Association of the Cell Cycle Regulatory Proteins p45 and CksHs1 FUNCTIONAL EFFECT ON CDK2 COMPLEX FORMATION AND KINASE ACTIVITY*

In mammalian cells, CDK2 is part of a multiprotein complex that includes Cyclin A or E and cell cycle regulatory proteins such as p21, PCNA, p27, p45, p19, 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 p45 associates directly with CksHs1 and that CksHs1 negatively regulated the interaction between p45 and CDK2. Moreover, we showed that overexpression of CksHs1 inhibits CDK2 kinase activity and that additional expression of p45 overcame this inhibition and restored CDK2 kinase activity. We proposed that the association of CksHs1 and p45 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 p45 SKP2 , CksHs1, and p19 SKP1 (3), but the mechanism of action of these proteins as regulatory molecules of CDK2 kinase activity is not fully understood. Human p45 SKP2 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 p45 SKP2 protein are cell cycle regulated, increasing during G 1 -S phases, accumulating during S phase, and dropping toward M phase (4,5). The level of p45 SKP2 is also increased in many transformed cells (3). Moreover, p45 SKP2 functional interference in cultured cells inhibits S phase entry (3) and p45 SKP2 ectopic expression in quiescent fibroblasts causes mitogen-independent S phase entry (6).
As an F-box containing protein, p45 SKP2 is involved in ubiquitin-mediated protein degradation by functioning as a substrate-specific receptor of the SCF SKP2 (p19 SKP1 -CDC53 (CUL1)-p45 SKP2 ) 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 G 1 -S transition; they consist of p19 SKP1 , 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 p45 SKP2 is growing in importance due to the number of cell cycle proteins found to interact with p45 SKP2 . There is evidence that the cell cycle-regulated transcription factor E2F-1 binds to SCF SKP2 and is ubiquitinated by this complex (7). It is also described that p45 SKP2 promotes p27 Kip1 ubiquitination and degradation (6,8,9). p45 SKP2 is also responsible for the ubiquitination and degradation of B-myb, a DNA-binding protein (10).
Taken together, the existing evidence suggests that p45 SKP2 plays a prominent role in progression of cells through phases G 1 -S and S of the cell cycle. The requirement of p45 SKP2 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 p45 SKP2 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 p45 SKP2 exerts other cell cycle regulatory functions involving protein-protein interactions through its carboxyl-terminal leucine-rich repeats. To investigate novel p45 SKP2 protein-protein interactions, a p45 SKP2 two-hybrid screening was performed. This study defines and characterizes the novel p45 SKP2 -CksHs1 interaction and analyzes its effects on the CDK2 complex formation and the CDK2 kinase function.

EXPERIMENTAL PROCEDURES
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 CLON-TECH Laboratories, Inc., (Palo Alto, CA). The various p45 SKP2 , CDK2, p19 SKP1 , 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-p45 SKP2 and pSK-p19 SKP1 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 p45 SKP2 deletion mutants were generated by PCR. For COS-7 cell transient transfections, different regions of p45 SKP2 , CDK2, p19 SKP1 , 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.
Cell Transfections-Transient transfection into COS-7 cells (ECACC 87021302) were performed by the DEAE-dextran/Me 2 SO method using 2 g of plasmid DNA per 2 ϫ 10 5 cells in a 9-cm 2 dish. The cells were harvested ϳ48 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 ϳ48 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 Ci of [␥-32 P]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 M protein kinase C inhibitor peptide, 2 M PKA inhibitor peptide, and 20 M compound R24571. The precipitates were resolved by SDS-PAGE in reducing conditions followed by autoradiography (6 -12 h).

RESULTS
The Carboxyl-terminal Domain of p45 SKP2 Interacts with CksHs1-To identify novel proteins that interact with p45 SKP2 , the interaction-trap system was used (11). DNA encoding p45 SKP2 (amino acids 3-436) was cloned into the pEG202 plasmid to create the LexA-p45 SKP2 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-p45 SKP2 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 10 6 independent transformants of the lymphoid cDNA library were screened. One of the clones isolated that had the desired phenotype, namely Leuϩ, lacZϩ 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 p45 SKP2 when we performed an interactiontrap 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 p45 SKP2 and CksHs1 (Fig. 1A). The interaction-trap assays showed that p45 SKP2 strongly interacted with full-length CksHs1 (696 ␤-gal units). p19 SKP1 , which was previously known to interact with p45 SKP2 , and was also obtained in the present screening, was used as a positive control (161 ␤-gal units).
Quantitative interaction-trap assays were also used to test the interaction between CDK2 and either p45 SKP2 or CksHs1.
FIG. 1. Identification of CksHs1 as a p45 SKP2 -interacting protein. The interaction-trap assay was performed as described previously (11). Numbers in parentheses are the amino acid residues of p45 SKP2 (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 p45 SKP2 is indicated as a solid black bar. p45 SKP2 baits were transfected into the yeast strain EGY191, while the CDK2 bait was transformed into the yeast strain EGY048.
p45 SKP2 Interacts with CksHs1 in Vivo-To confirm the interaction between p45 SKP2 and CksHs1 in vivo, co-precipitation assays were performed in the lymphoid cell lines Jurkat and Cem ( Fig. 2A). p45 SKP2 was immunoprecipitated from unstimulated Jurkat and Cem cells using an anti-p45 SKP2 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 p45 SKP2 . Similarly, co-precipitation experiments were done in COS-7 cells that were transfected with GST expression vectors encoding either CksHs1 or CksHs1⌬N, which corresponds to the initial clone obtained in the interaction-trap screening, and myc-tagged p45 SKP2 (myc-p45 SKP2 ). The precipitation of the corresponding lysates with glutathione-Sepharose beads and the immunoblot with an antimyc antibody resulted in a specific co-precipitation of p45 SKP2 with CksHs1 and CksHs1⌬N (Fig. 2B).
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 p45 SKP2 , 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-p45 SKP2 binding region to CksHs1-(36 -71) and confirmed that CksHs1 binds to p45 SKP2 or CDK2 through different regions (Fig. 3).
CksHs1 Inhibits the Interaction between p45 SKP2 and CDK2-To test whether CksHs1 has any effect on the p45 SKP2 -CDK2 interaction, a series of p45 SKP2 -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 g) were expressed in COS-7 cells, together with fixed amounts of GST-CDK2 and HA-p45. HA-p45 SKP2 co-precipitation with GST-CDK2 was detected by anti-HA immunoblotting of glutathione-Sepharose precipitates. As shown in Fig. 4A, less p45 SKP2 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 p45 SKP2 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 p45 SKP2 bound to CDK2. p45 SKP2 and CksHs1 Complex with CDK2 in a Mutually Exclusive Way-The possibility that p45 SKP2 binds to CDK2 indirectly through CksHs1 cannot be excluded based on either the previous data concerning p45 SKP2 and CksHs1 interaction with CDK2 or our results about the interaction between p45 SKP2 and CksHs1. Additionally, two-hybrid assays using CDK2 as bait did not result in its interaction with p45 SKP2 (Fig.  1B), suggesting that a p45 SKP2 -CDK2 interaction is indeed indirect.
To further assess the involvement of CksHs1 in the interaction of p45 SKP2 and CDK2, we tested the effect of increasing amounts of p45 SKP2 on CksHs1 binding to CDK2. Increasing amounts of myc-p45 SKP2 (0.4, 0.8, 1.6, and 3.2 g) 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 p45 SKP2 are present (Fig. 5A). This result argues against the hypothesis of CksHs1 acting like a bridge between p45 SKP2 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-p45 SKP2 . These precipitates were resolved by SDS-PAGE and immunoblotted using an anti-HA antibody, which allowed the amount of p45 SKP2 protein bound to CDK2 with and without the addition of His-CksHs1 protein to be determined. The results indicated that less p45 SKP2 protein co-precipitated with His-CksHs1-saturated CDK2 precipitates in comparison with the p45 SKP2 co-precipitated with CDK2 precipitates without the exogenous addition of His-CksHs1  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-p45 SKP2 Ab (lanes 4 and 6) or a control Ab (lanes 3 and 5). Immunoprecipitated proteins and total lysates (ϳ5% 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-p45 SKP2 (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, ϳ5% of immunoprecipitated lysates) were resolved by 12% SDS-PAGE and then transferred onto a PVDF membrane. (Fig. 5B). This result indicates that the exogenous His-CksHs1 blocks the interaction between CDK2 and p45 SKP2 , presumably by structural impediments, and that CksHs1 probably does not function as a bridge molecule between p45 SKP2 and CDK2.
Overexpression of CksHs1 Inhibits CDK2 Kinase Activity, and Overexpression of p45 SKP2 Restores Basal Kinase Activity-To examine whether CksHs1 or p45 SKP2 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-p45 SKP2 . 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 fulllength 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 fulllength 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 p45 SKP2 -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 g), and increasing amounts of myc-p45 SKP2 (0, 4, 0, 8 and 1, 6 g). Our results showed that the increasing amounts of p45 SKP2 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 p45 SKP2 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. DISCUSSION In this study we provide evidence for the interaction between p45 SKP2 and CksHs1. Both p45 SKP2 and CksHs1 were known to be associated with the Cyclin A-CDK2 complexes, but the direct interaction between p45 SKP2 and CksHs1 had never been reported. We identified CksHs1 as a candidate p45 SKP2 -interacting protein in an interaction-trap screening using p45 SKP2 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 p45 SKP2 and CksHs1. This interaction has been mapped to the carboxylterminal region of p45 SKP2 , which contains several leucine-rich repeats, while the amino-terminal F-box domain has proved not to be necessary for this association.
p45 SKP2 is an F-box/leucine-rich repeat-containing protein  (lanes 1-6). Lysates were prepared 48 h after transfection and used in precipitation studies. Coprecipitation analysis was performed using glutathione-Sepharose beads ( lanes  1-6, upper panel). Precipitated proteins (upper panel) and total lysates (middle and lower panels, ϳ5% 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 ex-  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, ϳ5% of immunoprecipitated lysates) were resolved by 12% SDS-PAGE and then transferred onto a PVDF membrane. that belongs to both the ubiquitin-protein ligase complexes SCF SKP2 (4,5,18,19) and the S phase Cyclin A-CDK2 complexes (3). The association between p45 SKP2 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 p45 SKP2 has been related to the SCF SKP2 E3 ubiquitin ligase, both CksHs1 and p45 SKP2 have been related to the S phase Cyclin A-CDK2 complexes. Since both CksHs1 and p45 SKP2 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-p45 SKP2 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-p45 SKP2 complexes in addition to their regulation of S phase kinase activity however.
The interaction between CksHs1 and p45 SKP2 and CDK2 as analyzed by co-precipitation studies indicated that different sites of CksHs1 interact with p45 SKP2 and CDK2. While the CksHs1 residues found to interact with CDK2 agreed with previous studies (24,33,34), we mapped the CksHs1-p45 SKP2 binding region to CksHs1 amino acids 36 -71. Furthermore, our results indicate that CksHs1 modulates the interaction between p45 SKP2 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 p45 SKP2 , blocks the interaction between p45 SKP2 and CDK2. This fact can be explained if p45 SKP2 and CDK2 interact through CksHs1 or if CksHs1 blocks the CDK2-interacting region of p45 SKP2 . To clarify the situation, we performed the inverse experiment, showing that excess amounts of p45 SKP2 blocked the interaction between CksHs1 and CDK2. This result indicated that p45 SKP2 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 p45 SKP2 pulled from a cell extract by the CDK2 alone, indicating that p45 SKP2 competes for a binding site on the cyclin-CDK2 complex with CksHs1 or that the p45 SKP2 -CksHs1 complex formation results in blocking of the  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, ϳ5% 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-p45 SKP2 and GST-CDK2.  1-6), and increasing amounts of myc-p45 SKP2 (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, ϳ5% 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 g/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-p45 SKP2 . Precipitated proteins (lanes 2-5) and total lysates (lane 1, ϳ5% of immunoprecipitated p45 SKP2 lysate) were resolved by 12% SDS-PAGE and then transferred onto a PVDF membrane. 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 p45 SKP2 or CksHs1. The two different cyclin-CDK2 complexes may be cyclin-CDK2-p45 SKP2 and cyclin-CDK2-CksHs1, where p45 SKP2 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 G 1 and S or between G 2 and M (24,(33)(34)(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 p21 Cip1 and p27 Kip1 , 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 p45 SKP2 and CksHs1 inhibits each of their respective interactions with cyclin-CDK2, the p45 SKP2 -CksHs1 complex also has functional implications regarding CDK2 activity. The results of our experiments show that p45 SKP2 restores basal kinase activity after CksHs1-mediated CDK2 kinase inhibition by blocking CksHs1 binding to CDK2. Thus our findings attribute a new role to p45 SKP2 in CDK2 kinase regulation and therefore S phase progression, which is different from the p45 SKP2 function in the ubiquitin degradation pathway. Finally, our data also suggest that factors that regulate the CksHs1-p45 SKP2 interaction may have an important role in cell cycle progression as they would regulate CksHs1-CDK2 interaction and hence kinase activity.