REQUIREMENT OF CYCLIN E-CDK2 INHIBITION IN P16(INK4A)-MEDIATED GROWTHSUPPRESSION

Loss-of-function mutations of p16(INK4a) have been identified in a lar ge number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in v itro, and this inhibition is believed to be the cause of the p16-media ted G(1) cell cycle arrest after reintroduction of p16 into p16-defici ent tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to sp ecifically inhibit cyclin D-dependent kinases through dominant negativ e interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050-2054, 1993). In thi s study, we determined functional differences between p16 and Cdk4(N15 8). We show that p16 and Cdk4(N158) inhibit the kinase activity of cel lular cyclin D1 complexes through different mechanisms. p16 dissociate d cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) formed complexes with cyclin D1 and p27. In cells induced t o overexpress p16, a higher portion of cellular p27 formed complexes w ith cyclin E-Cdk2, and Cdk2-associated kinase activities were correspo ndingly inhibited. Cells engineered to express moderately elevated lev els of cyclin E became resistant to p16-mediated growth suppression. T hese results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G(1) arrest in actively prolif erating tumor cells. Inhibition of cyclin E-dependent kinases is requi red in p16-mediated growth suppression.