title: Phosphorylation of ΔNp63α via a novel TGFβ/ALK5 signaling mechanism mediates the anti-clonogenic effects of TGFβ.
abstract: Genetic analysis of TP63 implicates ΔNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. ΔNp63α is also an oncogene and survival factor that mediates therapeutic resistance in squamous carcinomas. These diverse activities are the result of genetic and functional interactions between TP63 and an array of morphogenic and morphostatic signals that govern tissue and tumor stasis, mitotic polarity, and cell fate; however the cellular signals that account for specific functions of TP63 are incompletely understood. To address this we sought to identify signaling pathways that regulate expression, stability or activity of ΔNp63α. An siRNA-based screen of the human kinome identified the Type 1 TGFβ receptor, ALK5, as the kinase required for phosphorylation of ΔNp63α at Serine 66/68 (S66/68). This activity is TGFβ-dependent and sensitive to either ALK5-directed siRNA or the ALK5 kinase inhibitor A83-01. Mechanistic studies support a model in which ALK5 is proteolytically cleaved at the internal juxtamembrane region resulting in the translocation of the C-terminal ALK5-intracellular kinase domain (ALK5(IKD)). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5(IKD) mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of ΔNp63α is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFβ-signaling pathway that mediates the anti-clonogenic effects of TGFβ and the effects of cellular stress via ΔNp63α phosphorylation.
authors.affiliation: Department of Pharmacology and Toxicology, The Audrey and Theodor Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
authors.forename: Pratima
authors.initials: P
authors.lastname: Cherukuri
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: DeCastro
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Balboni
authors.forename: Sondra L
authors.initials: SL
authors.lastname: Downey
authors.forename: Jennifer Y
authors.initials: JY
authors.lastname: Liu
authors.forename: Justine A
authors.initials: JA
authors.lastname: Hutchinson
authors.forename: James
authors.initials: J
authors.lastname: DiRenzo
mesh_terms: D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; C115051:TP63 protein, human; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human