Phosphorylation of DNp63a via a Novel TGFb/ALK5 Signaling Mechanism Mediates the Anti-Clonogenic Effects of TGFb

Genetic analysis of TP63 implicates DNp63 isoforms in preservation of replicative capacity and cellular lifespan within adult stem cells. DNp63a 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 DNp63a. An siRNA-based screen of the human kinome identified the Type 1 TGFb receptor, ALK5, as the kinase required for phosphorylation of DNp63a at Serine 66/68 (S66/68). This activity is TGFb-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). In this study, we demonstrate that ALK5-mediated phosphorylation of DNp63a is required for the anti-clonogenic effects of TGFB and ectopic expression of ALK5 mimics these effects. Finally, we present evidence that ultraviolet irradiation-mediated phosphorylation of DNp63a is sensitive to ALK5 inhibitors. These findings identify a non-canonical TGFb-signaling pathway that mediates the anti-clonogenic effects of TGFb and the effects of cellular stress via DNp63a phosphorylation. Citation: Cherukuri P, DeCastro AJ, Balboni AL, Downey SL, Liu JY, et al. (2012) Phosphorylation of DNp63a via a Novel TGFb/ALK5 Signaling Mechanism Mediates the Anti-Clonogenic Effects of TGFb. PLoS ONE 7(11): e50066. doi:10.1371/journal.pone.0050066 Editor: Carol Feghali-Bostwick, University of Pittsburgh, United States of America Received July 12, 2012; Accepted October 19, 2012; Published November 16, 2012 Copyright: 2012 Cherukuri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part grants to J.D.R. from the National Cancer Institute (5RO1CA108539-05), the United States Department of Defense Breast Cancer Research Program (BC095560) and a Prouty Pilot Award from the Friends of the Norris Cotton Cancer Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]