Abstract 197: Phospholipids regulate the localization and oncogenic potential of protein tyrosine kinase 6 in prostate cancer

Abstract Background: Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular SRC-related tyrosine kinase that does not possess canonical localization signals. Lack of targeting yields flexibility in its intracellular localization and access to substrates. PTK6 is observed in the nuclei of prostate epithelial cells where it promotes differentiation and inhibits growth. PTK6 is activated at the plasma membrane in PTEN-deficient mouse prostate tumors where the concentration of PIP3 is high due to PTEN loss of function. Membrane-associated PTK6 induces cell transformation, the epithelial-mesenchymal transition, and tumor cell metastasis. In human prostate cancer, active PTK6 associates with the plasma membrane and its increased expression correlates with metastasis and poor survival. Our studies aimed to characterize how the mislocalization and activation of PTK6 in prostate cancer is regulated. Results: Based on our observation that loss of PTEN induces accumulation of PTK6 at the membrane, we hypothesized that PTK6 binds to the plasma membrane secondary messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Amino acid sequence alignment predicts H52, Y53, and K54 of PTK6 as a potential PIP3 binding site, which resembles the lipid-binding HYK domain of tyrosine kinases BTK and Abl. Using NMR saturation transfer difference (STD) experiment, we observed that the SH2 domain of PTK6 contains a binding site for inositol trisphosphate (IP3) a soluble PIP3 mimic. As predicted, statistical analysis identified Y53 of the PTK6 HYK motif within the SH2 domain as a residue with substantial chemical shift perturbations (CSPs) induced by independent titrations with PIP3 analogs IP3, IP4, and 1,2-dihexanoyl PIP3 in 15N HSQC, as compared to controls. Using autophosphorylated and unphosphorylated PTK6, NMR STD experiments show that only active (pY342) PTK6 binds to IP3. Treatment of prostate cancer cells with the PI3K inhibitor wortmannin reduces active PTK6 at the plasma membrane. Conclusions: Our data demonstrate that active PTK6 is recruited to the plasma membrane through direct interactions with PIP3. Membrane association places PTK6 in close proximity to its oncogenic substrates AKT, FAK and BCAR1, thereby promoting tumor progression. These findings may facilitate identification of novel PTK6 inhibitors that block its association with the membrane, while not affecting its tumor suppressive role in the nucleus. This work is funded by NIH grant 1R01CA188427 to ALT and VG. Citation Format: Darren J. Wozniak, Ben Hitchinson, Wenjun Bie, Vadim Gaponenko, Angela L. Tyner. Phospholipids regulate the localization and oncogenic potential of protein tyrosine kinase 6 in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 197.