Haas Michael, Wang Haojie, Tian Jiang, Xie Zijian
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614, USA.
J Biol Chem. 2002 May 24;277(21):18694-702. doi: 10.1074/jbc.M111357200. Epub 2002 Mar 20.
Binding of ouabain to Na(+)/K(+)-ATPase activates tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), Src, and p42/44 mitogen-activated protein kinases (MAPKs) in both cardiac myocytes and A7r5 cells. Here, we explored the roles of Src and the EGFR in the ouabain-invoked pathways that lead to the activation of MAPKs. Exposure of A7r5 and LLC-PK1 cells to ouabain caused a dose-dependent inhibition of Na(+)/K(+)-ATPase activity, which correlated well with ouabain-induced activation of Src and MAPKs in these cells. Immunoprecipitation experiments showed that ouabain stimulated Src binding to Na(+)/K(+)-ATPase in a dose- and time-dependent manner and increased phosphorylation of Src at Tyr(418) but had no effect on Tyr(529) phosphorylation. Ouabain failed to activate MAPKs in A7r5 cells that were pretreated with the Src inhibitor PP2 and in SYF cells in which Src family kinases are knocked out. Preincubation with AG1478, but not AG1295, also blocked the effects of ouabain on p42/44 MAPKs in A7r5 cells. Significantly, both herbimycin A and PP2 abrogated ouabain-induced but not epidermal growth factor-induced Src binding to the EGFR and the subsequent EGFR tyrosine phosphorylation. Ouabain also failed to affect tyrosine phosphorylation of the EGFR in SYF cells. In addition, unlike epidermal growth factor, ouabain did not increase EGFR autophosphorylation at Tyr(1173). These findings clearly indicate that ouabain transactivates the EGFR by activation of Src and stimulation of Src binding to the EGFR. Furthermore, we found that the transactivated EGFR was capable of recruiting and phosphorylating the adaptor protein Shc. This resulted in increased binding of another adaptor protein Grb2 to the Src-EGFR complex and the subsequent activation of Ras and MAPKs. Taken together, these new findings suggest that Src mediates the inter-receptor cross-talk between Na(+)/K(+)-ATPase and the EGFR to transduce the signals from ouabain to the Ras/MAPK cascade.
哇巴因与钠钾ATP酶的结合可激活心肌细胞和A7r5细胞中表皮生长因子受体（EGFR）、Src以及p42/44丝裂原活化蛋白激酶（MAPK）的酪氨酸磷酸化。在此，我们探究了Src和EGFR在哇巴因引发的导致MAPK激活的信号通路中的作用。将A7r5和LLC - PK1细胞暴露于哇巴因会导致钠钾ATP酶活性呈剂量依赖性抑制，这与哇巴因诱导这些细胞中Src和MAPK的激活密切相关。免疫沉淀实验表明，哇巴因以剂量和时间依赖性方式刺激Src与钠钾ATP酶结合，并增加Src在Tyr(418)位点的磷酸化，但对Tyr(529)位点的磷酸化没有影响。哇巴因未能在预先用Src抑制剂PP2处理的A7r5细胞以及Src家族激酶被敲除的SYF细胞中激活MAPK。用AG1478而非AG1295预孵育也可阻断哇巴因对A7r5细胞中p42/44 MAPK的作用。重要的是，除草霉素A和PP2都消除了哇巴因诱导的而非表皮生长因子诱导的Src与EGFR的结合以及随后的EGFR酪氨酸磷酸化。哇巴因也未能影响SYF细胞中EGFR的酪氨酸磷酸化。此外，与表皮生长因子不同，哇巴因不会增加EGFR在Tyr(1173)位点的自身磷酸化。这些发现清楚地表明，哇巴因通过激活Src并刺激Src与EGFR的结合来反式激活EGFR。此外，我们发现反式激活的EGFR能够募集并磷酸化衔接蛋白Shc。这导致另一种衔接蛋白Grb2与Src - EGFR复合物的结合增加，随后激活Ras和MAPK。综上所述，这些新发现表明Src介导钠钾ATP酶与EGFR之间的受体间串扰，以将哇巴因的信号转导至Ras/MAPK级联反应。
