Expression of the PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrate that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr336. Knockdown of Rak enhanced the binding of PTEN to its E3 ligase NEDD4-1 and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

Rak is a nuclear tyrosine kinase containing Src homology 2 and 3 domains at its NH2 terminus. We report here that the retinoblastoma tumor susceptibility gene product pRb associates with Rak in vivo and in vitro. Rak binds in the A/B pocket region of pRb, a region that is frequently mutated in human cancer, during the G1 and S phases of the cell cycle. Furthermore, Rak expression is elevated in G1, and transfection of Rak into NIH 3T3 cells results in a significant decrease in the number of emerging colonies. Thus, Rak is a tyrosine kinase with growth suppressing activity that may function, in part, through its interaction with pRb.

Rak is a nuclear tyrosine kinase containing Src homology 2 and 3 domains at its NH2 terminus. We report here that the retinoblastoma tumor susceptibility gene product pRb associates with Rak in vivo and in vitro. Rak binds in the A/B pocket region of pRb, a region that is frequently mutated in human cancer, during the G1 and S phases of the cell cycle. Furthermore, Rak expression is elevated in G1, and transfection of Rak into NIH 3T3 cells results in a significant decrease in the number of emerging colonies. Thus, Rak is a tyrosine kinase with growth suppressing activity that may function, in part, through its interaction with pRb.

We have identified a new tyrosine kinase from human breast cancer cells called Rak (a Russian word for cancer) that shares 51% identity with c-Src. Sequencing of the full-length complementary DNA revealed that Rak is a tyrosine kinase with a molecular weight of 54,000 that contains SH2 and SH3 domains, as well as tyrosine residues analogous to the autophosphorylation and regulatory tyrosines of the Src family. Biochemical and site-directed mutagenesis analyses revealed that a carboxy-terminal peptide of p54rak was phosphorylated by a cytoplasmic tyrosine kinase (CSK) and that, as in the Src family, it is the COOH-terminal tyrosine that is phosphorylated by CSK. However, there were some properties of Rak that are distinct from Src-like kinases: (a) expression of Rak was predominantly in epithelial-derived cell lines and tissues, especially normal liver and kidney, and cell lines of breast and colon origin; (b) Rak does not harbor the NH2-terminal glycine essential for myristylation and membrane localization; and (c) Rak possesses a putative bipartite nuclear localization signal in the SH2 domain, and subcellular fractionation studies revealed that p54rak resides predominantly in the nucleus. In addition, p54rak was overexpressed in subsets of primary human epithelial tumors, suggesting that p54rak may have a role in human cancer. Thus, Rak is a novel epithelial-associated nuclear tyrosine kinase that may represent a unique subfamily of the Src-related kinases.