Regulation of HEF1 Expression and Phosphorylation by TGF-β1 and Cell Adhesion

Mingzhe Zheng,Paula J Mckeown-Longo

doi:10.1074/jbc.m202263200

Mingzhe Zheng, Paula J Mckeown-Longo

Open Access

https://doi.org/10.1074/jbc.m202263200

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Abstract

Transforming growth factor-beta1 (TGF-beta1) is a multipotential cytokine, which regulates remodeling of tissue extracellular matrix during early tumorigenesis and wound healing. Human enhancer of filamentation-1 (HEF1), a multifunctional docking protein, is involved in integrin-based signaling, which affects cell motility, growth, and apoptosis. Our studies reveal that TGF-beta1 is a potent inducer of HEF1 gene transcription in human dermal fibroblasts. TGF-beta1 promoted HEF1 expression in a dose-dependent manner and resulted in a 16-fold increase in HEF1 protein level. TGF-beta1 had no effect on the stability of either HEF1 protein or mRNA. The TGF-beta1-induced HEF1 expression was independent of cell adhesion and resistant to cytoskeleton disruption. TGF-beta1 increased levels of both p105 and p115 HEF1 in adherent fibroblasts. Digestion with specific phosphatases indicated that the p115HEF1 resulted from serine/threonine phosphorylation of p105HEF1. The appearance of the p115HEF1 as well as tyrosine phosphorylation of p105HEF1 required cell adhesion and/or an organized cytoskeleton. An in vitro kinase assay indicated that p105HEF1 was a substrate for Src. PP1, a specific Src kinase inhibitor, was able to block adhesion-dependent tyrosine phosphorylation of p105HEF1. These findings suggest that TGF-beta1 regulates HEF1 gene expression and that HEF1 phosphorylation is dependent on cell adhesion and Src kinase activity.

Highlights

Remodeling of the tissue extracellular matrix occurs as a component of a number of physiological and pathological processes including wound healing as well as tumorigenesis
Transforming growth factor-␤1 (TGF-␤1) Induces Human enhancer of filamentation-1 (HEF1) Gene Expression—In order to examine the effects of growth factors on the expression and phosphorylation of HEF1, serum-starved human skin fibroblast monolayers were incubated with exogenous growth factors for various periods of time
Increased levels of p105HEF1 were seen within 2 h after exposure of fibroblasts to 2.5 ng/ml TGF-␤1, with maximum increases in both p105 and p115 HEF1 levels achieved after 4 h (Fig. 1)

Summary

Introduction

Remodeling of the tissue extracellular matrix occurs as a component of a number of physiological and pathological processes including wound healing as well as tumorigenesis. These findings suggest that TGF-␤1 regulates HEF1 gene expression and that HEF1 phosphorylation is dependent on cell adhesion and Src kinase activity. These findings demonstrate that in dermal fibroblasts, the levels of phosphorylated HEF1 are coordinately regulated by both growth factors and cell adhesion.

Results

Conclusion