Galandrini R, Palmieri G, Piccoli M, Frati L, Santoni A
Department of Experimental Medicine and Pathology, University La Sapienza, Rome, Italy.
J Exp Med. 1996 Jan 1;183(1):179-86. doi: 10.1084/jem.183.1.179.
The Src homology (SH) 2/SH3 domain-containing protein Grb2 and the oncoprotein Shc have been implicated in a highly conserved mechanism that regulates p21ras activation. We investigated the involvement of these adaptor proteins in the signaling pathway induced by CD16 or interleukin (IL) 2R triggering in human natural killer (NK) cells. Both p46 and p52 forms of Shc were rapidly and transiently tyrosine phosphorylated upon CD16 or IL-2 stimulation with different kinetics. Shc immunoprecipitates from lysates of CD16- or IL-2-stimulated NK cells contained Grb2 and an unidentified 145-kD tyrosine phosphoprotein. Grb2 immunoprecipitates from anti-CD16-stimulated NK cells contained not only Shc, but also a 36-kD tyrosine phosphoprotein (p36). The interaction between Grb2 and Shc or p36 occurred via the Grb2SH2 domain as indicated by in vitro binding assays using a bacteriologically synthesized glutathione S-transferase-Grb2SH2 fusion protein. We also present evidence that p21ras is activated by CD16 and IL-2R cross-linking. Accumulation of guanosine triphosphate-bound Ras was detected within 1 minute and occurred with kinetics similar to inductive protein tyrosine phosphorylation and Grb2 association of Shc and p36 adaptor proteins.
含有Src同源（SH）2/SH3结构域的蛋白Grb2和癌蛋白Shc参与了一种高度保守的调节p21ras激活的机制。我们研究了这些衔接蛋白在人自然杀伤（NK）细胞中由CD16或白细胞介素（IL）-2受体触发所诱导的信号通路中的作用。在用不同动力学的CD16或IL-2刺激后，Shc的p46和p52形式均迅速且短暂地发生酪氨酸磷酸化。从CD16或IL-2刺激的NK细胞裂解物中免疫沉淀得到的Shc含有Grb2和一种未鉴定的145-kD酪氨酸磷酸化蛋白。从抗CD16刺激的NK细胞中免疫沉淀得到的Grb2不仅含有Shc，还含有一种36-kD酪氨酸磷酸化蛋白（p36）。如使用细菌合成的谷胱甘肽S-转移酶-Grb2SH2融合蛋白进行的体外结合试验所示，Grb2与Shc或p36之间的相互作用是通过Grb2的SH2结构域发生的。我们还提供了证据表明p21ras可被CD16和IL-2R交联激活。在1分钟内检测到结合鸟苷三磷酸的Ras的积累，其动力学与诱导性蛋白酪氨酸磷酸化以及Shc和p36衔接蛋白与Grb2的结合相似。
