From the Servei d'Immunologia, Hospital Clínic i Provincial de Barcelona, Villarroel 170, Barcelona 08036, Spain
Received for publication, March 12, 2001, and in revised form, May 10, 2001
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ABSTRACT |
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In mammalian cells, CDK2 is part of a
multiprotein complex that includes Cyclin A or E and cell cycle
regulatory proteins such as p21Cip1, PCNA,
p27Kip1, p45SKP2, p19SKP1, and
CksHs1/CksHs2. While the role of some of these proteins has been well
studied, the function of other proteins in the complex remains unclear.
In this study, we showed that the carboxyl-terminal region of
p45SKP2 associates directly with CksHs1 and that CksHs1
negatively regulated the interaction between p45SKP2 and
CDK2. Moreover, we showed that overexpression of CksHs1 inhibits CDK2
kinase activity and that additional expression of p45SKP2
overcame this inhibition and restored CDK2 kinase activity. We proposed
that the association of CksHs1 and p45SKP2 prevented CksHs1
from binding CDK2 and negatively regulating the CDK2 kinase activity.
Cell cycle events are tightly controlled by the sequential
activation of the enzymes known as the cyclin-dependent
kinases (CDKs).1 In mammalian
cells, CDK2 complexed with Cyclin A acts as the main promoter for
progression through the S phase of the cell cycle. Therefore the
regulation of Cyclin A-CDK2 kinase activity is extremely important and
is accomplished by several mechanisms.
CDK2 structure consists of a central deep cleft positioned between the
amino-terminal and carboxyl-terminal lobes, which contains the
catalytic residues, the substrate-binding site, and the ATP-binding site. Mechanisms of CDK2 regulation involve protein-protein
interactions and phosphorylations, reviewed elsewhere (1). CDK2 kinase
activation is mainly achieved by cyclin binding and phosphorylation of
the conserved residue Thr-160. On the other hand, cyclin kinase
inhibitors binding to the Cyclin A-CDK2 complex inhibit
CDK2 kinase activity by direct blockade of the CDK2
substrate-binding site (2).
The active form of the Cyclin A-CDK2 enzyme complexes associates with
other proteins such as p45SKP2, CksHs1, and
p19SKP1 (3), but the mechanism of action of these proteins
as regulatory molecules of CDK2 kinase activity is not fully
understood. Human p45SKP2 is an F-box/leucine-rich
repeat-containing protein that was originally identified by its
association with Cyclin A-CDK2 (3), hence its designation as an S phase
kinase-associated protein (SKP2). Levels of p45SKP2 protein
are cell cycle regulated, increasing during G1-S phases, accumulating during S phase, and dropping toward M phase (4, 5). The
level of p45SKP2 is also increased in many transformed
cells (3). Moreover, p45SKP2 functional interference in
cultured cells inhibits S phase entry (3) and p45SKP2
ectopic expression in quiescent fibroblasts causes mitogen-independent S phase entry (6).
As an F-box containing protein, p45SKP2 is involved in
ubiquitin-mediated protein degradation by functioning as a
substrate-specific receptor of the SCFSKP2
(p19SKP1-CDC53 (CUL1)-p45SKP2)
ubiquitin-protein isopeptide ligase complex (E3). The SCF
complexes function as the main ubiquitin ligases controlling the
abundance of the cell cycle regulatory proteins at the G1-S
transition; they consist of p19SKP1, CUL1, and Rbx1/ROC1 as
the invariable components and the F-box protein as the variable
component and the substrate recognition subunit. The
ubiquitin-dependent cell cycle regulatory role of p45SKP2 is growing in importance due to the number of cell
cycle proteins found to interact with p45SKP2. There is
evidence that the cell cycle-regulated transcription factor E2F-1 binds
to SCFSKP2 and is ubiquitinated by this complex (7). It is
also described that p45SKP2 promotes p27Kip1
ubiquitination and degradation (6, 8, 9). p45SKP2 is
also responsible for the ubiquitination and degradation of B-myb, a
DNA-binding protein (10).
Taken together, the existing evidence suggests that p45SKP2
plays a prominent role in progression of cells through phases
G1-S and S of the cell cycle. The requirement of
p45SKP2 function may be partially explained by its
F-box-dependent implication in cell cycle protein
ubiquitination and proteasomal degradation. However, recent
experimental evidence suggests involvement of p45SKP2 in
several CDK2 regulatory functions, not only related to its amino-terminal F-box domain, but also to its carboxyl-terminal cyclin-protein kinase-binding domain, such as Cyclin A accumulation and
Cyclin A- and Cyclin E-associated kinase activation (6).
Consistent with these observations is the hypothesis that
p45SKP2 exerts other cell cycle regulatory functions
involving protein-protein interactions through its carboxyl-terminal
leucine-rich repeats. To investigate novel p45SKP2
protein-protein interactions, a p45SKP2 two-hybrid
screening was performed. This study defines and characterizes the novel
p45SKP2-CksHs1 interaction and analyzes its effects on the
CDK2 complex formation and the CDK2 kinase function.
Interaction-trap Assay--
Plasmid DNAs, yeast strains, and the
HeLa cell cDNA library used for the interaction-trap assay were
provided by Dr. R. Brent and colleagues and used as described (11, 12).
The human lymphocyte LexA cDNA library, derived from the
HTLV-1-transformed T-cell line SLB-I, used for the interaction-trap
assay was from CLONTECH Laboratories, Inc., (Palo
Alto, CA). The various p45SKP2, CDK2, p19SKP1,
and CksHs1 regions fused to LexA or the B42 transcription activation domain are shown in Fig. 1. The yeast strains EGY048 (MATa trp1 ura3 his3 LEU2::pLexAop6-LEU2) and EGY191 (MATa
trp1 ura3 his3 LEU2::pLexAop2-LEU2), used as hosts for
all the interaction assays, were kindly provided by Dr. E. Golemis.
Both yeast strains contain the plasmid pSH18-34, which includes the
reporter lacZ gene under the control of a modified
Gal1 promoter.
Plasmid Constructions--
The different expression constructs
were made using standard techniques and confirmed by DNA sequencing.
pSK-p45SKP2 and pSK-p19SKP1 were a gift from Dr
Beach. pCMV-CDK2 was a gift from Dr Harlow. All the CksHs1 cDNA
clones were isolated from the interaction-trap HeLa and human
lymphocyte cDNA libraries. The CksHs1 full coding region was
generated by PCR using the above mentioned lymphocyte cDNA library.
CksHs1 and p45SKP2 deletion mutants were generated by PCR.
For COS-7 cell transient transfections, different regions of
p45SKP2, CDK2, p19SKP1, and CksHs1 cDNAs
were cloned into pMT2 derived plasmids. These included pMT2-HA, which
encodes a HA epitope tag sequence immediately upstream of the cloning
site (12), pMT2-myc, which encodes a myc epitope tag sequence
immediately upstream of the cloning site (13) and pMT-GST, which
contains the glutathione S-transferase gene coding sequence
immediately upstream of the cloning site.
Antibodies--
The anti-p45SKP2 rabbit polyclonal
Ab was generated by immunizing rabbits with purified, Escherichia
coli-derived GST-p45SKP2 (amino acids 1-435). For
precipitation studies we used the rabbit polyclonal Ab
anti-p45SKP2 (H-435) from Santa Cruz Biotechnology (Santa
Cruz, CA). The anti-HA mAb HA11 was from Berkeley Antibody Co.
(Richmond, CA). The rabbit polyclonal Ab anti-CDK2 (sc-163) was from
Santa Cruz Biotechnology. The rabbit polyclonal Ab anti-CksHs1 (FL-79)
was from Santa Cruz Biotechnology. The anti-myc tag mAb 9E10 was
described previously (14).
Cell Transfections--
Transient transfection into COS-7 cells
(ECACC 87021302) were performed by the
DEAE-dextran/Me2SO method using 2 µg of plasmid DNA per 2 × 105 cells in a 9-cm2 dish.
The cells were harvested ~48 h after transfection (15).
Western Blot Analysis and
Immunoprecipitations--
Immunoblotting analysis was done essentially
as described previously (15). COS-7 cells that were transiently
transfected with various constructs were harvest ~48 h after
transfection, washed with phosphate-buffered saline, and lysed in
Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl,
50 mM Tris-HCl (pH 7.5), 1 mM EDTA) containing
1 mM phenylmethylsulfonyl fluoride and protease inhibitor
mixture complete from Roche Molecular Biochemicals (Mannheim, Germany).
Insoluble material was removed from the lysates by centrifugation in a
microcentrifuge. Lysates were resolved by SDS-PAGE or precipitated
using the indicated Abs and protein A-Sepharose beads (Amersham
Pharmacia Biotech) or using glutathione-Sepharose beads (Amersham
Pharmacia Biotech). Precipitates were washed with buffer containing
0.1% Nonidet P-40, 150 mM NaCl, and 50 mM
Tris-HCl (pH 7.5). Immunoprecipitated proteins were resolved by
SDS-PAGE and transferred to a polyvinylidene difluoride membrane
(PVDF)-Hybond-P (Amersham Pharmacia Biotech) and probed with various
mAbs and polyclonal antibodies followed by species-specific
peroxidase-labeled antibodies from Amersham Pharmacia Biotech and
visualized by fluorography with enhanced chemiluminescence reagent
(ECL), essentially as described by the supplier (Amersham Pharmacia
Biotech).
CDK2 Kinase Assays--
COS-7 cells expressing exogenous CDK2
were lysed and precipitated as described above. The reactions were
incubated for 10 min at 30 °C in the presence of 150 µCi of
[ The Carboxyl-terminal Domain of p45SKP2 Interacts with
CksHs1--
To identify novel proteins that interact with
p45SKP2, the interaction-trap system was used (11). DNA
encoding p45SKP2 (amino acids 3-436) was cloned into the
pEG202 plasmid to create the LexA-p45SKP2 bait. This
plasmid was transformed into the yeast strain EGY048, which contains
the LEU2 and lacZ reporter genes under the
control of the LexA operon. Unfortunately, this yeast strain could not be used for an interactor hunt, since the LexA-p45SKP2 bait
was a weak transcription activator. The plasmid was then transformed
into the less sensitive yeast strain EGY191, which contains one
operator instead of three upstream of the LEU2 gene and was
co-transfected with the lacZ reporter (pSH18-34). The resulting yeast strain was then transformed with a pJG4-5 based lymphoid cDNA library that conditionally expresses fusion proteins combining the B42 activation domain and the lymphoid proteins. A total
of 106 independent transformants of the lymphoid cDNA
library were screened. One of the clones isolated that had the desired
phenotype, namely Leu+, lacZ+ when grown using galactose, but not
glucose, was identified as CksHs1. This clone encoded amino acids
19-79 of the full-length CksHs1 protein. Several other clones encoding
the CksHs1 gene were also found to interact with p45SKP2
when we performed an interaction-trap screening with a HeLa cDNA library.
A cDNA encoding full-length CksHs1 was cloned by PCR, and
interaction-trap assays were also used to quantify the interactions between p45SKP2 and CksHs1 (Fig.
1A). The interaction-trap
assays showed that p45SKP2 strongly interacted with
full-length CksHs1 (696
To further characterize the p45SKP2 and CksHs1 interaction,
quantitative interaction-trap assays were performed using
amino-terminal and carboxyl-terminal deletion mutants of
p45SKP2. These deletions were cloned by PCR into the
appropriate vector. One construct consisted of amino acids 3-232,
which contains the F-box domain (p45SKP2
Quantitative interaction-trap assays were also used to test the
interaction between CDK2 and either p45SKP2 or CksHs1. As
shown in Fig. 1B, CDK2 strongly interacted with CksHs1 (483 p45SKP2 Interacts with CksHs1 in Vivo--
To confirm
the interaction between p45SKP2 and CksHs1 in
vivo, co-precipitation assays were performed in the lymphoid cell
lines Jurkat and Cem (Fig.
2A). p45SKP2 was
immunoprecipitated from unstimulated Jurkat and Cem cells using an
anti-p45SKP2 antibody. The precipitates were resolved by
SDS-PAGE, subjected to Western blotting, and the membranes probed using
an anti-CksHs1 antibody. In both cell lines CksHs1 was specifically
co-immunoprecipitated with p45SKP2. Similarly,
co-precipitation experiments were done in COS-7 cells that were
transfected with GST expression vectors encoding either CksHs1 or
CksHs1 CksHs1 Binds to CDK2 and p45SKP2 through Different
Sites--
Since CDK2-p45SKP2 and CDK2-CksHs1 interactions
were reported previously; this newly defined interaction of CksHs1 with
p45SKP2 brings into question whether the same domain of
CksHs1 is responsible for the interaction with both p45SKP2
and CDK2. Since the CksHs1 residues involved in the CDK2 interaction have been determined (amino acids 7-23 and 57-70) (16, 17), four
CksHs1 deletion mutants were constructed accordingly (Fig. 3A). The mutant
CksHs1-(19-79) did not include the first 18 amino acids and
therefore lacked some of the CDK2-binding sites. The three other
mutants constructed encoded CksHs1-(36-79), CksHs1-(1-71), and
CksHs1-(36-71). Of all the mutants tested, only CksHs1-(1-71) included all the described CDK2-binding sites.
Expression vectors encoding full-length CksHs1 and the four CksHs1
mutants mentioned above were fused to GST and were co-transfected in
COS-7 cells with either myc-p45 (Fig. 3B) or HA-tagged CDK2 (HA-CDK2; Fig. 3C). The lysates were incubated with
glutathione-Sepharose beads, and the precipitates were immunoblotted
using an anti-myc or anti-HA antibody, respectively. All the deletion
mutants tested were able to bind p45SKP2, but only the
full-length GST-CksHs1 and the mutant lacking the last 8 amino acids
was able to bind CDK2. These results mapped the
CksHs1-p45SKP2 binding region to CksHs1-(36-71) and
confirmed that CksHs1 binds to p45SKP2 or CDK2 through
different regions (Fig. 3).
CksHs1 Inhibits the Interaction between p45SKP2 and
CDK2--
To test whether CksHs1 has any effect on the
p45SKP2-CDK2 interaction, a series of
p45SKP2-CDK2 co-precipitation assays were performed in the
presence of varying the amounts of CksHs1. Increasing amounts of
HA-CksHs1 (0.4, 0.8, 1.6, and 3.2 µg) were expressed in COS-7 cells,
together with fixed amounts of GST-CDK2 and HA-p45.
HA-p45SKP2 co-precipitation with GST-CDK2 was detected by
anti-HA immunoblotting of glutathione-Sepharose precipitates. As shown
in Fig. 4A, less p45SKP2 is
bound to CDK2 when higher amounts of CksHs1 are present. Similarly, a
parallel co-precipitation assay was done using CksHs1-(19-79) instead
of full-length CksHs1. CksHs1-(19-79), as shown in Fig. 3, binds to
p45SKP2 but does not bind to CDK2. The results shown in
Fig. 4B indicate that the presence of increasing amounts of
CksHs1-(19-79) also resulted in less p45SKP2 bound to
CDK2.
p45SKP2 and CksHs1 Complex with CDK2 in a Mutually
Exclusive Way--
The possibility that p45SKP2 binds to
CDK2 indirectly through CksHs1 cannot be excluded based on either the
previous data concerning p45SKP2 and CksHs1 interaction
with CDK2 or our results about the interaction between
p45SKP2 and CksHs1. Additionally, two-hybrid assays using
CDK2 as bait did not result in its interaction with p45SKP2
(Fig. 1B), suggesting that a p45SKP2-CDK2
interaction is indeed indirect.
To further assess the involvement of CksHs1 in the interaction of
p45SKP2 and CDK2, we tested the effect of increasing
amounts of p45SKP2 on CksHs1 binding to CDK2. Increasing
amounts of myc-p45SKP2 (0.4, 0.8, 1.6, and 3.2 µg) were
expressed in COS-7 cells together with fixed quantities of GST-CDK2 and
myc-CksHs1. Glutathione-Sepharose precipitates were subjected to
anti-myc immunoblotting and showed that less CksHs1 is bound to CDK2
when higher amounts of p45SKP2 are present (Fig.
5A). This result argues
against the hypothesis of CksHs1 acting like a bridge between
p45SKP2 and CDK2. To further confirm this fact,
glutathione-Sepharose precipitates containing GST-CDK2 or GST protein
produced in COS-7 cells were incubated with saturating amounts of
His-CksHs1 protein produced in bacteria. The resulting precipitates
were then incubated with lysates from COS-7 cells transfected with
HA-p45SKP2. These precipitates were resolved by SDS-PAGE
and immunoblotted using an anti-HA antibody, which allowed the amount
of p45SKP2 protein bound to CDK2 with and without the
addition of His-CksHs1 protein to be determined. The results indicated
that less p45SKP2 protein co-precipitated with
His-CksHs1-saturated CDK2 precipitates in comparison with the
p45SKP2 co-precipitated with CDK2 precipitates without the
exogenous addition of His-CksHs1 (Fig. 5B). This result
indicates that the exogenous His-CksHs1 blocks the interaction between
CDK2 and p45SKP2, presumably by structural impediments, and
that CksHs1 probably does not function as a bridge molecule between
p45SKP2 and CDK2.
Overexpression of CksHs1 Inhibits CDK2 Kinase Activity, and
Overexpression of p45SKP2 Restores Basal Kinase
Activity--
To examine whether CksHs1 or p45SKP2 can
alter the CDK2 kinase activity in vitro, COS-7 cells were
co-transfected with GST-CDK2 and either myc-CksHs1, HA-CksHs1-(19-79),
or myc-p45SKP2. Subsequently the CDK2 kinase activity of
the GST-CDK2 precipitates was measured using histone H1 as a substrate.
The results showed that overexpression of the full-length CksHs1
substantially inhibited CDK2 kinase activity in vitro,
whereas overexpression of the mutant CksHs1-(19-79) failed to inhibit
the CDK2 kinase activity, consistent with its lack of binding to CDK2
(Fig. 6A). In a separate
experiment, the construct encoding GST-CDK2 transfected with increasing
amounts of either CksHs1 or CksHs1-(19-79) further illustrated the
inhibition that CksHs1 has over the CDK2 kinase activity (Fig.
6B). The presence of increasing amounts of full-length
CksHs1 corresponded with a progressive inhibition of the CDK2 kinase
activity, whereas in the case of CksHs1-(19-79), there was no effect
on kinase activity.
Once it was established that CksHs1 inhibited CDK2 kinase activity, we
investigated whether the p45SKP2-CksHs1 interaction could
modulate the effect of CksHs1 on the CDK2 kinase activity. The same
CDK2 kinase assay was done in COS-7 cells transfected with GST-CDK2, an
inhibitory concentration of myc-CksHs1 (0.8 µg), and increasing
amounts of myc-p45SKP2 (0, 4, 0, 8 and 1, 6 µg). Our
results showed that the increasing amounts of p45SKP2 were
able to progressively relieve CDK2 from the inhibitory effect of CksHs1
and restore CDK2 kinase activity (Fig. 6C). It is noteworthy that overexpression of p45SKP2 alone slightly increased
CDK2 kinase activity.
The same histone H1 kinase assay was performed in COS-7 cells
transfected with GST-CDK2 adding His-CksHs1 protein produced in
bacteria to the CDK2 precipitates. In this case CksHs1 failed to
inhibit CDK2 kinase activity (Fig. 6D), suggesting that
CksHs1 inhibits CDK2 kinase activity using an indirect mechanism.
In this study we provide evidence for the interaction between
p45SKP2 and CksHs1. Both p45SKP2 and CksHs1
were known to be associated with the Cyclin A-CDK2 complexes, but the
direct interaction between p45SKP2 and CksHs1 had never
been reported. We identified CksHs1 as a candidate
p45SKP2-interacting protein in an interaction-trap
screening using p45SKP2 as a bait. The interaction was
confirmed by co-precipitation studies in the lymphoid cell lines Cem
and Jurkat and using COS-7 transfected with various constructs encoding
p45SKP2 and CksHs1. This interaction has been mapped to
the carboxyl-terminal region of p45SKP2, which contains
several leucine-rich repeats, while the amino-terminal F-box domain has
proved not to be necessary for this association.
p45SKP2 is an F-box/leucine-rich repeat-containing protein
that belongs to both the ubiquitin-protein ligase complexes
SCFSKP2 (4, 5, 18, 19) and the S phase Cyclin A-CDK2
complexes (3). The association between p45SKP2 and Cyclin
A-CDK2 complexes has been well documented, but the exact nature of the
interaction remains unclear. We have not found any evidence in favor of
a direct protein-protein interaction, and in fact when we assayed this
in an interaction-trap assay the results were negative. On the other
hand, CksHs1 belongs to the Cks family of cell cycle regulatory
proteins composed of small proteins (9-18 kDa). Cks proteins are bound
to the mitotic cyclin-dependent kinase as described in
yeast (20-22), human cells (23), and frog eggs (24). Human cells
contain two isoforms of Cks proteins, namely CksHs1 and CksHs2, that
each can bind to CDK2 (25). The crystallographic structure of CksHs1 as
well as the human CDK2 kinase bound to CksHs1 has been described (17,
26, 27), but besides this knowledge in terms of sequence and structure, little is known about CksHs1 function (28). Even though Cks proteins
have been related to the cyclosome/APC (anaphase promoting complex)
(29-32), and p45SKP2 has been related to the
SCFSKP2 E3 ubiquitin ligase, both CksHs1 and
p45SKP2 have been related to the S phase Cyclin A-CDK2
complexes. Since both CksHs1 and p45SKP2 bind to the Cyclin
A-CDK2 complexes, it is possible that their interaction is important in
the structure and function of these complexes. Thus, once the
CksHs1-p45SKP2 interaction was characterized, our studies
focused on the role of this interaction in the structure and function
of the S phase cyclin-CDK2 complexes. This does not preclude different
roles for the CksHs1-p45SKP2 complexes in addition to their
regulation of S phase kinase activity however.
The interaction between CksHs1 and p45SKP2 and CDK2 as
analyzed by co-precipitation studies indicated that different sites of CksHs1 interact with p45SKP2 and CDK2. While the CksHs1
residues found to interact with CDK2 agreed with previous studies (24,
33, 34), we mapped the CksHs1-p45SKP2 binding region to
CksHs1 amino acids 36-71. Furthermore, our results indicate that
CksHs1 modulates the interaction between p45SKP2 and CDK2.
We have seen that excess amounts of either CksHs1 or CksHs1-(19-79),
an amino-terminal deletion of CksHs1 that does not bind to CDK2 but
does bind to p45SKP2, blocks the interaction between
p45SKP2 and CDK2. This fact can be explained if
p45SKP2 and CDK2 interact through CksHs1 or if CksHs1
blocks the CDK2-interacting region of p45SKP2. To clarify
the situation, we performed the inverse experiment, showing that excess
amounts of p45SKP2 blocked the interaction between CksHs1
and CDK2. This result indicated that p45SKP2 can bind
either CksHs1 or the CDK2 complex but not both simultaneously. In
further confirmation of this hypothesis, CksHs1 exogenously produced in
bacteria was added to a CDK2 precipitate, and the resulting complex
reduced the amount of p45SKP2 pulled from a cell extract by
the CDK2 alone, indicating that p45SKP2 competes for a
binding site on the cyclin-CDK2 complex with CksHs1 or that the
p45SKP2-CksHs1 complex formation results in blocking of the
cyclin-CDK2-binding sites on each protein. Taken together, these
results suggest the co-existence of at least two different cyclin-CDK2
complexes together with p45SKP2 or CksHs1. The two
different cyclin-CDK2 complexes may be cyclin-CDK2-p45SKP2
and cyclin-CDK2-CksHs1, where p45SKP2 and CksHs1 bind to
the complexes in a mutually exclusive way.
The structure of the cyclin-CDK2-CksHs1 complex is well determined, but
little is known about the role CksHs1 binding plays in CDK2 regulation.
Overexpression or depletion experiments performed on fission yeast,
budding yeast, and Xenopus eggs implicate the Cks family of
proteins in a wide variety of functions; namely entry into mitosis,
exit from mitosis, and transition between G1 and S or
between G2 and M (24, 33-35). According to most
experiments, Cks proteins modulate the tyrosine phosphorylation state
of the major mitotic CDK and may therefore have quite different effects on cell cycle progression depending upon when in the cell cycle the
interaction occurs (24, 36). When CksHs1 function was tested in our
experimental model, CksHs1 clearly inhibited CDK2 kinase activity in
mammalian cells transfected with both CDK2 and CksHs1. This result
demonstrates that CksHs1 complexed with cyclin-CDK2 exerts an
inhibitory effect over the CDK2 kinase activity. However, there was no
CDK2 kinase activity inhibition when exogenous CksHs1 protein was added
to mammalian cells transfected with CDK2. This result clearly indicates
that, in contrast to other CDK2 inhibitors like p21Cip1 and
p27Kip1, which inhibit due to their direct interaction with
the kinase (37, 38), CksHs1-mediated CDK2 kinase inhibition is probably through an indirect mechanism involving another regulatory protein.
Since binding between p45SKP2 and CksHs1 inhibits each of
their respective interactions with cyclin-CDK2, the
p45SKP2-CksHs1 complex also has functional implications
regarding CDK2 activity. The results of our experiments show that
p45SKP2 restores basal kinase activity after
CksHs1-mediated CDK2 kinase inhibition by blocking CksHs1 binding to
CDK2. Thus our findings attribute a new role to p45SKP2 in
CDK2 kinase regulation and therefore S phase progression, which is
different from the p45SKP2 function in the ubiquitin
degradation pathway. Finally, our data also suggest that factors that
regulate the CksHs1-p45SKP2 interaction may have an
important role in cell cycle progression as they would regulate
CksHs1-CDK2 interaction and hence kinase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and histone H1, using the CDK1/cdc2 kinase
kit from Upstate Biotechnology (Lake Placid, NY), according to the
manufacture's instructions. The reactions were done in the presence of
20 µM protein kinase C inhibitor peptide, 2 µM PKA inhibitor peptide, and 20 µM
compound R24571. The precipitates were resolved by SDS-PAGE in reducing
conditions followed by autoradiography (6-12 h).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal units). p19SKP1, which was
previously known to interact with p45SKP2, and was also
obtained in the present screening, was used as a positive control (161
-gal units).
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Fig. 1.
Identification of CksHs1 as a
p45SKP2-interacting protein. The interaction-trap
assay was performed as described previously (11).
Numbers in parentheses are the amino acid
residues of p45SKP2 (A) or CDK2 (B)
fused to the LexA DNA-binding domain. Measurements of -gal levels in
liquid cultures were done in duplicate from two independent isolates,
and the average values of
-gal units are shown. The F-box region of
p45SKP2 is indicated as a solid black bar.
p45SKP2 baits were transfected into the yeast strain
EGY191, while the CDK2 bait was transformed into the yeast strain
EGY048.
C3-232), while
the other consisted of amino acids 153-436, which contains the
leucine-rich domain (p45SKP2
N153-436). As detailed in
Fig. 1A, p45SKP2
N153-436 bound to CksHs1
(422
-gal units), while p45SKP2
C3-232 did not
interact with CksHs1 (6
-gal units) despite being able to bind to
p19SKP1.
-gal units) but not with p45SKP2 (2
-gal units).
N, which corresponds to the initial clone obtained in the
interaction-trap screening, and myc-tagged p45SKP2
(myc-p45SKP2). The precipitation of the corresponding
lysates with glutathione-Sepharose beads and the immunoblot with an
anti-myc antibody resulted in a specific co-precipitation of
p45SKP2 with CksHs1 and CksHs1
N (Fig.
2B).
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[in a new window]
Fig. 2.
Co-precipitation of p45SKP2
and CksHs1 from mammalian cells. A, immunoblot
analysis developed with an anti-CksHs1 Ab. Cem cells (lanes
1, 3, and 4) or Jurkat cells (lanes
2, 5, and 6) were lysed in a Nonidet
P-40-containing buffer, and proteins were immunoprecipitated using an
anti-p45SKP2 Ab (lanes 4 and 6) or a
control Ab (lanes 3 and 5). Immunoprecipitated
proteins and total lysates (~5% of immunoprecipitated lysates) were
resolved by 15% SDS-PAGE and then transferred onto PVDF membrane.
B, immunoblot analysis developed with an anti-myc mAb
(upper and middle panels) or anti-GST Ab
(lower panel). Nonidet P-40 cell lysates were prepared from
COS-7 cells that were transiently transfected with a mix of pMT2
expression vectors encoding GST (lane 1), GST-CksHs1
(lane 2), and GST-CksHs1 N (which encode the original
clone obtained in the interaction-trap screening that does not contain
the first 18 amino acids; lane 3) and
myc-p45SKP2 (lanes 1-3). Lysates were prepared
48 h after transfection and used for precipitation studies.
Co-precipitation analysis was performed using glutathione-Sepharose
beads (lanes 1-3, upper panel). Precipitated
proteins (upper panel) and total lysates (middle
and lower panels, ~5% of immunoprecipitated lysates) were
resolved by 12% SDS-PAGE and then transferred onto a PVDF
membrane.
View larger version (26K):
[in a new window]
Fig. 3.
Mapping of sequences required for
p45SKP2-CksHs1 binding. A, schematically
shown are the regions of CksHs1 used for the experiments described
below. Solid black bands represent the known CDK2
interacting regions. B, immunoblot analysis developed with
an anti-myc mAb (upper and middle panels) or
anti-GST Ab (lower panel). Nonidet P-40 cell lysates were
prepared from COS-7 cells that were transiently transfected with pMT2
expression vectors encoding GST (lane 1), GST-CksHs1
(lane 2), GST-CksHs1-(19-79) (lane 3),
GST-CksHs1-(36-79) (lane 4), GST-CksHs1-(1-71) (lane
5), GST-CksHs1-(36-71) (lane 6), and
myc-p45SKP2 (lanes 1-6). Lysates were prepared
48 h after transfection and used in precipitation studies.
Co-precipitation analysis was performed using glutathione-Sepharose
beads (lanes 1-6, upper panel). Precipitated
proteins (upper panel) and total lysates (middle
and lower panels, ~5% of immunoprecipitated lysates) were
resolved by 12% SDS-PAGE and then transferred onto a PVDF membrane.
C, immunoblot analysis developed with an anti-HA mAb
(upper and lower panels). Nonidet P-40 cell
lysates were prepared from COS-7 cells that were transiently
transfected with pMT2 expression vectors encoding GST (lane
1), GST-CksHs1 (lane 2), GST-CksHs1-(19-79)
(lane 3), GST-CksHs1-(36-79) (lane 4),
GST-CksHs1-(1-71) (lane 5), GST-CksHs1-(36-71) (lane
6), and HA-CDK2 (lanes 1-6). Lysates were prepared
48 h after transfection and used for precipitation studies.
Co-precipitation analysis was performed using glutathione-Sepharose
beads (lanes 1-6, upper panel). Precipitated
proteins (upper panel) and total lysates (lower
panel, ~5% of immunoprecipitated lysates) were resolved by 12%
SDS-PAGE and then transferred onto a PVDF membrane.
View larger version (35K):
[in a new window]
Fig. 4.
CksHs1 modulates the
p45SKP2-CDK2 binding. A,
immunoblot analysis developed with an anti-HA mAb (upper
panels) or an anti-GST Ab (lower panel). Nonidet P-40
cell lysates were prepared from COS-7 cells that were transiently
transfected with pMT2 expression vectors encoding GST (lane
1), GST-CDK2 (lanes 2-6), HA-p45SKP2
(lanes 1-6), and increasing amounts of HA-CksHs1
(lanes 3-6). Lysates were prepared 48 h after
transfection and used for precipitation studies. Co-precipitation
analysis was performed using glutathione-Sepharose beads (lanes
1-6, upper panel). Precipitated proteins (upper
panel) and total lysates (lower panels, ~5% of
immunoprecipitated lysates) were resolved by 12% SDS-PAGE and then
transferred onto a PVDF membrane. B, the experiment was
performed as in A; however, HA-CksHs1-(19-79) rather than
full-length HA-CksHs1 was co-transfected with HA-p45SKP2
and GST-CDK2.
View larger version (35K):
[in a new window]
Fig. 5.
CksHs1 and p45SKP2 bind to CDK2
in a mutually exclusive way. A, immunoblot analysis
developed with an anti-myc mAb (upper panels) or an anti-GST
Ab (lower panel). Nonidet P-40 cell lysates were prepared
from COS-7 cells that were transiently transfected with pMT2 expression
vectors encoding GST (lane 1), GST-CDK2 (lanes
2-6), myc-CksHs1 (lanes 1-6), and increasing amounts
of myc-p45SKP2 (lanes 1, 3, and
6). Lysates were prepared 48 h after transfection and
used for precipitation studies. Co-precipitation analysis was performed
using glutathione-Sepharose beads (lanes 1-6, upper
panel). Precipitated proteins (upper panel) and total
lysates (lower panels, ~5% of immunoprecipitated lysates)
were resolved by 12% SDS-PAGE and then transferred onto a PVDF
membrane. B, immunoblot analysis developed with an anti-HA
mAb. COS-7 cells were transiently transfected with pMT2 expression
vectors encoding GST (lanes 2 and 3) or GST-CDK2
(lanes 4 and 5). Lysates were prepared 48 h
after transfection and precipitated using glutathione-Sepharose beads.
The precipitates were incubated with saturating amounts of His-CksHs1
produced in bacteria (10 µg/ml) for 3 h (lanes 3 and
5). After washing the lysates were incubated for 3 h
with a lysate from COS-7 cells transfected with HA-p45SKP2.
Precipitated proteins (lanes 2-5) and total lysates
(lane 1, ~5% of immunoprecipitated p45SKP2
lysate) were resolved by 12% SDS-PAGE and then transferred onto a PVDF
membrane.
View larger version (28K):
[in a new window]
Fig. 6.
CDK2 kinase assays in COS-7 cells transfected
with p45SKP2 and CksHs1. A, analysis of
CDK2 kinase activity using histone H1 as a substrate. COS-7 cells were
transiently transfected with pMT2 expression vectors encoding GST
(lane 1) or GST-CDK2 (lanes 2-5) and
myc-p45SKP2 (lane 3), myc-CksHs1 (lane
4), or HA-CksHs1-(19-79) (lane 5). Lysates were
prepared 48 h after transfection. Precipitation studies were
performed using glutathione-Sepharose beads, and the precipitates were
incubated 10 min at 30 °C in the presence of 150 µCi of
[ -32P]ATP and histone H1. Precipitates were resolved
by 12% SDS-PAGE followed by autoradiography. As a transfection
control, ~5% of the lysates were resolved by 12% SDS-PAGE,
transferred onto a PVDF membrane, and then developed with an anti-CDK2
Ab (lower panel). B, analysis of CDK2 kinase
activity using histone H1 as a substrate. COS-7 cells that were
transiently transfected with pMT2 expression vectors encoding GST
(lane 1) or GST fused to CDK2 (lanes 2-8) and
pMT2-myc (lane 2), myc-CksHs1 at various concentrations
(lanes 3-6), or myc-CksHs1(19-79) at various
concentrations (lanes 7-8). Lysates were prepared and
processed as described in A. As a transfection control, an
approximate 5% of the lysates was resolved by 12% SDS-PAGE,
transferred onto a PVDF membrane, and then developed with an anti-CDK2
Ab (lower panel). C, analysis of CDK2 kinase
activity using histone H1 as a substrate. COS-7 cells were transiently
transfected with pMT2 expression vectors encoding GST (lane
1) or GST-CDK2 (lanes 2-7) and pMT2-myc (lane
2), myc-CksHs1 (lanes 4-7), and
myc-p45SKP2 at various concentrations (lanes 3 and 5-7). Lysates were prepared and processed as described in
A. As a transfection control, an approximate 5% of the
lysates was resolved by 12% SDS-PAGE, transferred onto a PVDF
membrane, and then developed with an anti-CDK2 Ab (lower
panel). D, shown is the analysis of CDK2 kinase
activity using histone H1 as a substrate. COS-7 cells were transiently
transfected with pMT2 expression vectors encoding GST (lane
1) or GST-CDK2 (lanes 2-6). Lysates were prepared
48 h after transfection. Precipitation studies were performed
using glutathione-Sepharose beads, and the precipitates were incubated
10 min at 30 °C in the presence of 150 µCi of
[
-32P]ATP and histone H1 and various concentrations of
protein His-CksHs1 produced in bacteria. Precipitates were resolved by
12% SDS-PAGE followed by autoradiography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Q. Medley and J. Pratt for critical review of the manuscript; Drs. R. Brent, E. Golemis, and colleagues for plasmid DNAs and yeast strains used for the interaction-trap assay; and Drs. D. Beach and E. Harlow for various cDNA constructs.
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FOOTNOTES |
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* This work was supported in part by Grant 00/3010 from the Fundació Marató TV3, Grant SAF1996-0355 from the Comisión Interministerial de Ciencia y Tecnología, Spain, and Grant PM-98/0025 from the Ministerio de Educación Ciencio.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: The Babraham Institute, Babraham, Cambridge CB2
4AT, United Kingdom.
§ These authors contributed equally to this work.
¶ To whom the correspondence may be addressed: Servei d'Immunologia, ICII, Hospital Clínic i Provincial de Barcelona, Villarroel 170, Barcelona 08036, Spain. Tel.: 34-93-454-4920; Fax: 34-93-451-8038; E-mail: spages@medicina.ub.es.
Published, JBC Papers in Press, May 10, 2001, DOI 10.1074/jbc.M102184200
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ABBREVIATIONS |
---|
The abbreviations used are:
CDK, cyclin-dependent kinase;
Ab, antibody;
GST, glutathione
S-transferase;
HA, hemagglutinin;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain
reaction;
E3, ubiquitin-protein isopeptide ligase;
PVDF, polyvinylidene difluoride;
-gal,
-galactosidase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Morgan, D. O. (1995) Nature 374, 131-134[CrossRef][Medline] [Order article via Infotrieve] |
2. | Morgan, D. O. (1996) Curr. Opin. Cell Biol. 8, 767-772[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zhang, H., Kobayashi, R., Galaktionov, K., and Beach, D. (1995) Cell 82, 915-925[Medline] [Order article via Infotrieve] |
4. |
Lisztwan, J.,
Marti, A.,
Sutterluty, H.,
Gstaiger, M.,
Wirbelauer, C.,
and Krek, W.
(1998)
EMBO J.
17,
368-383 |
5. | Michel, J. J., and Xiong, Y. (1998) Cell Growth Differ. 9, 435-449[Abstract] |
6. | Sutterluty, H., Chatelain, E., Marti, A., Wirbelauer, C., Senften, M., Muller, U., and Krek, W. (1999) Nat. Cell Biol. 1, 207-214[CrossRef][Medline] [Order article via Infotrieve] |
7. | Marti, A., Wirbelauer, C., Scheffner, M., and Krek, W. (1999) Nat. Cell Biol. 1, 14-19[CrossRef][Medline] [Order article via Infotrieve] |
8. | Carrano, A. C., Eytan, E., Hershko, A., and Pagano, M. (1999) Nat. Cell Biol. 1, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
9. | Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H., and Zhang, H. (1999) Curr. Biol. 9, 661-664[CrossRef][Medline] [Order article via Infotrieve] |
10. | Charrasse, S., Carena, I., Brondani, V., Klempnauer, K. H., and Ferrari, S. (2000) Oncogene 19, 2986-2995[CrossRef][Medline] [Order article via Infotrieve] |
11. | Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791-803[Medline] [Order article via Infotrieve] |
12. | Serra-Pages, C., Kedersha, N. L., Fazikas, L., Medley, Q., Debant, A., and Streuli, M. (1995) EMBO J. 14, 2827-2838[Abstract] |
13. |
Medley, Q. G.,
Serra-Pages, C.,
Iannotti, E.,
Seipel, K.,
Tang, M.,
O'Brien, S. P.,
and Streuli, M.
(2000)
J. Biol. Chem.
275,
36116-36123 |
14. | Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Medline] [Order article via Infotrieve] |
15. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology , John Wiley & Sons |
16. | Bourne, Y., Watson, M. H., Arvai, A. S., Bernstein, S. L., Reed, S. I., and Tainer, J. A. (2000) Struct. Fold Des. 8, 841-850[CrossRef][Medline] [Order article via Infotrieve] |
17. | Bourne, Y., Watson, M. H., Hickey, M. J., Holmes, W., Rocque, W., Reed, S. I., and Tainer, J. A. (1996) Cell 84, 863-874[Medline] [Order article via Infotrieve] |
18. |
Lyapina, S. A.,
Correll, C. C.,
Kipreos, E. T.,
and Deshaies, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7451-7456 |
19. |
Yu, Z. K.,
Gervais, J. L.,
and Zhang, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11324-11329 |
20. | Hayles, J., Beach, D., Durkacz, B., and Nurse, P. (1986) Mol. Gen. Genet. 202, 291-293[Medline] [Order article via Infotrieve] |
21. | Brizuela, L., Draetta, G., and Beach, D. (1987) EMBO J. 6, 3507-3514[Abstract] |
22. | Hadwiger, J. A., Wittenberg, C., Mendenhall, M. D., and Reed, S. I. (1989) Mol. Cell. Biol. 9, 2034-2041[Medline] [Order article via Infotrieve] |
23. | Draetta, G., Brizuela, L., Potashkin, J., and Beach, D. (1987) Cell 50, 319-325[Medline] [Order article via Infotrieve] |
24. | Patra, D., and Dunphy, W. G. (1996) Genes Dev. 10, 1503-1515[Abstract] |
25. | Richardson, H. E., Stueland, C. S., Thomas, J., Russell, P., and Reed, S. I. (1990) Genes Dev. 4, 1332-1344[Abstract] |
26. | Jeffrey, P. D., Russo, A. A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., and Pavletich, N. P. (1995) Nature 376, 313-320[CrossRef][Medline] [Order article via Infotrieve] |
27. | Arvai, A. S., Bourne, Y., Hickey, M. J., and Tainer, J. A. (1995) J. Mol. Biol. 249, 835-842[CrossRef][Medline] [Order article via Infotrieve] |
28. | Pines, J. (1996) Curr. Biol. 6, 1399-1402[Medline] [Order article via Infotrieve] |
29. |
Patra, D.,
and Dunphy, W. G.
(1998)
Genes Dev.
12,
2549-2559 |
30. |
Patra, D.,
Wang, S. X.,
Kumagai, A.,
and Dunphy, W. G.
(1999)
J. Biol. Chem.
274,
36839-36842 |
31. |
Kaiser, P.,
Moncollin, V.,
Clarke, D. J.,
Watson, M. H.,
Bertolaet, B. L.,
Reed, S. I.,
and Bailly, E.
(1999)
Genes Dev.
13,
1190-1202 |
32. | Shteinberg, M., and Hershko, A. (1999) Biochem. Biophys. Res. Commun. 257, 12-18[CrossRef][Medline] [Order article via Infotrieve] |
33. | Moreno, S., Hayles, J., and Nurse, P. (1989) Cell 58, 361-372[Medline] [Order article via Infotrieve] |
34. | Tang, Y., and Reed, S. I. (1993) Genes Dev. 7, 822-832[Abstract] |
35. |
Reynard, G. J.,
Reynolds, W.,
Verma, R.,
and Deshaies, R. J.
(2000)
Mol. Cell. Biol.
20,
5858-5864 |
36. | Dunphy, W. G., and Newport, J. W. (1989) Cell 58, 181-191[Medline] [Order article via Infotrieve] |
37. | Chen, J., Jackson, P. K., Kirschner, M. W., and Dutta, A. (1995) Nature 374, 386-388[CrossRef][Medline] [Order article via Infotrieve] |
38. | Russo, A. A., Jeffrey, P. D., Patten, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331[CrossRef][Medline] [Order article via Infotrieve] |