Phosphorylation-induced unfolding regulates p19^INK4d during the human cell cycle

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19^INK4d functions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19^INK4d controls cell cycle progression. We delineate how the stepwise phosphorylation of p19^INK4d Ser66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19^INK4d This dissociates the CDK6-p19^INK4d inhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19^INK4d is ubiquitinated and degraded. Our findings reveal how signaling-dependent p19^INK4d unfolding contributes to the irreversibility of G1/S transition.