Substrate specificity of CDK2-cyclin A. What is optimal?

The optimal amino acid sequence of substrates for recognition by the cyclin-dependent kinases is well established as -Ser/Thr0-Pro+1-Lys+2-Lys+3-. The catalytic efficiency of CDK2-cyclin A is impaired 2000-, 10-, and 150-fold, when Pro+1, Lys+2, or Lys+3, respectively, is substituted with Ala in a short synthetic peptide substrate. Yet, in physiological substrates of both CDK2-cyclin A and CDK2-cyclin E, it is found that Lys+2, and, occasionally, both Lys+2 and Lys+3 together are replaced with suboptimal determinants. Such suboptimal phosphorylation site motifs are invariably associated with a distinct cyclin-binding (Cy) motif, which has been shown to compensate for otherwise poor catalysis. Here we have investigated the kinetic basis for substrate recognition by CDK2-cyclin A. In the optimal motif, Pro+1 serves to dramatically enhance both substrate binding affinity as well as the rate of chemical phosphotransfer, whereas Lys+2 and Lys+3 both serve to enhance mainly substrate binding. When linked to a suboptimal phosphorylation site sequence (Lys+2 --> Pro) the Cy motif increases catalytic efficiency (kcat/Km) by increasing affinity without affecting turnover (kcat). When fused to the optimal sequence, however, catalytic efficiency is only minimally enhanced, because the resulting high substrate affinity impedes the rate of the phosphoryl transfer reaction. Our results provide kinetic insight into the basis for selecting suboptimal specificity determinants for the phosphorylation of cellular substrates.