Phosphorylation of ΔNp63α via a Novel TGFβ/ALK5 Signaling Mechanism Mediates the Anti-Clonogenic Effects of TGFβ

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 (ALK5IKD). In this study, we demonstrate that ALK5-mediated phosphorylation of ΔNp63α is required for the anti-clonogenic effects of TGFΒ and ectopic expression of ALK5IKD 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.对TP63的遗传分析显示，ΔNp63亚型（ΔNp63 isoforms）参与维持成体干细胞的复制能力与细胞寿命。ΔNp63α同时也是癌基因与存活因子，可介导鳞状细胞癌的治疗抵抗。上述多样的生物学功能，源于TP63与一系列调控组织及肿瘤稳态、有丝分裂极性与细胞命运的形态发生与形态稳态信号之间的遗传与功能相互作用；但目前学界对介导TP63特定功能的细胞信号仍未完全阐明。为解决这一科学问题，本研究旨在筛选可调控ΔNp63α表达、稳定性或活性的信号通路。通过对人类激酶组（human kinome）开展基于小干扰RNA（small interfering RNA, siRNA）的筛选，本研究鉴定出转化生长因子βⅠ型受体（Transforming Growth Factor β Type 1 Receptor, TGFβRⅠ）ALK5为可使ΔNp63α在丝氨酸66/68位点（Serine 66/68, S66/68）发生磷酸化的必需激酶。该磷酸化活性依赖于TGFβ，且可被靶向ALK5的siRNA或ALK5激酶抑制剂A83-01所阻断。机制研究支持如下模型：ALK5在其内部近膜区域发生蛋白水解切割，进而导致其C端ALK5胞内激酶结构域（ALK5 intracellular kinase domain, ALK5IKD）发生转位。本研究证实，ALK5介导的ΔNp63α磷酸化是TGFβ发挥抗克隆形成效应所必需的，且异位表达ALK5IKD可模拟该效应。最后，本研究提供证据表明，紫外线照射（ultraviolet irradiation）介导的ΔNp63α磷酸化可被ALK5抑制剂所抑制。本研究结果揭示了一条非经典TGFβ信号通路，该通路通过ΔNp63α磷酸化介导TGFβ的抗克隆形成效应以及细胞应激相关生物学效应。