van Hoek M L, Allen C S, Parsons S J
Department of Microbiology, University of Virginia, Charlottesville 22908, USA.
Biochem J. 1997 Aug 15;326 ( Pt 1)(Pt 1):271-7. doi: 10.1042/bj3260271.
Chromaffin cells, which secrete catecholamines in response to acetylcholine, express high levels of the Src-family tyrosine kinases. These kinases contain protein-protein interaction domains which bind signal transduction proteins that participate in a variety of cellular processes. To determine if signalling proteins bind c-Src in chromaffin cells, we examined c-Src immunocomplexes for co-precipitating proteins. We discovered a phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) activity which associates with specific subcellular pools of c-Src in vivo and which preferentially binds the SH2 (Src homology 2) domain of c-Src in vitro. Known PTPases were not identified by blotting of c-Src immunocomplexes with a panel of anti-PTPase antibodies, suggesting that the PTPase may be a novel family member. The c-Src-PTPase complex is enriched in the plasma membrane fraction and exists in several large complexes, as revealed by gel-filtration analysis. This PTPase activity is altered rapidly following stimulation by secretagogues, decreasing within 30 s and returning to basal levels by 60 s of stimulation. Both the subcellular localization and rapid activity changes suggest that the c-Src-associated PTPase may function in early signalling events emanating from the nicotinic acetylcholine receptor. In support of this is the co-precipitation of a PTPase activity with the nicotinic acetylcholine receptor and co-chromatography of this receptor with one or the c-Src-PTPase complexes.
嗜铬细胞可响应乙酰胆碱分泌儿茶酚胺，其Src家族酪氨酸激酶表达水平较高。这些激酶含有蛋白质-蛋白质相互作用结构域，可结合参与多种细胞过程的信号转导蛋白。为了确定信号蛋白是否在嗜铬细胞中与c-Src结合，我们检测了c-Src免疫复合物中共同沉淀的蛋白。我们发现了一种磷酸酪氨酸磷酸酶（PTPase；EC 3.1.3.48）活性，其在体内与c-Src的特定亚细胞池相关联，并且在体外优先结合c-Src的SH2（Src同源2）结构域。用一组抗PTPase抗体对c-Src免疫复合物进行印迹分析未鉴定出已知的PTPase，这表明该PTPase可能是一个新的家族成员。凝胶过滤分析显示，c-Src-PTPase复合物在质膜组分中富集，并以几种大复合物的形式存在。促分泌剂刺激后，这种PTPase活性迅速改变，在30秒内降低，刺激60秒后恢复到基础水平。亚细胞定位和快速的活性变化均表明，与c-Src相关的PTPase可能在烟碱型乙酰胆碱受体引发的早期信号事件中发挥作用。支持这一观点的是，PTPase活性与烟碱型乙酰胆碱受体共同沉淀，且该受体与一种或多种c-Src-PTPase复合物共色谱。
