p27^Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes

Abstract

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.

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

“…8,16,20 On the other hand, as first exemplified by T187 phosphorylation of p27, 21 phosphorylations of Cip/Kip proteins, including by oncogenic tyrosine kinases, have also emerged as other potential mechanisms for CDK regulation. [22][23][24] Consistent with this idea, we have recently demonstrated that S130 phosphorylation of p21 inside the cyclin D-CDK4/6 complexes is catalyzed by other active CDK4/6 and CDK2 complexes and is required for the activating T172 phosphorylation of p21-bound CDK4 complexes. 15 Later at G1/S transition, S130 phosphorylation of p21 leads to its recognition by the SCF/Skp2 ubiquitin ligase complex and proteasomal degradation of cyclin/CDK-bound p21, hence contributing to CDK2 activation.…”

Regulation of p27Kip1by mitogen-induced tyrosine phosphorylation

“…63 As a consequence, ATP is able to bind to the catalytic cleft of the kinase and restores partial kinase activity to the p27-bound cyclin/Cdk complex. 6,15,18,53,63,64 Tyrosine 88 phosphorylation partially restored phosphorylation of histone H1 by p27-Cdk2/cyclin A and pRb by p27-cyclin A/Cdk2 or p27-cyclin D1/Cdk4. 63 Thus, Y88 phosphorylation impairs the ability of p27 to inactivate Cdks.…”

“…13 In addition, binding of p27 can prevent the activating phosphorylation of the Cdk subunit by Cdk-activating kinase (CAK). 14,15 While inactivation of cyclin A/Cdk2 by p27 has been resolved at the molecular level, the mechanisms of p27 regulating the activity of cyclin D/Cdk4 or cyclin D/ Cdk6 complexes has long remained an issue of debate, as both, kinase-active and kinase-inactive complexes with p27 exist. 6,16,17 A recent thermodynamic analysis of the interactions of p27 with cyclin D1/Cdk4 and cyclin A/Cdk2 suggests that, even though the K d values and IC 50 of p27 for both Cdk complexes are essentially identical, different thermodynamic forces govern p27 subdomain binding.…”

“…Phosphorylation on Y89 was observed to permit activation of p27-bound Cdk4 kinase by enabling the activating phosphorylation of the T-loop at T172 of the Cdk subunit. 15 Significant activation of cyclin D/Cdk4 complexes requires this T-loop phosphorylation by a Cdk-activating kinase (CAK).…”

The function of p27KIP1during tumor development

“…Arguing for a two state mechanism, Dr. Blain's group has assiduously sought-after, and shown that p27 KIP1 can be both a CDK4 bound inhibitor, and a bound non-inhibitor, depending on the growth state of the cell (Ray et al, 2009). They also have discovered that p27 KIP1 associates with cyclin D/CDK4 constitutively, and that a specific tyrosine phosphorylation converts p27 KIP1 from a bound inhibitor to a bound non-inhibitor under different growth conditions.…”