Serine 10 phosphorylation in p27Kip1 metabolism: studies on wild type protein and glycine 9 arginine oncogenic mutant

p27Kip1 is a Cyclin-dependent Kinase Inhibitor (CKI) belonging to CIP/Kip protein family. It is essentially known for its inhibitory action on several cyclin/CDK complexes (specifically cyclin E(A)/CDK2 and cyclin A(B)/CDK1), suggesting a role as tumor suppressor. However, when localized in cytosol, p27Kip1 has a number of CDK-independent functions, including the regulation of apoptosis, cell motility and differentiation. Some of these activities can enhance malignant transformation and/or metastasization under specific conditions. p27Kip1 is characterized by the lack of a stable tertiary structure that favors its “adaptability” to bind different targets and contributes to the heterogeneity of its functions. Because of this peculiar structure, the presence of several post translational modifications (especially phosphorylation) has a key relevance for the CKI cellular localization, metabolism and functions. In this study, we have investigated the turn-over and cyclins/CDK interactions of phosphoserine10-p27Kip1 (pSer10p27Kip1), the main CKI phosphoisoform. We also examined the localization, metabolism, phosphoisoforms pattern and interaction of a cancer-associated mutant form of the CKI (namely G9Rp27Kip1) in which glycine 9 is substituted by an arginine. Our attention focused on this p27Kip1 mutant since the residue change (i.e. glycine 9) occurs in the amino acid preceding serine 10 thus allowing the hypothesis that the mutation affects Ser10 post translational modification.