Abstract LB-123: PTK6/BRK modulates tyrosine phosphorylation of p27Kip1 and the activity of the oncogene cyclin D-cdk4.

Abstract

The oncogenes Cyclin D and cdk4 are overexpressed in a variety of tumors, but the levels of these proteins are not always accurate indicators of oncogenic activity because p27Kip1 is required to assemble this otherwise unstable dimer. However, p27’s association activates or alternatively inhibits cyclin D-cdk4, serving as a bona fide ON/OFF “switch.” Tyrosine (Y) phosphorylation of residues Y88/89 in p27 displaces its C-terminus from the cdk4 active site, permitting both ATP binding and CAK phosphorylation of cdk4’s T loop. This model leads to the following hypothesis: modulation of p27 Y phosphorylation controls cdk4 activity, which in turn regulates efficient cell cycle passsage, and in cancers where cdk4 activity is deregulated, p27 may be constitutively switched ON. Deregulated Src Family Kinase (SFK) signaling in cancer may increase p27 Y phosphorylation, constitutively activating oncogenic cdk4, causing continuous cell cycling. Using our p27 Y88 phosphospecific antibody, we have shown in primary tumors, that p27 Y phosphorylation is not detected in benign tissue regions, but is detected in grade 1 and progressively higher grade tumors, suggesting that p27 Y phosphorylation may be a marker for increased oncogenic cdk4 activity and cdk4 inhibitor sensitivity. Although SFKs have been implicated in p27 Y phosphorylation, little is known about the domains involved on either the SFK or p27. We identified two SH3 recruitment domains within p27 that modulate Y88 phosphorylation, thereby modulating cdk4 activity. Mutation of these domains results in loss of Y88 phosphorylation, while the prior addition of an SH3 peptide is able to prevent Y88 phosphorylation. Using a phage-ELISA assay, we identified PTK6/Brk, (Protein Tyrosine Kinase 6/Breast Tumor Kinase), that functions as a high-affinity kinase, able to phosphorylate p27 in vitro and associate with phosphorylated p27 in vivo. Overexpression of PTK6 in vivo increases Y88 phosphorylation and increases resistance to specific cdk4 inhibition by the chemical inhibitor, PD0332991, in a kinase-dependent fashion. As PTK6/Brk is overexpressed in more than 60% of human breast carcinomas, our data suggest that PTK6/Brk overexpression facilitates cell cycle progression by increasing cdk4 activity through direct p27 Y phosphorylation. As PD0332991 moves into the clinic, p27 Y phosphorylation could serve as a marker to identify tumors sensitive to cdk4 inhibition, while blocking the PTK6:p27 interaction represents a novel therapeutic option to inhibit cdk4 activation. Citation Format: Stacy W. Blain, Cindy Gomez, Elina Shteyn, Priyank Patel, Susan R.S. Gottesman, Benedikt Asbach, Ralf Wagner, Angela L. Tyner. PTK6/BRK modulates tyrosine phosphorylation of p27Kip1 and the activity of the oncogene cyclin D-cdk4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-123. doi:10.1158/1538-7445.AM2013-LB-123