Vinexin beta regulates the phosphorylation of epidermal growth factor receptor on the cell surface.
Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration and oncogenesis. In this study, we found that exogenous expression of vinexin beta enhanced the phosphorylation of 180-kDa proteins in an EGF-dependent manner in Cos-7 cells. Western blot analysis using phospho-specific antibodies against EGFR identified EGFR as a phosphorylated 180-kDa protein. Vinexin beta did not stimulate the phosphorylation of EGFR but suppressed the dephosphorylation, resulting in a sustained phosphorylation. Mutational analyses revealed that both the first and third SH3 domains were required for a sustained phosphorylation of EGFR. Small interfering RNA-mediated knockdown of vinexin beta reduced the phosphorylation of EGFR on the cell surface in HeLa cells. The sustained phosphorylation of EGFR induced by vinexin beta was completely abolished by adding the EGFR-specific inhibitor AG1478 even after EGF stimulation, suggesting that the kinase activity of EGFR is required for the sustained phosphorylation induced by vinexin beta. We also found that E3 ubiquitin ligase c-Cbl is a binding partner of vinexin beta through the third SH3 domain. Expression of wild-type vinexin beta but not a mutant containing a mutation in the third SH3 domain decreased the cytosolic pool of c-Cbl and increased the amount of membrane-associated c-Cbl. Furthermore, over-expression of c-Cbl suppressed the sustained phosphorylation of EGFR induced by vinexin beta. These results suggest that vinexin beta plays a role in maintaining the phosphorylation of EGFR on the plasma membrane through the regulation of c-Cbl.
Mitsushima
Masaru
M
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Ueda
Kazumitsu
K
Kioka
Noriyuki
N
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D009124:Muscle Proteins; C486523:SORBS3 protein, human; D050721:Proto-Oncogene Proteins c-cbl; D010770:Phosphotransferases; D066246:ErbB Receptors