Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclin-dependent kinases

Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) u tilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind direc tly to the cyclin subunit, Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (cons tituting the conserved RXL sequence) was important for p21's ability to inh ibit cyclin E-cdk2 activity, Further analysis of mutant Cy motifs showed, h owever, that RXL was neither necessary nor sufficient for a functional cycl in-binding motif. Replacement of either of these two residues with small hy drophobic residues such as valine preserved p21's inhibitory activity on cy clin E-cdk2, while mutations in either polar or charged residues dramatical ly impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequ ences inhibited growth of mammalian cells, providing in vivo confirmation t hat RXL was not necessary for a functional Cy motif, We also show that the variant Cy motifs identified in this study can effectively target substrate s to cyclin-cdk complexes for phosphorylation, providing additional evidenc e that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the associa tion of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demon strate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.