Abstract
Deregulated cell proliferation, resulting from disruption of cell cycle control, is characteristic of many cancers. In normal cells, cell cycle progression is mediated by a family of cyclin dependent kinases (Cdks) that are positively regulated by associated cyclins. The activities of these cyclin-Cdk complexes are regulated by two protein families: the inhibitors of Cdk4 (INK4) and the kinase inhibitor proteins (KIP). p27 is a KIP family member that can inhibit cyclin E-Cdk2 activity. It also plays a role in the assembly and nuclear import of cyclin D-Cdk4 in early G1. p27 has been shown to be deregulated in human cancers by accelerated proteolysis, sequestration in cyclin D-Cdk complexes, and mislocalization to the cytoplasm. The causes of these alterations are not fully understood, but result, at least in part, from changes in signal transduction pathways that alter p27 phosphorylation and function. Activation of both the Ras/Raf/ mitogen activated protein kinase (MAPK) and the phospho-inositol 3' kinase (PI3K) pathways have been shown to alter p27 function and to activate p27 degradation in different cell types. In this thesis, I have investigated the roles played by two kinases downstream of PI3K, protein kinase B (PKB) and p90 ribosomal S6 kinase (RSK1), in regulation of p27 function. I observed that PKB-mediated phosphorylation of p27 promotes p27-cyclin D1-Cdk4 assembly. p27 phosphorylation by RSK1 alters the interaction of p27 with cytoskeleton proteins to promote cell motility. I observed that PKB activation and the appearance of p27pT157 and p27pT198 in early G1 precede p27-cyclin D1-Cdk4 assembly. PI3K/PKB inhibition dissociates cellular p27-cyclin D1-Cdk4 and p27T157A, p27T198A and p27T157A/T198A bind cellular cyclin D1 and Cdk4 poorly. Cellular p27pT157 and p27pT198 co-precipitate with Cdk4 but not Cdk2. p27 phosphorylation by PKB increases the ability of p27 to assemble cyclin D1-Cdk4 in vitro, but yields inactive Cdk4. While Src does not affect p27's ability to assemble cyclin D1-Cdk4, Src treatment yields catalytically active p27-cyclin D1-Cdk4. Thus, while PKB dependent p27 phosphorylation promotes p27-cyclin D1-Cdk4 assembly, tyrosine phosphorylation of p27 is required for activation of p27-cyclin D1-Cdk4 complexes. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process. While RSK1 has been shown to phosphorylate p27, the key phosphorylation sites and the consequence of this phosphorylation event were not fully elucidated. I have shown that RSK1 activation in early G1 precedes p27 phosphorylation at T157 and T198 in synchronized cell populations. Overexpression of RSK1 causes resistance to G1 arrest by TGF-รข. Moreover, cells overexpressing RSK1 show an increase in p27 phosphorylation at T198, increased p27 stability, and an increase in p27 binding to Cdk4. In addition, RSK1-transfectants have increased cytoplasmic p27, associated with increased cell motility and inhibition of RhoA. p27 phosphorylation by recombinant RSK1 increases p27 binding to RhoA, while p27T157A/T198A shows reduced association with RhoA in cells. Thus, phosphorylation of p27 at T198 by RSK1 promotes its binding to RhoA and loss of actin stress fiber stability. Oncogenic RSK1 activation may promote increased cancer cell migration and cancer metastasis. Taken together our results indicate that oncogenic activation of the PI3K pathway can contribute to loss of cyclin E-Cdk2 inhibitory action of p27 by at least two mechanisms. Activation of PKB and RSK1 signaling would promote cytoplasmic mislocalization of p27, p27-RhoA binding and inhibition of the RhoA pathway to augment cell motility. In addition, these phosphorylation events on p27 would increase the assembly of p27-cyclin D1-Cdk4 as a first step in a chain of events that would promote that nuclear import and activation of D-type cyclin Cdk complexes, shifting the equilibrium away from the Cdk2 inhibitory action of p27.