Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin.
Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin.
复制标题
大鼠肠细胞系中 pp62 和 pp54 src 样蛋白响应胃泌素的磷酸化。
DOI：
10.1152/ajpgi.1994.267.2.g235
复制
发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Adiga,RB
中科院分区：
文献类型：
--
作者：
Singh,P;Narayan,S;Adiga,RB
调节胃泌素促有丝分裂作用的细胞内机制在很大程度上仍不清楚。本研究旨在探讨蛋白酪氨酸激酶(PTKs)是否介导胃泌素对大鼠肠上皮细胞系(IEC-6细胞)的生长影响。胃泌素(10 NM)对IEC-6细胞有丝分裂作用。0.01-10.0 nM和0.05-10.0微米胃泌素以双相方式刺激细胞膜PTK活性。用胃泌素刺激H3(32)PO4标记的细胞，用磷酸酪氨酸抗体免疫沉淀细胞蛋白。内源性蛋白质以剂量(100%有效剂量=0.1-1.0 nM)和时间依赖的方式磷酸化；在胃泌素10 nM时，在完整细胞中未检测到第二反应峰。因此，完整细胞对胃泌素的生长和磷酸化反应是相似的。IEC-6细胞上存在高[解离常数(Kd)=1 nM]和低(Kd=约0.1微米)亲和力的胃泌素结合位点。本研究的结果表明，占据高亲和力和低亲和力的胃泌素结合位点都可以潜在地激活膜相关的PTK。然而，在完整的细胞中，低亲和力位点的占据明显减弱了激酶的活性，从而降低了蛋白质的磷酸化。完整细胞对0.1-1.0 nM胃泌素有8条蛋白质带[相对分子质量(M(R))为32-145 kDa]发生酪氨酸磷酸化，其中包括2条pp60src样蛋白(M(R)为54 kDa和62 kDa)。因此，靶细胞对胃泌素的生长反应模式可能取决于激酶和其他因素(磷酸酶？)的刺激。以剂量依赖的方式磷酸化和/或去磷酸化几种蛋白质，包括c-src样蛋白。
Intracellular mechanisms that mediate mitogenic effects of gastrin remain largely unknown. The present studies were designed to examine if protein tyrosine kinases (PTKs) mediate growth effects of gastrin on a rat intestinal epithelial cell line (IEC-6 cells). Gastrin (< 10 nM) was mitogenic for IEC-6 cells. PTK activity of cell membranes was stimulated in response to 0.01-10.0 nM and 0.05-10.0 microM gastrin in a double biphasic manner. Cells labeled with H3(32)PO4 were stimulated with gastrin and cellular proteins immunoprecipitated with phosphotyrosine antibodies. Endogenous proteins were phosphorylated in a dose- (100% effective dose = 0.1-1.0 nM) and time-dependent manner; at > 10 nM gastrin, the second peak of response was not measured in intact cells. Thus the growth and phosphorylation response of intact cells to gastrin was similar. Both high [dissociation constant (Kd) = 1 nM]- and low (Kd = approximately 0.1 microM)-affinity gastrin binding sites are present on IEC-6 cells. The results of the present study suggest that occupancy of both high- and low-affinity gastrin-binding sites can potentially activate membrane-associated PTKs. However, in intact cells, occupancy of low-affinity sites apparently attenuates kinase activity resulting in reduced protein phosphorylation. Eight protein bands [with relative molecular weight (M(r)) of 32-145 kDa] were tyrosine phosphorylated in intact cells in response to 0.1-1.0 nM gastrin, including two pp60 src-like proteins (with M(r) of 54 and 62 kDa). Thus the growth response pattern of a target cell to gastrin may depend on the stimulation of kinases and other factors (phosphatases?) that phosphorylate and/or dephosphorylate several proteins including c-src-like proteins in a dose-dependent manner.