Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of β-catenin-responsive transcription (β-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of β-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/β-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.
This work was supported, in whole or in part, by National Institutes of Health Grant K99/R00 1K99HL103768-01 from the NHLBI (to R. G. J.), Grant K08CA128565 from the NCI (to A. J. C.), a grant from the NCI (to R. M. K.), and Training Grant T32AR056969 from the NIAMS (to T. L. B.). This work was also supported by University of Washington's Proteomics Resource Grant UWPR 95794.
