Action of the p53 Effector, p21, on its Targets: Cyclin-CDK & PCNA
Annual rept. 22 Sep 1998-21 Sep 1999
BRIGHAM AND WOMEN'S HOSPITAL BOSTON MA
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Our aim is to dissect the interactions of p21 a downstream effector of the tumor suppressor protein p53 with its targets, cyclin-cdks and PCNA. In year 2 we have focused on the cyclin-cdks. We have quantitated the contribution of the cyclin-cdks binding Cy motif on cdk substrates to their interaction with the cyclin-cdks. We find that a Cy motif improves the Km of a substrate by 5OO-1000 fold without any effect on the Vmax of the peptide or the Km of ATP. Such a large contribution of the Cy motif to substrate recognition explains why a peptide based on the Cy motif of p21 inhibits phosphorylation by cdk. A small chemical that mimics the structure of the Cy motif would be a specific inhibitor of cdks and suppress breast cancer cell growth. Towards this end, site directed mutagenesis has been used to generate a library of mutants in the Cy motif of p21. Using this library we find that the first arginine R1 and the third leucine L3 of the Cy motif RRLFG are critical for docking to cyclins. The R cannot be efficiently substituted by other basic amino acids and the L cannot be efficiently substituted by other non-polar or hydrophobic residues. The R and L need to be separated by a distance of at least two peptide bonds. These results should help design chemicals that mimic the structure and cdk inhibitory activity of Cy peptides.
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