Murphy-Ullrich J E, Pallero M A, Boerth N, Greenwood J A, Lincoln T M, Cornwell T L
Department of Pathology, University of Alabama at Birmingham 35294-0019, USA.
J Cell Sci. 1996 Oct;109 ( Pt 10):2499-508. doi: 10.1242/jcs.109.10.2499.
Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.
粘着斑是细胞膜的特殊区域，是细胞内外信号传递的焦点。细胞内信号事件对这些结构的组织和稳定性很重要。在之前的工作中，我们发现抗粘着性细胞外基质蛋白血小板反应蛋白、腱生蛋白和骨连接蛋白能诱导粘着斑的解体，并且我们确定了这些蛋白的活性区域。为了确定抗粘着性基质蛋白调节细胞骨架组织和粘着斑完整性的机制，我们研究了蛋白激酶在介导这些蛋白导致粘着斑丧失中的作用。这些研究的数据表明，环磷酸鸟苷依赖性蛋白激酶是介导由血小板反应蛋白或腱生蛋白触发的粘着斑解体所必需的，但骨连接蛋白则不需要。在使用各种蛋白激酶抑制剂的实验中，我们观察到环磷酸鸟苷依赖性蛋白激酶的选择性抑制剂KT5823和Rp-8-Br-cGMPS，能阻断血小板反应蛋白1的活性序列（hep I）和腱生蛋白C的可变剪接片段（TNfnA-D）对粘着斑解体的影响。此外，与传代的环磷酸鸟苷依赖性蛋白激酶缺陷细胞不同，早期传代的大鼠主动脉平滑肌细胞具有高水平的环磷酸鸟苷依赖性蛋白激酶，对hep I处理敏感，传代的环磷酸鸟苷依赖性蛋白激酶缺陷细胞对hep I或TNfnA-D处理无反应，但对骨连接蛋白敏感。用PKG Iα的催化结构域转染传代的平滑肌细胞可恢复对hep I和TNfnA-D的反应性。虽然这些研究表明环磷酸鸟苷依赖性蛋白激酶活性是血小板反应蛋白和腱生蛋白介导的粘着斑解体所必需的，但仅激酶活性不足以诱导解体，因为在没有额外刺激的情况下转染激酶的催化结构域不会导致粘着斑丧失。
