Graness A, Hanke S, Boehmer F D, Presek P, Liebmann C
Institute of Biochemistry & Biophysics, Biological and Pharmaceutical Faculty, Friedrich-Schiller-University Jena, Philosophenweg 12, D-07743 Jena, Germany.
Biochem J. 2000 Apr 15;347(Pt 2):441-7. doi: 10.1042/0264-6021:3470441.
Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR.
表皮生长因子（EGF）受体（EGFR）的反式激活被认为是多种细胞类型中G蛋白偶联受体与丝裂原活化蛋白激酶（MAPK）途径之间的重要联系。然而，在本研究中我们发现，在A431细胞中，缓激肽可使EGFR反式失活，并独立于EGFR酪氨酸磷酸化刺激MAPK活性。缓激肽的这两种作用均由百日咳毒素不敏感的G蛋白介导。三条证据表明缓激肽可激活蛋白酪氨酸磷酸酶（PTP）：（i）用缓激肽处理A431细胞可降低基础和EGF诱导的EGFR酪氨酸磷酸化，（ii）缓激肽的这种作用可被两种不同的PTP抑制剂阻断，（iii）体外测定时，缓激肽可显著增加A431细胞总裂解物中的PTP活性。跨膜受体PTP sigma被确定为缓激肽诱导的EGFR自磷酸化下调的假定介质。缓激肽刺激后MAPK的激活对EGFR酪氨酸激酶的特异性抑制剂AG 1478不敏感，但被渥曼青霉素或双吲哚马来酰亚胺阻断，分别为磷脂酰肌醇3激酶（PI3-K）和蛋白激酶C（PKC）的抑制剂。这些结果还表明，缓激肽诱导的MAPK激活独立于EGFR，并表明存在一条涉及PI3-K和PKC的途径。此外，缓激肽可引起Src激酶活性的快速短暂增加。虽然Src不参与缓激肽诱导的PTP活性刺激，但4-氨基-5-（4-甲基苯基）-7-（叔丁基）吡唑并（3,4-d）嘧啶对Src的抑制导致缓激肽对MAPK激活的增加。我们的结果表明，在A431细胞中，G（q/11）蛋白偶联的缓激肽B（2）受体可能刺激PTP活性，从而使EGFR反式失活，并可能同时通过可绕过EGFR的替代信号通路激活MAPK。
