Schlaepfer D D, Broome M A, Hunter T
Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Mol Cell Biol. 1997 Mar;17(3):1702-13. doi: 10.1128/MCB.17.3.1702.
The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.
粘着斑激酶（FAK）是一种蛋白酪氨酸激酶（PTK），它与整合素受体相关联，并通过细胞与细胞外基质蛋白（如纤连蛋白（FN））结合而被激活。FAK在酪氨酸397位点的自身磷酸化促进了Src家族PTK的Src同源2（SH2）结构域的结合，而FAK在酪氨酸925位点的c-Src磷酸化则为Grb2 SH2-SH3衔接蛋白创造了一个SH2结合位点。FN刺激的Grb2与FAK的结合可能会促进细胞内信号传导至诸如ERK2-丝裂原活化蛋白激酶等靶点。我们研究了FN刺激的向ERK2的信号传导，发现与稳定重新表达野生型c-Src的Src-成纤维细胞相比，Src-成纤维细胞中ERK2的激活降低了10倍。FN刺激的FAK磷酸酪氨酸（P.Tyr）以及Grb2与FAK的结合减少，而在Src-细胞中未检测到另一种信号蛋白p130cas的酪氨酸磷酸化。在Src-细胞中稳定表达Src的1至298位残基（Src 1-298，其包含c-Src的SH3和SH2结构域）可阻断Grb2与FAK的结合；但令人惊讶的是，与Src-细胞中的水平相比，Src 1-298的表达还导致p130cas P.Tyr水平升高以及FN刺激的ERK2活性增加两到三倍。Src 1-298与FAK和p130cas都结合，并在体内促进FAK与p130cas的结合。在体外观察到FAK可磷酸化p130cas，因此在FN刺激表达Src 1-298的细胞时，FAK可以磷酸化p130cas。FAK诱导的Src 1-298细胞中p130cas的磷酸化促进了Nck衔接蛋白与p130cas的SH2结构域依赖性结合，这可能有助于向ERK2的信号传导。这些结果表明，存在不涉及Grb2与FAK结合的额外的FN刺激的向ERK2的信号传导途径。
