CHARACTERIZATION OF THE CYCLIN-DEPENDENT KINASE INHIBITORY DOMAIN OF THE INK4 FAMILY AS A MODEL FOR A SYNTHETIC TUMOR-SUPPRESSOR MOLECULE

We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16(CDKN2/INK4a) (p16) tumour suppre ssor protein (residues 84-103 of the human p16 protein) can bind to cd k4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as wel l as block cell cycle progression through G1. Substitution of two vali ne residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC0.5 for kinase inhibition approximately threefold when linked to th e Antennapedia homeodomain carrier sequence. The same mutations increa se the IC0.5 approximately fivefold in the p16 protein. Substitution o f aspartic acid 92 by alanine instead increases the binding of the pep tide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 pept ide blocks S-phase entry in nonsynchronized human HaCaT cells by appro ximately 90% at a 24 mu M concentration. The V95A and V96A double subs titution minimizes the cell cycle inhibitory capacity of the peptide w hereas the D92A substitution increases its capacity to block cell cycl e progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cyc le inhibitory activity. The p16 peptide inhibited S-phase entry in fiv e cell lines tested, varying between 47-75%, but had only a limited (1 1%) inhibitory effect in the pRb negative Saos-2 cells at a concentrat ion of 24 mu M. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These d ata suggest that acute inhibition of CDK-cyclin D activity by a peptid e derived from the INK4 family mill stop cells in late G1 in a pRb dep endent fashion.