Skp1 and Skp2 are believed to be important in several different cellular processes. First, the level of Skp2 is high in S phase, and its association with cyclin A at this time may be important for cell cycle regulation. Second, the level of Skp2 is typically elevated in transformed cells; hence, overexpression of Skp2 can either play a role in or be a consequence of transformation. Third, Skp1 is a component of the kinetochore, suggesting that it may link kinetochore function with the cell cycle. Finally, degradation of Sic1 in S. cerevisiae is mediated by the E3 complex containing Skp1 and the F-box protein Cdc4 (SCFCdc4), and it is possible that proteolysis of many key cell cycle regulators is mediated by SCF complexes containing other F-box proteins such as Skp2.
We found that the kinase activity of recombinant cyclin A-Cdk2 can be inhibited by Skp2 in vitro. The kinase activity of endogenous cyclin A-Cdk2 in cell lysates can also be inhibited by Skp2. Other explanations for the inhibition of kinase activity by Skp2 are conceivable; for instance, there could be a phosphatase copurified with Skp2 that removes phosphates from histone H1, or Skp2 could bind to histone H1 and sequester them from Cdk2. The fact that Skp2 binds directly to cyclin A-Cdk2 is consistent with the idea that the inhibition could be due to Skp2 directly. Apart from directly inhibiting cyclin A-Cdk2 kinase activity, Skp2 also inhibited the activation of cyclin A-Cdk2 by Thr160 phosphorylation by CAK. Similarly, the p21Cip1/WAF1
families of CDK inhibitors also block the phosphorylation by CAK (1
). These data are in contrast to a previous report that the kinase activity of cyclin A-Cdk2 was not affected by Skp2 in a study using baculovirus-expressed proteins (40
). In contrast to cyclin A-Cdk2, the kinase activity of the neuronal p25-Cdk5 complexes was not affected by Skp2 (Fig. ). Similarly, it was also found that Cdk5 was not inhibited by p21Cip1/WAF1
). The parallel between Skp2 and p21Cip1/WAF1
is further seen by the fact that Skp2 and p21Cip1/WAF1
appears to bind to cyclin A-Cdk2 in a mutually exclusive manner (Fig. ). This may partly explain why Skp2 and p21Cip1/WAF1
are not found together binding to cyclin A-Cdk2 in different cell lines (40
). However, in the experiments described here, we do not exclude the possibility that Skp2 interacts with p21Cip1/WAF1
As for p21Cip1/WAF1
), there was kinase activity associated with Skp2 which could be reduced by addition of more Skp2 (Fig. ). This finding suggests that higher stoichiometry of Skp2 may be required for the inhibition of one molecule of Cdk2 or that Cdk2 dissociates from Skp2 immunoprecipitates at a high rate. Alternatively, there may be two separate binding conformations between cyclin A-Cdk2 and Skp2, one that inhibits the kinase activity and another that does not. The last possibility is supported by the fact that both the N-terminal and C-terminal regions of Skp2 are found to be involved in binding to cyclin A-Cdk2 (Fig. ). In this connection, p21Cip1/WAF1
also contains two cyclin binding sites, one at the N terminus and the other at the C terminus of the protein.
While Skp2 can inhibit the kinase activity of cyclin A-Cdk2 toward histone H1, Skp2 itself served as a substrate for cyclin A-Cdk2 at the same time (Fig. and ). We found that Ser76 was the major site in Skp2 phosphorylated by cyclin A-Cdk2, and phosphorylation caused a mobility shift of Skp2 on SDS-PAGE (Fig. ). Furthermore, S191A, but neither S76A nor S76A S191A, exhibited a mobility shift on SDS-PAGE as did wild-type Skp2 (data not shown). We have evidence that normally in the cell, all endogenous Skp2 is present in the Ser76-phosphorylated, shifted form (unpublished data). This scenario is similar to that for the CDK inhibitors p21Cip1/WAF1
, which inhibit Cdk2 but are phosphorylated by Cdk2 at the same time (32
). This may in part be due to the requirement of multiple molecules or multiple binding sites models of Cdk2 inhibition by p21Cip1/WAF1
described above. Given that Skp2 was phosphorylated by cyclin A-Cdk2, it is possible that when Skp2 bound to cyclin A-Cdk2, Skp2 became the preferred substrate of cyclin A-Cdk2 and thus less phosphate was incorporated into substrates like histone H1. This may be similar to the possibility suggested for the inhibition of cyclin A-Cdk2 by p107 and p130 (37
). However, mutants of Skp2 that were not phosphorylated by cyclin A-Cdk2 (S76A and S76A S191A) can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1 (Fig. ), suggesting that Skp2 is not a mere substrate that competed with histone H1 in these assays.
We analyzed that regions of Skp2 that are important for binding to Skp1 and cyclin A-Cdk2 (Fig. ). The regions of Skp2 that are important for binding to cyclin A-Cdk2 were also important for inhibition of the kinase activity, mainly at the region N terminal to the F box, although the C-terminal sequences also have some affinity for cyclin A-Cdk2. As expected, the F-box motif of Skp2 is required for the binding to Skp1. When we mutated the conserved proline residue in the F-box to alanine to create the P113A mutants we observed that binding between Skp1 and Skp2 decreased about 20% (data not shown); in contrast, in the case of another F-box protein, Cdc4, binding to Skp1 of the proline-to-alanine mutant was completely abolished (4
). It is interesting that in Skp2, at least one of the binding site for cyclin A-Cdk2 is close to the F box. This finding suggests that Cdk2 may be in close proximity to Skp1 in the complex and that the direct interaction between Skp1 and Cdk2 that we described could also be present within the cyclin A-Cdk2-Skp2-Skp1 complex.
The stimulation effect of Skp1 on Cdk2 kinase activity is intriguing (Fig. ). Skp1 alone did not contain kinase activity, which suggests that it is unlikely that the increase in kinase activity was due to a contaminated kinase in the Skp1 preparation. Boiling of Skp1 abolished the kinase stimulation, suggesting that the effect was likely to be due to a protein factor and not the buffer. In contrast to Skp2, which associated with both the Cdk2 and cyclin A subunits, Skp1 interacted with Cdk2 but not the cyclin A subunit (Fig. ). We suspect that the binding between cyclin A-Cdk2 and Skp1 was weaker than that between cyclin A-Cdk2 and Skp2, or between Skp2 and Skp1, because the stimulation of cyclin A-Cdk2 by Skp1 was abolished in the presence of Skp2. There are precedents that proteins can promote the activity of cyclin-CDK complexes. Cdc37 stabilizes and promotes the folding of Cdk4 and Cdk6 (34
), leading to the formation of more active cyclin D-CDK complexes. It has also been suggested that at low concentrations, p21Cip1/WAF1
, and p57Kip2
promote the assembly of cyclin D-Cdk4 (15
). However, we do not think that Skp1 (or Skp2) promotes the assembly of cyclin A-Cdk2 complex in vitro. Rapid complex formation between cyclin A and Cdk2 was observed when these proteins purified from bacteria were mixed together (reaching a maximum in ~5 min). In contrast, the binding of Skp1 or Skp2 to cyclin A-Cdk2 was much slower (reached a maximum in ~30 min). We did not detect any change in the rate of cyclin A-Cdk2 assembly in the presence of Skp1 or Skp2 (unpublished data). One possibility is that some chaperones from bacteria were carried over with the Skp1 preparation, which in turn may assist the folding of Cdk2.
The arrest of the cell cycle after overexpression of Skp2 in mammalian cells was in good agreement with the findings that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 and can block the activation of Cdk2 by CAK. We found that after gel filtration fractionation of HeLa cell extracts, Skp1 consisted of two populations, an apparent monomeric form and a complexed form (unpublished data). This finding suggests that Skp1 is likely to be in excess over its partners like Skp2 in the cell. This may explain why further expression of extra Skp1 in the cell has little effect on the cell cycle distribution. In contrast, gel filtration fractionation suggests that all of the Skp2 molecules are complexed to other proteins in the cell, suggesting that overexpression of Skp2 may disrupt the equilibrium between cyclin-CDK and cyclin-CDK-Skp2. It should be noted that there are ample potential problems underlying these kinds of ectopic expression experiments, where the expression levels of Skp2 and Skp1 must be exceedingly high. One alternative explanation of the action of Skp2 is that the overexpressed Skp2 may bind to Skp1 and cyclin A-Cdk2 separately, and the usual cyclin A-Cdk2-Skp2-Skp1 complexes (which may be required for G1-S transition) are disrupted. Furthermore, if Skp2 is involved in mediating the proteolysis of cyclin A, the destruction of cyclin A upon expression of Skp2 may also arrest the cell cycle. One useful experiment is to express a mutant of Skp2 that is defective in Skp1 or cyclin A-Cdk2 binding in cells and observe the effect on the cell cycle. We found that expression of the Skp2 CΔ96 and CΔ131 mutants (which can bind to cyclin A-Cdk2 but not to Skp1) in mammalian cells has no effect on the cell cycle (data not shown). However, one problem is that both N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2 (Fig. ).
Perhaps a more important question is whether the proteolysis of cyclin A is regulated through binding to Skp2 and Skp1 (SCFSkp2). It is at present unclear whether Skp1 and Skp2 are involved in cyclin A destruction. The actions of Skp2 serving as a mediator of cyclin A proteolysis or an inhibitor of cyclin A-Cdk2 kinase activity would presumably serve the same end—turning off cyclin A-Cdk2 kinase activity. It is not inconceivable that the cyclin A that is targeted for destruction would be inhibited by Skp2 before proteolysis actually occurs.
One intriguing question is why Skp2 is generally expressed at higher levels in transformed cells than in normal fibroblasts (Fig. ). In contrast, the levels of cyclin A and Skp1 do not vary significantly between normal and cancer cells. It should be noted that not all cancer cell lines have elevated levels of Skp2; one example is the human neuroglioma cell line H4 shown in Fig. . It is possible that overexpression of Skp2, acting either as an inhibitor of cyclin A-Cdk2 or as a mediator of cyclin A proteolysis, would lead to cyclin A-Cdk2 being turned on later or turned off earlier during S phase and hence may contribute to transformation. Another possibility is that the increase in Skp2 in transformed cells is a mechanism used by the cells to compensate for the loss of negative regulators of cyclin-CDK such as p21Cip1/WAF1
. Consistent with this, there is an inverse relationship between the level of p21Cip1/WAF1
and Skp2 in cultured cells (38
), and there is a correlation between the higher expression level of cyclin A and that of Skp2 in hepatocellular carcinoma (5