Differential Modification of p27^Kip1 Controls Its Cyclin D-cdk4 Inhibitory Activity

Abstract

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

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

“…Collectively, these results highlight a previously unknown role for c-Abl in the regulation of p27 KIP1 stability in vivo and support the conclusion that inhibition of c-Abldependent phosphorylation of p27 KIP1 on tyrosine residues is the mechanism of the imatinib-mediated stabilization of p27 KIP1 in neuroblastoma cells. To further test the effects of the loss of p27 KIP1 phosphorylation on tyrosine residues, neuroblastoma cells were transfected with a plasmid coding for a mutated form of p27 KIP1 (p27 Y88F/Y89F ) that cannot be phosphorylated on Tyr-88 and Tyr-89, the major phospho-acceptor sites of p27 KIP1 targeted by c-Abl [4,5]. Transfection with p27 Y88F/Y89F but not with a plasmid coding for wild type p27 KIP1 , resulted in the expression of a stable protein which lacked phosphorylated Thr-187.…”

“…Tyr-88 and Tyr-89 are major phospho-acceptor sites of p27 KIP1 targeted by c-Abl [4,5]. To further test the effects of the loss of p27 KIP1 phosphorylation on tyrosine residues, neuroblastoma cells were transfected with a plasmid coding for a mutant form of p27 KIP1 (p27 Y88F/Y89F ) which cannot be phosphorylated on Tyr-88 and Tyr-89 or wild type p27 KIP1 .…”

“…Loss of phosphorylation on tyrosine residues converts p27 KIP1 from a bound non-inhibitor to a bound inhibitor of cyclin D-CDK4 complexes [5]. To evaluate the effects of imatinib on CDK4 activity, the phosphorylation status of pRb at the CDK4-specific Ser-780 [30] was investigated.…”

“…Upon tyrosine phosphorylation by Abl-or Src-family non-receptor tyrosine kinases, the inhibitory 3 10 helix is ejected from the ATP-binding site of CDK2, partially restoring CDK2 activity. This event would allow cyclin E-CDK2 to phosphorylate Thr-187 leading to p27 KIP1 proteolytic degradation and complete CDK2 activation [4,5]. c-Abl is a %130 kDa protein localized to the plasma membrane, cytosol and nucleus.…”

Tyrosine Phosphorylation of the p21 Cyclin-dependent Kinase Inhibitor Facilitates the Development of Proneural Glioma

“…Some reactions were supplemented with 10 Ci of radiolabeled [ 32 P]ATP as well. Phosphorylated proteins were incubated with cyclin D1-CDK4 complexes produced in Hi5 cells (11), and p21 and the associated proteins were affinitypurified on TALON beads. The amounts of p21-associated CDK4, His-tagged p21, and p21-associated Rb kinase activities were measured by immunoblotting and autoradiography (11).…”