A cancer‐associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss‐of‐function

Abstract

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27Kip1 variant, namely glycine9‐>arginine‐p27Kip1 (G9R‐p27Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27Kip1 lacks the major tumor suppressor activities of p27Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27Kip1 on S12, a residue normally never found modified in p27Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27Kip1 since S12AG9R‐p27Kip1 recovers most of the p27Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27Kip1 recapitulates G9R‐p27Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.

Keywords

• CDK inhibitors
• haploinsufficiency
• p27 post‐translational modifications
• tumor suppressor gene