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A5761 - Tyrosine Phosphorylation of Tollip on C2 Domain Is Important for TGF-beta Pathway Inhibition Through Modulation of Smad7 Expression
Author Block: X. Li1, H. Bahudhanapati1, S. Yu2, J. Zhao3, D. Kass1, Y. Zhang1; 1Division of Pulmonary, Allergy, and Critical Care Medicine and the Dorothy P. and Richard P. Simmons, University of Pittsburgh, Pittsburgh, PA, United States, 2PUH-Histology Lab, UPMC, Pittsburgh, PA, United States, 3University of Pittsburgh, Pittsburgh, PA, United States.
Rationale: Toll interacting protein, also known as TOLLIP, is an inhibitory adaptor protein in TGF-beta pathway. Genetic variations of the TOLLIP gene are associated with the development and disease progression of idiopathic pulmonary fibrosis (IPF). Repetitive injury of the alveolar epithelium and aberrant wound healing are considered triggering events of IPF. TGF-beta1 plays a pivotal role in this abnormal re-epithelialization process by targeting many cellular processes such as epithelial proliferation and migration. TOLLIP is known to be a tyrosine kinase substrate during cancer development. Tyrosine phosphorylation of TOLLIP in IPF is unknown. We hypothesized that tyrosine phosphorylation of TOLLIP protein is important for its inhibitory role on TGF-beta/Smad3 pathway by regulating inhibitory Smad7. Methods: Wild type and Y83A mutation of TOLLIP were cloned into pcDNA3.1/V5-His-TOPO vector. TOLLIP WT and Y83A mutant were transiently transfected in A549 cells along with a TGF-beta luciferase reporter. Cells were lysed for protein analysis and luciferase assays. Scratch assays were used to evaluate the effects of WT and Y83A TOLLIP on cell migration in A549 cells. The expression of Smad7 was detected by western blot. Results: We have found the tyrosine residue at position 83 in the C2 domain of TOLLIP protein is phosphorylated. TOLLIP overexpression with Y83A mutation in A549 cells led to decreased protein levels of Smad7. Additionally, overexpression of the TOLLIP Y83A mutant partially abolished the inhibitory effects of TOLLIP on a TGF-beta luciferase reporter. Furthermore, cell migration of the A549 cells in a wound healing scratch assay model was impaired when TOLLIP Y83A mutant was overexpressed. Conclusion: Tyrosine phosphorylation of amino acid 83 in the C2 domain of TOLLIP is important for its inhibitory role of TGF-beta pathway by modulating Smad7 expression levels in lung epithelial cells. Our study suggests that TOLLIP may have a critical role in modulating TGF-beta signaling in the respiratory epithelium in IPF. Funding: None.