Whether p27 is a cyclin D-cdk4/6 inhibitor or not is controversial, and how it might switch between these two modes is unknown. Arguing for a two-state mechanism, we show that p27 bound to cyclin D-cdk4 can be both inhibitory and noninhibitory, due to its differential-growth-state-dependent tyrosine phosphorylation. We found that p27 from proliferating cells was noninhibitory but that p27 from arrested cells was inhibitory, and the transition from a bound noninhibitor to a bound inhibitor was not due to an increase in p27 concentration. Rather, two tyrosine residues (Y88 and Y89) in p27's cdk interaction domain were phosphorylated preferentially in proliferating cells, which converted p27 to a noninhibitor. Concordantly, mutation of these sites rendered p27 resistant to phosphorylation and locked it into the bound-inhibitor mode in vivo and in vitro. Y88 was directly phosphorylated in vitro by the tyrosine kinase Abl, which converted p27 to a cdk4-bound noninhibitor. These data show that the growth-state-dependent tyrosine phosphorylation of p27 modulates its inhibitory activity in vivo.Cell cycle progression through the G 1 phase is regulated by the action of cyclin D-cdk4, cyclin D-cdk6, and cyclin E-cdk2 (30, 49). These serine/threonine kinases phosphorylate and inactivate substrates, such as the tumor suppressor retinoblastoma (Rb), which prevents S-phase entry (16,54). cdk activity is tightly regulated by a combination of mechanisms, including changes in the cyclin level, mitogen-dependent assembly, the phosphorylation of positive and negative regulatory sites on the cdk partner, cellular localization, and interaction with stoichiometric cyclin kinase inhibitors (CKIs), such as p27 Kip1

Cell cycle progression is regulated by cyclin-dependent kinases (cdk's), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27 Kip1 . Although p27 associates with cyclin D-cyclindependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4's active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contactarrested cells, p27's association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27's occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.Cyclin-cyclin-dependent kinase (cyclin-cdk) complexes drive progression through the different phases of the cell cycle by acquiring catalytic activity only at specific points (29, 36). These serine/threonine kinases phosphorylate the substrates that promote these transitions, and therefore, their activity must be tightly regulated to ensure orderly cell cycle progression. Cyclin-dependent kinase 4 (cdk4) and its homologue cdk6 serve as regulators of early G 1 and appear particularly important in the G 0 -to-G 1 transition. Multiple steps are required for the activation of these kinases.