Chrzanowska-Wodnicka M, Burridge K
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
J Cell Sci. 1994 Dec;107 ( Pt 12):3643-54. doi: 10.1242/jcs.107.12.3643.
Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phosphorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phosphorylation is important, other tyrosine kinases may also be involved.
已知酪氨酸磷酸化可调节细胞与细胞外基质（ECM）黏附时黏着斑的形成。我们研究了酪氨酸磷酸化和黏着斑激酶（FAK）在血清或溶血磷脂酸（LPA）诱导的静止瑞士3T3成纤维细胞细胞骨架变化中可能发挥的作用。如其他人之前所示，用血清或LPA刺激静止细胞会导致黏着斑和应力纤维迅速重新出现。在此我们表明，这伴随着黏着斑中磷酸酪氨酸的增加，特别是FAK酪氨酸磷酸化的增加。LPA刺激引起的黏着斑和应力纤维重新出现被磷脂酶C抑制剂阻断，但不被百日咳毒素（PTX）阻断，这表明该LPA信号通路是由磷脂酶C激活介导的，不涉及PTX敏感的G蛋白。在无血清或LPA的情况下，这些细胞骨架效应和FAK的酪氨酸磷酸化可被原钒酸钠与过氧化氢模拟，这两种试剂可抑制蛋白酪氨酸磷酸酶，从而提高磷酸酪氨酸水平。两种酪氨酸激酶抑制剂，埃博霉素和染料木黄酮可阻断血清诱导的FAK酪氨酸磷酸化以及黏着斑和应力纤维的组装。另外两种酪氨酸激酶抑制剂，酪氨酸磷酸化抑制剂47和25，先前已证明可抑制FAK，但未能阻止FAK磷酸化或响应血清时黏着斑和应力纤维的重新组装。然而，这些抑制剂确实阻止了响应溶血磷脂酸（LPA）时的FAK磷酸化和细胞骨架组装，LPA是血清的一种成分，先前已证明可刺激黏着斑和应力纤维的组装。我们的研究结果表明，对血清的反应很复杂，虽然FAK磷酸化很重要，但其他酪氨酸激酶可能也参与其中。
