title: SH3 domain of c-Src governs its dynamics at focal adhesions and the cell membrane.
abstract: We studied the role of the Src SH3 domain in its dynamics at the cell membrane using site-directed mutagenesis and live cell imaging. Physiologically, cell proliferation and migration require the expression of Src family kinases. Hyperactivation of Src molecules has been detected in various cancer cells. Although the activation mechanism of Src has been intensively studied, the dynamics of Src at the cell membrane are still unclear. Although Src molecules also exist at various cellular locations, we found that activated Src molecules are mainly localized at peripheral cell adhesion sites. Src phosphorylation status and subdomain conformations are thought to regulate Src activation and translocation. In this study, we analyzed the single-molecule dynamics of wild-type Src and SH2- and SH3-mutated Src at the cell membrane. Introducing mutations in the SH3 domain resulted in reduced Src motility at the cell membrane, both inside and outside of focal adhesions. Disruption of the actin cytoskeleton resulted in less diffusive Src movement at the cell membrane. We demonstrate that, inside focal adhesions, the SH3 domain enhanced dissociation of Src from the adhesion site and disruption of the SH3 domain altered the distribution of Src at the cell membrane. Inside focal adhesions, kinase activity of Src was essential for the Src mobility reduction by SH3 domain mutation, suggesting that rapid mobility of Src at focal adhesions mediated by the SH3 domain is catalytic-activity-dependent. These findings show that the SH3 domain of Src governs the dynamics of Src at the cell membrane and may be involved in rapid signal transduction in cells.
authors.affiliation: Immunology Frontier Research Center, Osaka University, Suita, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Machiyama
authors.affiliation: Immunology Frontier Research Center, Osaka University, Suita, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Research Institute, National Rehabilitation Center for Persons with Disabilities, Saitama, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Sawada
authors.affiliation: Immunology Frontier Research Center, Osaka University, Suita, Japan.|Quantitative Biology Center, Riken, Suita, Osaka, Japan.
authors.forename: Tomonobu M
authors.initials: TM
authors.lastname: Watanabe
authors.affiliation: Immunology Frontier Research Center, Osaka University, Suita, Japan.|Quantitative Biology Center, Riken, Suita, Osaka, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Fujita
mesh_terms: D019943:Amino Acid Substitution; D015415:Biomarkers / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D002462:Cell Membrane / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D008164:Luminescent Proteins; D050505:Mutant Proteins; C496061:PXN protein, human; D051419:Paxillin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: SH3 domain; Src; TIRF microscopy; focal adhesion; phosphorylation