Differential post-transcriptional regulation of two Ink4 proteins, p18 Ink4c and p19 Ink4d.

Cyclin(-D-)-dependent kinase (Cdk) inhibitors of the Ink4 family specifically bind to Cdk4 and Cdk6, but not to other Cdks. Ink4c and Ink4d mRNAs are maximally and periodically expressed during the G(2)/M phase of the cell division cycle, but the abundance of their encoded proteins is regulated through distinct mechanisms. Both proteins undergo polyubiquitination, but the half life of p18(Ink4c) (approximately 10 hours) is much longer than that of p19(Ink4d) (approximately 2.5 hours). Lysines 46 and 112 are preferred sites of ubiquitin conjugation in p18(Ink4c), although substitution of these and other lysine residues with arginine, particularly in combination, triggers protein misfolding and accelerates p18(Ink4c) degradation. When tethered to either catalytically active or inactive Cdk4 or Cdk6, polyubiquitination of p18(Ink4c) is inhibited, and the protein is further stabilized. Conversely, in competing with p18(Ink4c) for binding to Cdks, cyclin D1 accelerates p18(Ink4c) turnover. In direct contrast, polyubiquitination of p19(Ink4d) is induced by its association with Cdks, whereas cyclin D1 overexpression retards p19(Ink4d) degradation. Although it has been generally assumed that p18(Ink4c) and p19(Ink4d) are biochemically similar Cdk inhibitors, the major differences in their stability and turnover are likely key to understanding their distinct biological functions.