Abstract 2507: Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer.
Authors: Ling Liu, Howard Chan, David Croucher, Falco Hochgräfe, Luxi Zhang, Carole Tactacan, Roger Daly
Journal: Cancer Research (2013)

Abstract The human genome encodes 518 protein kinases which can be mined for potential cancer drug targets and biomarkers. Previously, we performed SILAC labelling in combination with mass spectrometry-based phosphoproteomics to identify seven proteins exhibiting decreased tyrosine phosphorylation upon Lyn knockdown in the BT549 and MDA-MB-231 basal breast cancer cell lines. From these we selected the atypical kinase SgK269 (also known as PEAK1) for further study. SgK269 is classified as a pseudokinase since it contains critical substitutions in one of the three conserved motifs important for kinase activity. The primary objective of this study was to functionally characterize SgK269 and to identify its signaling role through proteomic and phosphoproteomic profiling in mammary epithelial cells. Initial pull-down experiments using SgK269 recombinant protein were performed to identify binding partners of SgK269 in MDA-MB-231 cells. Functional annotation of the identified binding partners subsequently indicated that SgK269 may be involved in MAPK signaling, regulation of the actin cytoskeleton and focal adhesions, and ubiquitin-mediated proteolysis. To further interrogate the role of SgK269 in breast cancer, SgK269 was overexpressed in the human mammary epithelial cell line MCF-10A. We also expressed a mutant form of SgK269 where the Lyn phosphorylation site, Y635, was altered to phenylalanine (F). Overexpression of SgK269 resulted in a change in cellular morphology. In monolayer, cells were elongated resembling a mesenchymal phenotype and exhibited a decreased expression of E-cadherin and an increase in N-cadherin, indicative of epithelial-to-mesenchymal transition (EMT). When grown in Matrigel, the SgK269-overexpressing cells formed large, aberrant acini, while the SgK269 Y635F mutant was unable to enhance acinar size. In terms of signaling, overexpression of SgK269 increased Erk phosphorylation in three-dimensional culture, an effect associated with SgK269/Grb2 binding and enhanced interaction between the EGFR and Grb2. Since SgK269 showed strong binding with Stat3 in pull-down studies, we also characterized Stat3 signalling in this model. SgK269 overexpression led to enhanced Stat3 tyrosine phosphorylation and transcriptional activity whilst the Y635F mutation abolished this effect. The global tyrosine phosphorylation signature associated with SgK269 overexpression in MCF-10A cells is currently under investigation. SgK269 is a poorly characterized kinase that may contribute to the progression of breast and other cancers. This study provides novel and highly significant insights into the signaling mechanism and function of SgK269 that highlights potential strategies for the improved treatment of basal breast cancers, as well as other cancers where SgK269 is overexpressed. Citation Format: Ling Liu, Howard Chan, David Croucher, Falco Hochgräfe, Luxi Zhang, Carole Tactacan, Roger Daly. Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer. [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 2507. doi:10.1158/1538-7445.AM2013-2507 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.