Mutant B-RAF signaling and cyclin D1 regulate Cks1/S-phase kinase-associated protein 2-mediated degradation of p27Kip1 in human melanoma cells

Abstract

Levels of cyclins and cyclin-dependent kinase (Cdk) inhibitors are tightly controlled during normal cell proliferation and are frequently dysregulated in cancerous cells. In melanoma, cyclin D1 is highly expressed and downregulation of the Cdk inhibitor, p27^Kip1, is associated with a poor prognosis. Mutant B-RAF is frequently expressed in melanoma and overrides growth factor and matrix adhesion control of cyclin D1 and p27^Kip1 levels in human melanocytes. Here, we demonstrate that p27^Kip1 expression is regulated by multiple mechanisms in melanoma cells. B-RAF regulates p27^Kip1 mRNA abundance independently of cyclin D1. Additionally, B-RAF and cyclin D1 control the levels of S-phase kinase-associated protein 2 (Skp2) that directs ubiquitin-mediated proteolysis of p27^Kip1. The cofactor for Skp2, Cdc kinase subunit 1 (Cks1) controls levels of Skp2 in melanoma cells and acts jointly with Skp2 to regulate p27^Kip1 levels. Importantly, expression of Cks1 is regulated by B-RAF and cyclin D1 at the mRNA level. Reduced Cks1 or Skp2 expression and enhanced p27^Kip1 levels inhibit melanoma cell growth. In summary, p27^Kip1 expression in melanoma is regulated by B-RAF at the mRNA level, and via B-RAF and cyclin D1 control of Cks1/Skp2-mediated proteolysis.