ABSTRACT
In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Cell Movement/physiology , Keratinocytes/physiology , Muscle Proteins/physiology , Wound Healing/physiology , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Base Sequence , Cell Line , Cell Proliferation , Cells, Cultured , ErbB Receptors/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , Keratinocytes/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/deficiency , Muscle Proteins/genetics , RNA, Small Interfering/genetics , Skin/injuries , Skin/pathology , Skin/physiopathologyABSTRACT
Vinexin is an adaptor-type focal adhesion protein that interacts with vinculin. Here, we report the tyrosine phosphorylation of vinexin alpha in v-Src-transformed NIH3T3 cells. Point mutational analysis of vinexin alpha clarified that three tyrosine residues in vinexin alpha were phosphorylated. A non-phosphorylatable mutant of vinexin alpha had higher binding affinity for vinculin than its wild-type counterpart. In conclusion, vinexin alpha is tyrosine phosphorylated in v-Src-transformed cells, and this tyrosine phosphorylation of vinexin alpha attenuates the association of vinexin alpha with vinculin.
Subject(s)
Muscle Proteins/metabolism , Tyrosine/metabolism , Vinculin/metabolism , Animals , Cell Line, Transformed , Mice , Muscle Proteins/genetics , Mutation , NIH 3T3 Cells , Oncogene Protein pp60(v-src)/genetics , Oncogene Protein pp60(v-src)/metabolism , Phosphorylation , Tyrosine/geneticsABSTRACT
Expression of focal adhesion protein vinexin is reported to be altered in several cancer tissues; however, the mechanism of expressional change in vinexin is not known. Here we report the suppression of vinexin expression according to cellular transformation by v-Src. We found that vinexin expression was down-regulated both at the mRNA level and at the post-transcriptional level in v-Src-transformed cells. Both mTOR and MEK/ERK signals were involved in the suppression. Inhibition of these pathways by pharmacological treatment partially restored both vinexin protein and mRNA expression. Moreover, re-expression of vinexin in v-Src-transformed cells suppressed cell migration. Taken together, these observations suggest that cellular transformation by v-Src suppressed vinexin expression and that down-regulation of vinexin may be associated with oncogenic transformation.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Cell Transformation, Viral/genetics , Focal Adhesions/metabolism , Genes, src , Muscle Proteins/metabolism , Animals , Carrier Proteins/metabolism , Cell Movement , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Dose-Response Relationship, Drug , Down-Regulation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesions/drug effects , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Muscle Proteins/genetics , NIH 3T3 Cells , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/metabolism , TOR Serine-Threonine Kinases , Time Factors , TransfectionABSTRACT
Although vinexin was originally identified as a protein binding to the proline-rich hinge region of vinculin, the functions and biochemical properties of the vinexin-vinculin interaction are not known. Here, we determined the affinity of the vinexin-vinculin interaction using surface plasmon resonance measurements and found that vinexin beta interacts with the C-terminal half of vinculin, which mimics an activated "open" form, with a threefold higher affinity than with the full-length "closed" vinculin. Coimmunoprecipitation experiments showed that cell adhesion on fibronectin enhances the vinexin-vinculin interaction. We also show that the interaction with vinculin is necessary for the efficient localization of vinexin alpha and beta at focal adhesions. These observations suggest a model that "activated" vinculin localized at focal adhesions recruits vinexins to focal adhesions.
