Scott G, Liang H
Department of Dermatology, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Exp Cell Res. 1995 Jul;219(1):197-203. doi: 10.1006/excr.1995.1219.
Focal adhesion kinase (pp125FAK) is a nonreceptor tyrosine kinase which colocalizes with integrins to focal contacts, sites where multiple proteins interact to regulate the assembly of the actin cytoskeleton. Autophosphorylation and activation of pp125FAK occur after integrin clustering or cell adhesion to ligands through cognate integrin receptors and are postulated to mediate integrin signaling events. In this report we examined pp125FAK expression and phosphorylation in normal human melanocytes, an adherent human metastatic melanoma cell line (SKMEL28), and a nonadherent human metastatic melanoma cell line (SKMEL1). We show that SKMEL28 cells express constitutively phosphorylated pp125FAK and that pp125FAK phosphorylation in melanocytes is induced by phorbol esters and growth factors present in melanocyte growth medium. Focal adhesion kinase phosphorylation could be enhanced by b1 integrin-activating antibodies in human melanocytes, but not in SKMEL28 cells. In contrast with SKMEL28 cells, constitutive phosphorylation of pp125FAK was not observed in SKMEL1 cells, and incubation with activating b1 integrin antibodies had no effect on pp125FAK phosphorylation. Absence of pp125FAK phosphorylation in SKMEL1 cells was not due to lack of expression of pp125FAK, as shown by immunoprecipitation of the pp125FAK protein from cell lysates. However, b1 integrin expression was significantly less in SKMEL1 cells than in human melanocytes and SKMEL28 cells. This study further supports the importance of integrins in pp125FAK-mediated signaling and indicates that transformation-related changes in pp125FAK phosphorylation exist in human melanocytes and melanoma cells.
粘着斑激酶（pp125FAK）是一种非受体酪氨酸激酶，它与整合素共定位于粘着斑，即多种蛋白质相互作用以调节肌动蛋白细胞骨架组装的位点。pp125FAK的自磷酸化和激活发生在整合素聚集或细胞通过同源整合素受体与配体粘附之后，并被认为介导整合素信号事件。在本报告中，我们检测了正常人黑素细胞、一种贴壁的人转移性黑素瘤细胞系（SKMEL28）和一种非贴壁的人转移性黑素瘤细胞系（SKMEL1）中pp125FAK的表达和磷酸化情况。我们发现SKMEL28细胞组成性表达磷酸化的pp125FAK，并且黑素细胞生长培养基中存在的佛波酯和生长因子可诱导黑素细胞中pp125FAK的磷酸化。人黑素细胞中，β1整合素激活抗体可增强粘着斑激酶的磷酸化，但在SKMEL28细胞中则不然。与SKMEL28细胞相反，在SKMEL1细胞中未观察到pp125FAK的组成性磷酸化，并且用激活的β1整合素抗体孵育对pp125FAK的磷酸化没有影响。如从细胞裂解物中免疫沉淀pp125FAK蛋白所示，SKMEL1细胞中缺乏pp125FAK磷酸化并非由于pp125FAK表达缺失。然而，SKMEL1细胞中β1整合素的表达明显低于人黑素细胞和SKMEL28细胞。本研究进一步支持了整合素在pp125FAK介导的信号传导中的重要性，并表明人黑素细胞和黑素瘤细胞中存在与转化相关的pp125FAK磷酸化变化。
