Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway

The CDK inhibitor, p21(WAF1/Cip1) blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, wherea s the C-terminus binds and inhibits PCNA (proliferating cell nuclear antige n) function. PCNA is essential for processivity of both DNA polymerase delt a and epsilon. We have performed a detailed analysis of growth inhibition b y the N- and C-terminal regions of p21, and determined whether the N- and C -terminal regions mediate this effect by different mechanisms. Expression o f either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21, The effectiveness o f the two p21 domains is dependent on their stability which is determined b y the ubiquitin-proteasome pathway. The stabilization of the N- and C-termi nal region of p21 increases their effectiveness as inhibitors of DNA synthe sis to levels comparable to full length p21, Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In cont rast, inhibition by the C-terminal region of p21 is not accompanied by supp ression of E2F activity, but is mediated via PCNA binding. The C-terminal r egion of p21 therefore inhibits cell growth by a mechanism distinct from th at of the N-terminal region containing the CDK-cyclin inhibitory domain.