Zhang Z, Hernandez-Lagunas L, Horne W C, Baron R
Departments of Cell Biology and Orthopaedics and the Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520-8044, USA.
J Biol Chem. 1999 Aug 27;274(35):25093-8. doi: 10.1074/jbc.274.35.25093.
HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.
HEF1是一种最近被描述的类p130(Cas)对接蛋白,它含有一个SH3结构域和多个SH2结合基序。在B细胞中,HEF1通过整合素连接触发的细胞骨架依赖性机制发生磷酸化。然而,尚未有关于G蛋白偶联受体诱导HEF1磷酸化的报道。我们发现,在稳定表达于HEK - 293细胞中的兔C1a降钙素受体受到刺激后,HEF1而非p130(Cas)发生酪氨酸磷酸化。降钙素诱导的HEF1酪氨酸磷酸化呈时间和剂量依赖性增加。二丁酰环磷腺苷和福斯可林对HEF1磷酸化几乎没有影响,蛋白激酶A抑制剂H89也未能显著抑制对降钙素的反应,这表明G(s)/环磷腺苷/蛋白激酶A途径不介导降钙素的作用。选择性阻断G(i/o)信号传导的百日咳毒素也没有作用。用离子霉素增加胞质Ca(2 +)刺激了HEF1磷酸化,而通过BAPTA和细胞外EGTA的组合阻止降钙素诱导的胞质钙变化则完全阻断了降钙素诱导的HEF1酪氨酸磷酸化。佛波酯12 - 肉豆蔻酸酯13 - 乙酸盐也诱导HEF1酪氨酸磷酸化,蛋白激酶C抑制剂钙泊三醇C完全抑制了降钙素和佛波酯12 - 肉豆蔻酸酯13 - 乙酸盐刺激的HEF1磷酸化。降钙素还诱导桩蛋白和粘着斑激酶的酪氨酸磷酸化,以及这两种蛋白与HEF1的结合。用细胞松弛素D预处理破坏肌动蛋白微丝,可阻止降钙素诱导的HEF1和桩蛋白磷酸化。总之,降钙素刺激的HEF1酪氨酸磷酸化由钙和蛋白激酶C依赖性机制介导,并且需要肌动蛋白细胞骨架的完整性。