title: Protein kinase PKN1 represses Wnt/β-catenin signaling in human melanoma cells.
abstract: 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.
authors.affiliation: From the Departments of Pharmacology and.
authors.forename: Richard G
authors.initials: RG
authors.lastname: James
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Bosch
authors.forename: Rima M
authors.initials: RM
authors.lastname: Kulikauskas
authors.forename: Peitzu T
authors.initials: PT
authors.lastname: Yang
authors.forename: Nick C
authors.initials: NC
authors.lastname: Robin
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Toroni
authors.forename: Travis L
authors.initials: TL
authors.lastname: Biechele
authors.forename: Jason D
authors.initials: JD
authors.lastname: Berndt
authors.forename: Priska D
authors.initials: PD
authors.lastname: von Haller
authors.forename: Jimmy K
authors.initials: JK
authors.lastname: Eng
authors.forename: Alejandro
authors.initials: A
authors.lastname: Wolf-Yadlin
authors.forename: Andy J
authors.initials: AJ
authors.lastname: Chien
authors.forename: Randall T
authors.initials: RT
authors.lastname: Moon
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D051157:Frizzled Receptors / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering; D015398:Signal Transduction; D060449:Wnt Signaling Pathway / Q000235:genetics*; D060509:Wnt3A Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
chemical_list: C115810:FZD7 protein, human; D051157:Frizzled Receptors; D034741:RNA, Small Interfering; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D051176:beta Catenin; C061570:protein kinase N; D011493:Protein Kinase C
keywords: Apoptosis; GSK3-β; Melanoma; PKN1; Phosphorylation; Wnt Signaling; β-Catenin