, E. (1991). Antibodies specific for the human retinoblastoma protein identify a family of related polypeptides.
Mol. Cell. Biol.
7 7 ( 5792-5799.
Huang, S., Lee, W.-H., and Lee, E. Y.-H. P. (1991). A cellular protein
that competes with SV40T antigen for binding the retinoblastoma
gene
product. Nature 350, 160-162.
Cdl
176
Kaelin, W. G., Jr., Pallas. D. C., DeCaprio, J. A., Kaye, F. J., and
Livingston,
D. M. (1991). identification
of cellular proteins that can
interact specifically
with the T/ElA-binding
region of the retinoblastoma gene product. Cell 64, 521-532.
Kalderon, D., and Smith, A. E. (1964). In vitro mutagenesis
of a putative
DNA binding domain of SV40 large-T. Virology 739, 109-137.
Koff, A., Cross, F., Fisher, A., Schumacher,
J., Leguellec, K., Philippe,
M., and Roberts, J. hf. (1991). Human cyclin E, a new cyclin that
interacts with two members of the CDCP gene family. Cell 66, 12171226.
Labbe, J. C., Picard, A., Peaucellier,
G., Cavadore, J. C., Nurse, P.,
and Doree, M. (1969). Purification of MPF from starfish: identification
as the Hl histone kinase ~34~
and a possible mechanism
for its
periodic activation.
Cell 57, 253-263.
Lee, M. G., and Nurse, P. (1987). Complementation
used to clone a
human homologue of the fission yeast cell cycle control gene cdc2.
Nature 327, 31-35.
Leopold, P., and O’Farrell, P. H. (1991). An evolutionarily
conserved
cyclin homolog from Drosophila rescues yeast deficient in Gl cyclins.
Cell 66, 1207-1216.
Lew, D. J., Dulic, V., and Reed, S. I. (1991). Isolation of three novel
human cyclins by rescue of Gl cyclin (Cln) function in yeast. Cell 66,
1197-l 206.
Lillie, J. W., Loewenstein,
P. M., Green, M. Ft., and Green, M. (1967).
Functional domains of adenovirus
type 5 Ela proteins. Cell 50,10911100.
Marshall,
C. J. (1991).
Tumor
suppressor
genes.
Cell 64. 313-326.
Matsushime,
H., Roussel,
M. F., Ashmun, R. A., and Sherr, C. J.
(1991). Colony-stimulating
factor 1 regulates novel cyclins during the
Gl phase of the cell cycle. Cell 65. 701-713.
Meijer, L., Arion, D., Golsteyn, R., Pines, J., Brizuela, L., Hunt, T., and
Beach, D. (1969). Cyclin is a component
of the sea urchin egg M-phase
specific histone Hl kinase. EMBO J. 8, 2275-2262.
Meyerson,
M., Faha, B., Harlow,
dependent kinase family. In The
Quant. Biol. 58) D. Beach, B.
Spring Harbor, New York: Cold
E., and Tsai, L.-H. (1991). The
Cell Cycle (Cold Spring Harbor
Stillman, and J. Watson, eds.
Spring Harbor Laboratory),
in
cyclinSymp.
(Cold
press.
Moran, E. (1966). A region of SV40 large T antigen can substitute for
a transforming
domain of the adenovirus
EIA products.
Nature 334,
166-170.
Moran, E., Zerler, B., Harrison, T. M., and Mathews,
M. B. (1966).
Identification
of separate domains in the adenovirus
El A gene for
immortalization
activity and the activation of virus early genes. Mol.
Cell. Biol. 6, 3470-3480.
Mudryj, M., Devoto, S. H., Hiebert, S. W., Hunter, T., Pines, J., and
Nevins, J. R. (1991). Cell cycle regulation of the E2F transcription
factor involves an interaction with cyclin A. Cell 65, 1243-1253.
Munger, K.,
Howley, P.
E7 proteins
EMBO J. 8,
Werness,
8. A., Dyson, N., Phelps, W. C., Harlow, E., and
M. (1969). Complex formation of human papillomavirus
with the retinoblastoma
tumor suppressor
gene product.
4099-4105.
Murray, A. W., and Kirschner,
M. W. (1969). Dominoes and clocks:
union of two views of the cell cycle. Science 246, 614-621.
the
Murray, A. W., Solomon, M. J., and Kirschner,
M. W. (1969). The
role of cyclin synthesis
and degradation
in the control of maturation
promoting factor activity. Nature 339. 260-266.
Nash, R., Tokiwa, G., Anand, S., Erickson,
K., and Futcher,
A. 8.
(1966). The WHll gene of S. cwevisiae tethers cell division to cell size
and is a cyclin homolog. EMBO J. 7, 4335-4346.
Nurse, P. (1990). Universal
control
M-phase. Nature 344, 503-506.
mechanism
regulating
onset
of
Nurse, P., and B&et, Y. (1961). Gene required in Gl for commitment
to the cell cycle and in 62 for control of mitosis in fission yeast. Nature
292, 556-560.
Paris, J., Le Guellec. R., Couturier, A., Le Guellec, K., Omilli, F., Camonis, J., MacNeil, S., and Philippe, M. (1991). Cloning by differential
screening of a Xerropus cDNA coding for a protein highly homologous
to cd&
Proc. Natl. Acad. Sci. USA 88, 1039-1043.
Piggot, J. R., Rai, R., and Carter, 8. L. A. (1962). A bifunctional gene
product involved in two phases of the yeast cell cycle. Nature 298,
391-393.
Pines, J., and Hunter,
Biologist 2, 369-401.
T. (199Oa).
~34~“~: the S and M kinase?
New
Pines, J., and Hunter, T. (1990b). Human cyclin A is adenovirus
ElAassociated
protein p60 and behaves differently from cyclin B. Nature
346, 760-763.
Pines, J., and Hunter,T.
(1991a). Human cyclinsAand
El aredifferentially located in the cell and undergo cell cyclindependent
nuclear
transport.
J. Cell. Biol. 115, 1-17.
Pines, J., and Hunter, T. (1991 b). Human cell division: the involvement
of cyclins A and Bi and multiple cdc2s. In The Cell Cycle (Cold Spring
Harbor Symp. Quant. Biol. 58) D. Beach, B. Stillman, and J. Watson,
eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory),
in press.
Reed, S. I., Hadwiger, J. A., and Lorincz, A. T. (1965). Protein kinase
activity associated with the product of the yeast cell division cycle gene
CDC26. Prod. Natl. Acad. Sci. USA 82, 4055-4059.
Richardson,
An essential
1133.
H. E., Wittenberg,
C., Cross, F., and Reed, S. I. (1969).
Gl function for cyclin-like proteins in yeast. Cell 59,1127-
Roy, L. M., Swenson, K. I., Walker, D. H., Gabrielli,
Piwnica-Worms,
H., and Mailer, J. L. (1991). Activation
by cyclin A. J. Cell Biol. 113, 507-514.
Scheffner,
The state
carcinoma
B. G., Li, R. S.,
of ~34~ kinase
M., Munger, K., Byrne, J. C., and Howley, P. M. (1991).
of the ~53 and retinoblastoma
genes in human cervical
cell lines. Proc. Natl. Acad. Sci. USA 88, 5523-5527.
Schneider,
J. F., Fisher, F., Goding, C. R., and Jones, N. C. (1967).
Mutational analysis of the adenovirus
Ela gene: the role of transcriptional regulation in transformation,
EMBO J. 6, 2053-2060.
Simanis. V., and Nurse, P. (1966). The cell cycle control gene cd@
of fission yeast encodes a protein kinase potentially regulated by phosphorylation.
Cell 45, 261-266.
Smith, D. H.. and Ziff, E. B. (1966). The amino-terminal
region of the
adenovirus
serotype 5 ElA protein performs two separate functions
when expressed
in primary baby rat kidney cells. Mol. Cell. Biol. 8,
3662-3690.
Solomon, M. J., Glotzer, M., Lee, T. H., Phillippe, M., and Kirschner.
M. W. (1990). Cyclin activation of ~34~~. Cell 63, 1013-1024.
Stein, R. W., Corrigan,
M., Yaciuk, P., Whelan, J., and Moran, E.
(1990). Analysisof
El A-mediated growth-regulating
functions: binding
of the 300-kilodalton
cellular product correlates
with ElA enhancer
repression
function and DNA synthesis-inducing
activity. J. Virol. 84,
4421-4427.
Swenson, K. I., Farrell, K. M., and Ruderman,
J. V. (1966). The clam
embryo protein cyclin A induces entry into M phase and resumption
of meiosis in Xenopus oocytes. Cell 47, 661-670.
Tsai, L.-H., Harlow, E., and Meyerson,
M. (1991). Isolation of the human cd/Q gene that encodes the cyclin A- and adenovirus
El A-associated p33 kinase. Nature 353, 174-177.
Walker, D. H., and Mailer, J. L. (1991). Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature 354, 314317.
Whyte, P., Buchkovich,
K. J., Horowitz, J. M., Friend, S. H., Raybuck,
M., Weinberg,
R. A., and Harlow, E. (1966). Association
between an
oncogene and an anti-oncogene:
the adenovirus
ElA proteins bind to
the retinoblastoma
gene product. Nature 334, 124-129.
Whyte, P., Williamson,
N. M., and Harlow, E. (1969). Cellular targets
for transformation
by the adenovirus
ElA proteins. Cell 56, 67-75.
Yee, A. S., Raychaudhuri,
P., Jakoi, L., and Nevins, J. R. (1969). The
adenovirus-inducible
factor E2F stimulates transcription
after specific
DNA binding. Mol. Cell. Biol. 9, 576-565.
Yee, S.-P., and Branton, P. E. (1965). Detection
associated
with human adenovirus
type 5 early
tides. Virology 147. 142-153.
of cellular proteins
region IA polypep