Maa M C, Leu T H, Trandel B J, Chang J H, Parsons S J
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.
Mol Cell Biol. 1994 Aug;14(8):5466-73. doi: 10.1128/mcb.14.8.5466-5473.1994.
p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.
p62是一种高度酪氨酸磷酸化的蛋白质，最初是在由致癌非受体酪氨酸激酶转化或通过酪氨酸激酶受体刺激的细胞中，从p21ras的GTP酶激活蛋白（GAP）的免疫沉淀物中鉴定出来的（C.埃利斯、M.莫兰、F.麦考密克和T.鲍森，《自然》343:377 - 381，1991）。在本文中，我们描述了一种高度相关的62 kDa蛋白质，在用表皮生长因子（EGF）刺激或被v - src转化的C3H10T1/2细胞中，它会发生酪氨酸磷酸化并与磷脂酶Cγ（PLCγ）结合。与GAP相关的p62和与PLCγ相关的p62在一维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中迁移率相同，并且呈现出几乎相同的磷酸化胰蛋白酶肽图谱。通过以下几点证明了p62与PLCγ的结合是直接的，并非通过GAP - p62与PLCγ或EGF受体的结合（以及受体与PLCγ的共沉淀）介导：（i）在PLCγ免疫复合物中无法检测到GAP，在GAP免疫复合物中也无法检测到PLCγ；（ii）在没有EGF刺激的情况下，v - src转化细胞中p62与PLCγ的结合；（iii）p62与含有PLCγ的Src同源2（SH2）结构域的谷胱甘肽S - 转移酶融合蛋白的体外溶液结合和直接印迹。与GAP不同，GAP的N端SH2介导GAP与p62之间的相互作用，而发现PLCγ需要其N端和C端SH2区域才能与p62结合。这些研究表明，一种与GAP相关的p62相同或高度相关的蛋白质与PLCγ结合，并提示了一种可能发生在PLCγ和GAP介导的信号传导之间“串扰”的方式。
