Hu X Q, Singh N, Mukhopadhyay D, Akbarali H I
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1998 Feb 27;273(9):5337-42. doi: 10.1074/jbc.273.9.5337.
There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.
越来越多的证据表明，平滑肌收缩以及通过电压依赖性L型钙通道的Ca2+内流受酪氨酸激酶调节；然而，具体涉及的激酶大多未知。在兔结肠肌黏膜细胞中，使用抗磷酸酪氨酸抗体进行免疫印迹时，观察到分子量约为60 kDa和125 kDa的酪氨酸磷酸化蛋白，通过用特异性抗体进行免疫印迹鉴定为c-Src和粘着斑激酶（FAK）。FAK与c-Src共免疫沉淀，血小板衍生生长因子（PDGF）BB显著增强了c-Src.FAK复合物的磷酸化。通过用识别自身磷酸化位点（P416Y）的抗体进行免疫印迹，在细胞裂解物中鉴定出未刺激细胞中存在活化的c-Src。在全细胞膜片钳研究中，Src底物肽以及抗c-Src和抗FAK抗体的细胞内透析分别使Ca2+电流抑制了60%、62%和43%。相比之下，抗小鼠IgG或抗Kv1.5抗体的细胞内透析并未抑制Ca2+电流。抗c-Src和抗FAK抗体的共同透析抑制Ca2+电流（63%），等同于单独使用抗c-Src抗体透析。PDGF-BB使Ca2+电流增强了43%，这被抗c-Src和抗FAK抗体消除。MEK抑制剂PD 098059和抗Ras抗体均未抑制基础Ca2+电流或PDGF刺激的Ca2+电流。L型钙通道的α1C亚基与抗c-Src和抗磷酸酪氨酸抗体共免疫沉淀，表明c-Src激酶与钙通道直接相关。这些数据表明，在分化的平滑肌细胞中，c-Src和FAK而非Ras/丝裂原活化蛋白激酶级联反应调节基础钙通道活性并介导PDGF诱导的L型Ca2+电流增强。
