Fernandez R, Boxer L A, Suchard S J
Department of Pediatrics, University of Michigan, Ann Arbor 48109, USA.
J Immunol. 1997 Dec 1;159(11):5568-75.
Although many of the receptors mediating neutrophil (polymorphonuclear leukocyte (PMN)) adhesion to extracellular matrix (ECM) proteins have been identified, the signal transduction pathways leading to cell spreading are not clearly defined. Studies showing that protein tyrosine phosphorylation increases with PMN adhesion suggest that phosphorylation is critical for adhesion-dependent processes. In other cells, increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin are thought to play pivotal roles in ECM-mediated signaling. In the present study we compared the tyrosine phosphorylation of FAK and paxillin in PMNs plated onto thrombospondin (TSP) or laminin (LN). Increased tyrosine phosphorylation of paxillin correlated with adhesion and spreading of PMNs plated onto LN or TSP. In PMNs adherent to LN, the kinetics of FAK phosphorylation preceded the kinetics of paxillin phosphorylation, suggesting that paxillin may be a downstream target of FAK. In contrast, FAK was not phosphorylated in PMNs spread on TSP. These findings suggested activation of different signaling pathways in TSP- vs LN-adherent PMNs. Others have proposed that paxillin phosphorylation requires beta2 integrins. Therefore, we used PMNs from a patient with leukocyte adhesion deficiency (LAD), lacking beta2 integrins, to determine whether these receptors were necessary for paxillin phosphorylation. LAD PMNs did not adhere to LN, and consequently, there was no tyrosine phosphorylation of FAK or paxillin. In contrast, paxillin, but not FAK, was phosphorylated in LAD PMNs adherent and spread on TSP. These results indicate that ECM-beta2 integrin-mediated signaling initiates the FAK/paxillin signaling pathway(s), while TSP-mediated signaling results in paxillin phosphorylation independent of FAK phosphorylation.
尽管介导中性粒细胞（多形核白细胞，PMN）与细胞外基质（ECM）蛋白黏附的许多受体已被鉴定，但导致细胞铺展的信号转导途径尚不清楚。研究表明，随着PMN黏附，蛋白酪氨酸磷酸化增加，这表明磷酸化对于依赖黏附的过程至关重要。在其他细胞中，黏着斑激酶（FAK）和桩蛋白酪氨酸磷酸化增加被认为在ECM介导的信号传导中起关键作用。在本研究中，我们比较了接种到血小板反应蛋白（TSP）或层粘连蛋白（LN）上的PMN中FAK和桩蛋白的酪氨酸磷酸化情况。接种到LN或TSP上的PMN中，桩蛋白酪氨酸磷酸化增加与细胞黏附和铺展相关。在黏附于LN的PMN中，FAK磷酸化动力学先于桩蛋白磷酸化动力学，这表明桩蛋白可能是FAK的下游靶点。相比之下，铺展在TSP上的PMN中FAK未被磷酸化。这些发现提示，在TSP黏附与LN黏附的PMN中激活了不同的信号通路。其他人提出桩蛋白磷酸化需要β2整合素。因此，我们使用了一名白细胞黏附缺陷（LAD）患者缺乏β2整合素的PMN，以确定这些受体对于桩蛋白磷酸化是否必要。LAD PMN不黏附于LN，因此，FAK或桩蛋白没有酪氨酸磷酸化。相比之下，黏附并铺展在TSP上的LAD PMN中，桩蛋白而非FAK被磷酸化。这些结果表明，ECM-β2整合素介导的信号传导启动了FAK/桩蛋白信号通路，而TSP介导的信号传导导致桩蛋白磷酸化，且不依赖于FAK磷酸化。
