1. Protein Kinase PKN1 Represses Wnt/β-Catenin Signaling in Human Melanoma Cells*
- Author
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Richard G. James, Priska D. von Haller, Katherine A. Bosch, Andy J. Chien, Jason D. Berndt, Nicholas C. Robin, Pei-Tzu Yang, Rima M. Kulikauskas, Randall T. Moon, Travis L. Biechele, Alejandro Wolf-Yadlin, Jimmy K. Eng, and Rachel A. Toroni
- Subjects
Frizzled ,β-Catenin ,Apoptosis ,Biochemistry ,MAP2K7 ,PKN1 ,03 medical and health sciences ,GSK3-β ,0302 clinical medicine ,Cell Line, Tumor ,Wnt3A Protein ,Humans ,ASK1 ,c-Raf ,Phosphorylation ,RNA, Small Interfering ,education ,Molecular Biology ,Melanoma ,Wnt Signaling Pathway ,Protein Kinase C ,beta Catenin ,030304 developmental biology ,0303 health sciences ,education.field_of_study ,biology ,Cyclin-dependent kinase 2 ,Wnt signaling pathway ,LRP6 ,Cell Biology ,Frizzled Receptors ,3. Good health ,Cell biology ,Protein kinase N1 ,Gene Expression Regulation, Neoplastic ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Wnt Signaling ,Signal Transduction - Abstract
Background: Wnt/β-catenin signaling inhibits melanoma cell viability. Results: Integration of phosphoproteomics and RNA interference screens querying the Wnt/β-catenin pathway reveals protein kinase N1 as an inhibitor of signaling. Conclusion: Protein kinase N1 inhibits Wnt/β-catenin signaling and apoptosis in melanoma cells. Significance: This study identifies a kinase that inhibits Wnt/β-catenin signaling, a pathway critical to melanoma cell viability., 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.
- Published
- 2013