The phosphorylation of vinculin on tyrosine residues 100 and 1065, mediated by SRC kinases, affects cell spreading.

Department of Surgery and the Cancer Institute of New Jersey, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 10/2004; 15(9):4234-47. DOI: 10.1091/mbc.E04-03-0264
Source: PubMed


Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin is tyrosine phosphorylated in platelets spread on fibrinogen and that the phosphorylation is Src kinases dependent. The phosphorylation of vinculin on tyrosine was reconstituted in vanadate treated COS-7 cells coexpressing c-Src. The tyrosine phosphorylation sites in vinculin were mapped to residues 100 and 1065. A phosphorylation-specific antibody directed against tyrosine residue 1065 reacted with phosphorylated platelet vinculin but failed to react with vinculin from unstimulated platelet lysates. Tyrosine residue 1065 located in the vinculin tail domain was phosphorylated by c-Src in vitro. When phosphorylated, the vinculin tail exhibited significantly less binding to the vinculin head domain than the unphosphorylated tail. In contrast, the phosphorylation did not affect the binding of vinculin to actin in vitro. A double vinculin mutant protein Y100F/Y1065F localized to focal adhesion plaques. Wild-type vinculin and single tyrosine phosphorylation mutant proteins Y100F and Y1065F were significantly more effective at rescuing the spreading defect of vinculin null cells than the double mutant Y100F/Y1065F. The phosphorylation of vinculin by Src kinases may be one mechanism by which these kinases regulate actin filament assembly and cell spreading.

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Available from: Gonzalo Izaguirre
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    • "Vinculin is an important FA protein that is tyrosine phosphorylated (pTyr). This post-translational modification is important for maintaining its conformation, activity, and localization [24]. "
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    ABSTRACT: Focal adhesions (FAs), integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147) is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC) cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001), length/width ratios (P<0.0001), and mean intensities (P<0.0001). CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2) recovery times (22.7±3.3 s) of vinculin-mediated focal adhesions (P<0.0001). In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2). Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "Vinculin is a binding partner for many FA proteins, and focal adhesion maturation is highly dependent on a process of vinculin activation, disrupting its head–tail interaction. Position Y100 and Y1065 [9] [10] have been linked to scr-kinase phosphorylation, while S1033 and S1045 [11] were associated with PKC phosphorylation in vitro. Y822 has recently been described as a src-kinase phosphorylation target in adherens junctions [12]. "
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    ABSTRACT: This study evaluates the influence of S1033 vinculin phosphorylation on the mechanical properties of cells. We demonstrate that MEFvcl KO cells transfected with the non-phosphorylatable eGFP-vinculin mutant S1033A are of lower stiffness compared to MEFvcl Rescue and phospho-mimicking mutant S1033D cells, which were of similar stiffness. Analogous, 2D traction microscopy indicates that MEFvcl Rescue and MEF mutant S1033D cells generate similar strain energy, but mutant S1033A display ∼50% less strain energy. Fluorescence recovery after photobleaching demonstrates that the recovery time for mutant S1033A was significantly lower compared to MEFvcl Rescue and mutant S1033D and that the mobile fraction was smaller for MEFvcl Rescue and mutant S1033D than for mutant S1033A cells. This indicates that serine phosphorylation is required for the activation of vinculin and force transmission in focal adhesions.
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    • "Reference SHP-1 protein tyrosine phosphatase [85] Protein tyrosine phosphatase-1B [86] Nonreceptor type 1 [87] SH2 (149–239) Dual-adaptor for phosphotyrosine and 3-phosphoinositides-1 [88] Heterogeneous nuclear ribonucleoprotein K-1 [89] CRK-associated substrate [90] Disabled-1 [91] Cyclin-dependent kinase-5 [92] KCNB1 [93] p21-activated kinase-2 [94] SH3 (87–144) CRK-associated substrate [90] Vinculin [95] Fragile histidine triad protein [96] GRB2 [87] "
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