Regulation of the human cell division cycle by the p16(Ink4a) cyclin-dependent kinase inhibitor and cyclin E.

摘要

In human cells, the p16Ink4a and cyclin E cell cycle regulators contribute to replicative decisions by affecting cyclin-dependent kinase (cdk) activity. Because p16Ink4a is a tumor suppressor frequently inactivated in cancer, I have studied the biochemical effects of its expression in cultured human cells. By inhibiting G1 phase cdk activity, p16Ink4a expression causes cellular growth arrest, strongly suggesting that its inactivation during tumor development can confer a growth advantage. I have shown that p16Ink4a can function both directly, by binding and inhibiting cdk4/6, and indirectly, by promoting the binding of other cdk inhibitors to cdk2. The combined effect of cdk4/6 and cdk2 inhibition with p16Ink4a expression is G1 phase cellular growth arrest, associated with dephosphorylation of the retinoblastoma protein, pRb. The hypophosphorylated, active form of pRb prevents G1 phase exit by transcriptionally repressing genes whose products are required for S phase entry and DNA synthesis. Other than regulating S phase entry by inactivating pRb, cdk2 activity is required for cellular processes linked to cell cycle progression, implying that its inhibition may be important for stable growth arrest during p16Ink4a expression.; While p16Ink4a indirectly inhibits cdk2 during cell cycle arrest, cyclin E is an activator of cdk2 during G1 phase. Cyclin E protein levels accumulate during G1 and peak at the G1/S phase transition, by a combination of regulated synthesis and degradation. Cyclin E expression is transcriptionally regulated by pRb phosphorylation during G1 phase. Cdk inhibition and pRb dephosphorylation during antimitogenic responses leads to the transcriptional repression of cyclin E and other E2F-regulated genes. During both G1 phase arrest and quiescence (G0), pRb is dephosphorylated and cyclin E is transcriptionally repressed, yet cyclin E protein levels do not decrease. Cells arrested by either p16 Ink4a expression or mitogen withdrawal show cdk2 inhibition, although cyclin E remains associated with its catalytic partner. Normally an unstable protein, cyclin E persistence during cellular growth arrest and transcriptional repression implies that post-transcriptional mechanisms can regulate its cellular abundance. Though inactive, cyclin E-cdk2 complexes may have non-catalytic roles during cellular growth arrest.