Structural and Biochemical Characterization of the p27-cyclin D-cdk4 complex.

Abstract

The cell cycle is controlled by the cyclin-cdks, which phosphorylate substrates required for progression. These kinases are regulated in part by their interaction with the stoichiometric inhibitor p27Kip1. We demonstrated for the first time that p27 could exist as a cyclin D-cdk4 bound-inhibitor and a bound, non-inhibitor, dependent on the growth state of the cell. In asynchronously growing cells (A), p27’s interaction with cyclin D-cdk4 was non-inhibitory, while in contact arrested cells (G0) p27 bound and inhibited cyclin D-cdk4 activity. The p27-cyclin D-cdk4 complex was assembled under both A and G0 conditions, as shown by immunoprecipitation-immunoblot analysis. However, the outcome of p27’s association with cyclin D-cdk4 was different. p27 levels in contact arrested cells, suggested that an increase in the level of p27 might lead to the inhibition of the cyclin D-cdk4 complex in G0 cells. Using a tetracycline repressible p27 induction system (Tetp27), we showed that overexpression of p27 did not inhibit cyclin D-cdk4 complexes, suggesting that p27’s ability to inhibit or alternatively not inhibit was not concentration dependent. We demonstrated p27’s ability to switch from a cyclin D-cdk4 bound inhibitor to a bound non-inhibitor was due to the absence or presence of tyrosine phosphorylation in the 3-10 helix of p27, respectively. We demonstrated that the association of non-tyrosine phosphorylated p27 with cyclin D-cdk4 prevented the required, activating phosphorylation of cdk4’s T-loop by cyclin H-cdk7 (CAK). Tyrosine phosphorylation of p27, which occurs preferentially in proliferating cells, permitted cdk7’s phosphorylation of cdk4. In vitro, the tyrosine kinase Abl was capable of phosphorylating pre-formed p27-cyclin D-cdk4 complexes, permitting both cdk7 phosphorylation of the T-loop and direct access to the cdk4 active site. Our data suggested that activation of cdk4 in vivo may occur in a temporal manner with p27 becoming phosphorylated by a tyrosine kinase in order for cyclin H-cdk7 to gain access to the T-loop. We additionally studied the p27-cyclin D-cdk4 interaction with a series of p27 peptide fragments: Tet p27 (25-106) and Tet p27 (25-73). In vitro, it was previously shown that peptide fragment p27 (22-73) bound without inhibiting cdk4, but bound and inhibited cdk2 activity. In vivo, peptide fragment p27 (25-73) continued to associate with cdk4 in a non-inhibitory manner but now was unable to bind to cdk2. This demonstrated that the interaction of p27 with cdk4 and cdk2 was different both in vivo and in vitro. Our findings help to elucidate the interaction domains of p27 with cyclin D-cdk4.