Cyclin- and CDK-interaction mutants of p27Kip1: implications for cyclin–CDK2 function and specificity
We have constructed two mutants of murine p27Kip1 specifically deficient in interacting with cyclins (p27c−) and CDKs (p27k−), and the corresponding double mutant (p27ck−) defective in both interactions. Using these mutants, we demonstrated that contacts with both cyclin and CDK subunits are required for the optimal function of p27 as a kinase inhibitor. Two residues were mutated to alanines in each mutant: arginine 30/leucine 32 in p27c− and phenylalanines 62/64 in p27k− (Figure 1A). These residues lie within the previously mapped cyclin–CDK-binding domains of mammalian Kip/Cip family proteins, and are part of conserved sequence motifs in all CKIs of this family, including p21Cip1, p27Kip1, p57Kip2, Xenopus p27Xic1, two putative C.elegans gene products, as well as the Drosophila CKI Dacapo (Figure 1A, see references in first paragraph of Results). The crystal structure of p27–cyclin A–CDK2 ternary complexes showed that the targeted residues contact the cyclin and CDK subunits, as predicted (Russo et al., 1996). We propose in Figure 1A a possible alignment of the yeast CKI SIC1 with the Kip/Cip family. It will be of interest to address whether mutations equivalent to those in p27c− or p27k− affect interaction of SIC1 with its cyclin and CDK partners in yeast.
The cyclin-binding motif (or RXL motif) mutated in p27c− is found in proteins other than CKIs, including the retinoblastoma family members p107 and p130, as well as E2F-1, -2 and -3 (Figure 1B) (Zhu et al., 1995; Adams et al., 1996). Several observations suggest that these different RXL motifs indeed bind a common determinant on cyclins. Peptides spanning the RXL motif from representative members of all groups shown in Figure 1 can compete with recombinant GST–p21 or GST–E2F-1 for interaction with cyclins. Mutations of the conserved Arg or Leu to Ala in E2F-1-derived peptides, which correspond to the two substitutions introduced here in p27c−, eliminated their competing activity (Adams et al., 1996). Besides the RXL motif, p107, p130 or E2F proteins lack any obvious CDK-contact motif and can act as targets or tethers for active CDK2. For example, binding of cyclin A–CDK2 to E2F-1/DP-1 dimers results in their phosphorylation and suppression of DNA-binding activity, a mechanism thought to play an important role in downregulation of E2F function during S-phase (Dynlacht et al., 1994; Krek et al., 1994).
Consistent with the notion that the RXL motif can target substrates to active cyclin–CDK complexes, the CDK-contact mutant p27k− (in which the RXL motif was intact; Figure 1A) bound to active cyclin E–CDK2 and was efficiently phosphorylated on Thr187. Further mutation of the RXL motif, such as in the double mutant p27ck−, led to a loss of phosphorylation at limiting substrate concentrations. At high concentrations, p27ck− was phosphorylated by both cyclin E–CDK2 and cyclin A–CDK2, analogous to histone H1. Interestingly, p27k− also associated with cyclin A–CDK2, but was not significantly phosphorylated by this complex. Thus, recruitment of p27k− through the cyclin was inhibitory for Thr187 phosphorylation in the case of cyclin A, but stimulatory in the case of cyclin E. This stimulatory effect became evident at low p27k− concentrations or in the presence of excess histone H1, indicating that interaction with cyclin E became rate-limiting for phosphorylation under these conditions. In our experiments, neither cyclin E–CDK2 nor cyclin A–CDK2 phosphorylated p27wt, correlating with the formation of inactive ternary complexes. Cyclin D1–CDK4 phosphorylated p27wt, p27c−, p27k−, p27ck or the Ala187 derivative of p27k− (p27k−V) to identical, albeit very low levels, which may bear no physiological relevance.
While our work was in revision, Sheaff et al. (1997) reported that wild-type p27 (p27wt) could be phosphorylated by cyclin E–CDK2 in vitro on Thr187. Analysis of phosphorylation kinetics and of the effects of ATP concentration led these authors to propose that p27 bound to cyclin E–CDK2 in two conformations: first in a ‘loose’ state, under which CDK2 phosphorylated p27, and secondly in a ‘tight’ state, under which the kinase was inhibited. The loose state may be analogous to that adopted by our mutant p27k−, which does not undergo the transition to the inhibitory conformation. The tight state most likely is equivalent to the described structure of p27–cyclin A–CDK2 complexes, in which ATP binding to CDK2 is precluded (Russo et al., 1996). The residues mutated in p27k− (Figure 1A) play an important role in this structure. These observations altogether imply that p27 must transiently exist in a cyclin E-bound non-inhibitory conformation in order to become an effective CDK2 substrate.
The cyclin-contact mutant p27c− also formed stable ternary complexes with cyclin A–CDK2 and cyclin D1–CDK4, in this case through its intact CDK-contact domain. In contrast, the presence of cyclin E reduced interaction of p27c− with CDK2. This suggests either that CDK2 adopts slightly different conformations when bound to cyclin A or E, or that cyclin A can sterically hinder interactions of CDK2 with p27c−. Thus, interaction of CDK2 with other proteins (exemplified here by p27c−) may be differentially affected by cyclin E or A.
Recruitment of p27 to active cyclin E–CDK2 complexes triggers its degradation in vivo
When expressed in Rat1 cells with a retroviral vector, p27wt inactivated cyclin E–CDK2 and arrested cells in G1 (Vlach et al., 1996). All of the p27 mutants described here had lost these properties. While p27c− and p27ck− were expressed in vivo at levels equal to, or slightly higher than those of p27wt, the CDK-contact mutant p27k− accumulated to much lower levels in vivo. 35S-pulse–chase studies showed that this was due to enhanced degradation of p27k−. Treatment of cells with the proteasome inhibitor LLNL elevated p27k− levels, but had no effect on the other recombinant p27 proteins, showing that p27k− was degraded by the proteasome, as previously shown for cellular p27 (Pagano et al., 1995).
From our in vitro data, we predicted that association of p27k− with active cyclin E–CDK2 in cells would lead to phosphorylation on Thr187, and that this effect would trigger the enhanced turnover of the protein. We were able to prove the validity of this hypothesis. First, consistent with our in vitro data, we observed that p27k− recovered from cells was associated with catalytically active CDK2. No significant kinase activity was recovered with p27wt, p27c− or p27ck− from cells, even though those proteins were expressed at higher levels. Second, mutation of threonine 187 to valine in p27k− (mutant p27k−V) led to restabilization of the protein, without preventing its stable association with active cyclin–CDK2 complexes. Thus, although we could not biochemically detect phosphorylation of Thr187 in vivo (see Results), our data demonstrated that this phosphosite was required for degradation. Third, degradation of p27k− was strictly dependent upon interaction with cyclin E, since the double mutant p27ck− which did not bind cyclin E–CDK2 complexes was not degraded by the proteasome. In addition, we showed in vitro that although both cyclin E and cyclin A recruited p27k− onto CDK2, only cyclin E–CDK2 efficiently phosphorylated p27k−. Cyclin D1–CDK4 phosphorylated p27wt and all the mutants very inefficiently and with no discrimination. This implies that cyclin E, rather than cyclin A or D1, was responsible for the turnover of p27k− in vivo.
Our results with p27k− imply that cellular p27, in order to be degraded in vivo, must be phosphorylated by CDK2 on Thr187. This model predicts two possible, alternative outcomes upon expression of recombinant p27wt in cells. In the first, p27wt would form inactive ternary complexes with cyclin E–CDK2, would not become phosphorylated on Thr187 and as a consequence would not be targeted to degradation by the proteasome. Consistent with this scenario, retrovirally expressed p27wt associated with inactive cyclin E–CDK2, was stable in our pulse–chase analysis, and was not further stabilized by proteasome inhibitors or by mutation of Thr187. In this system, co-expression of cyclin E with p27wt at moderate levels was not sufficient to bypass CDK2 inhibition, destabilize p27wt or overcome G1 arrest (Vlach et al., 1996). In the second outcome, ectopically expressed p27wt would be phosphorylated by cyclin E–CDK2 on Thr187 and would be rapidly degraded. This was observed in the parallel work of Sheaff et al. (1997), who showed that p27wt expressed by transient transfection was eliminated from cells by co-expression of cyclin E. By generating a high and sudden burst of cyclin E activity, these authors were able to demonstrate that phosphorylation of p27wt on Thr187 preceded its elimination in vivo. As expected, cyclin E suppressed p27-induced G1 arrest in those experiments. Thus, the two complementary approaches used by ourselves and by Sheaff et al. (1997) verify the two major predictions of the proposed model, and establish that phosphorylation of p27 on its carboxy-terminal TPKK site is a prerequisite for its degradation through the proteasome. It is particularly noteworthy that the TPKK site lies within a conserved homology domain found at the carboxy-termini of p27 and p57 (Lee et al., 1995; Matsuoka et al., 1995), suggesting that the two proteins may be subject to similar control mechanisms.
The half-life of p27 increases upon contact-inhibition in HS68 cells and, conversely, decreases upon S-phase arrest in HeLa cells (Hengst et al., 1994). In vitro studies showed that proliferating cell extracts ubiquitinated p27 at higher rates than quiescent extracts (Pagano et al., 1995), and that S-phase extracts degraded p27 whereas mid-G1 extracts did not (Brandeis and Hunt, 1996). In yeast, G1-specific CLN–CDK1 activity is required for ubiquitination and degradation of SIC1, most likely through direct SIC1 phosphorylation (Schneider et al., 1996; Tyers, 1996). SIC1 inhibits S-phase-specific CLB–CDK1 complexes. Thus, through the degradation of SIC1, G1-cyclin–CDK complexes allow activation of S-phase complexes (reviewed by King et al., 1996; Nasmyth, 1996). Our data suggest that degradation of p27 induced by cyclin E–CDK2 may have in part a similar role in preventing inhibition of cyclin A–CDK2 by p27 during S-phase.
Similarly to p27, cyclin E is degraded by the ubiquitin/proteasome pathway following phosphorylation by CDK2 (Clurman et al., 1996; Won and Reed, 1996). A cyclin E mutant deficient in interaction with CDK2 was not phosphorylated, but remained free and was degraded (Clurman et al., 1996). In contrast, our p27ck− mutant, which did not bind cyclin E–CDK2 complexes, was not targeted to degradation. This suggests that the primary effect of phosphorylation is different for cyclin E and p27: to induce release from CDK2 in the case of cyclin E, and to generate a recognition site for components of the degradatory machinery in the case of p27. Alternatively, both mechanisms may be involved in p27 degradation. In yeast, G1 cyclins and both identified CKIs, SIC1 and FAR1, are targeted to the ubiquitin/proteasome pathway in a phosphorylation-dependent manner (see Introduction). Thus, phosphorylation-dependent degradation of G1-specific cyclins and CKIs is a fundamentally conserved process.
One question arising here concerns the relationship between the different mechanisms that regulate p27 function in cells. In particular, p27 can be sequestered away fron cyclin E–CDK2 complexes, either by D-type cyclins (Polyak et al., 1994a; Poon et al., 1995; Reynisdottir et al., 1995; Sherr and Roberts, 1995; Soos et al., 1996) or by an as yet uncharacterized Myc-induced activity (Vlach et al., 1996). Our own observations suggest that Myc-induced sequestration may be mechanistically unlinked from phosphorylation-induced degradation. First, Myc overrode cell cycle arrest by p27 without decreasing p27 levels or half-life in cells. Secondly, Myc could overcome G1-arrest induced by the phosphosite mutant p27V187 (Vlach et al., 1996 and unpublished data). Thus, rescue of p27-induced G1 arrest by Myc does not require Thr187 phosphorylation.
Recent immunohistochemical studies showed that a decrease in p27 levels is an indicator of poor prognosis in human breast and colon carcinomas (Catzavelos et al., 1997; Fredersdorf et al., 1997; Loda et al., 1997; Porter et al., 1997). It was further suggested that this decrease may be due to enhanced p27 degradation in high-grade tumor cells (Loda et al., 1997). Thus, although p27 itself is not the product of a tumor suppressor gene (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996; reviewed by Harper and Elledge, 1996), deregulation of p27 degradation and/or sequestration (induced by Myc or D-type cyclins) may play a major role in tumorigenesis. The precise interplay between these p27-regulatory pathways remains to be elucidated, and will require identification of the cellular proteins that associate with p27 in response to either activation of Myc or phosphorylation by CDK2.