Reedquist K A, Fukazawa T, Panchamoorthy G, Langdon W Y, Shoelson S E, Druker B J, Band H
Lymphocyte Biology Section, Division of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Apr 5;271(14):8435-42. doi: 10.1074/jbc.271.14.8435.
We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl associates with all three forms of the human Crk protein, predominantly CrkL, following T cell receptor activation of Jurkat T cells. Association between Cbl and Crk proteins was confirmed in normal human peripheral blood-derived T cells. In vitro, Cbl was able to interact with the Crk SH2 domain but not the SH3 domain. A phosphopeptide corresponding to a potential Crk SH2 domain-binding motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk SH2 domain. Anti-Cbl antibody completely immunodepleted the CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the recently described p130cas-related Crk-associated p116 of T cells may be Cbl. Consistent with this possibility, the 4F4 antibody used to characterize the p116 polypeptide cross-reacted with Cbl protein when it was resolved on one- or two-dimensional gels. CrkL was constitutively associated with a substantial amount of the guanine nucleotide exchange protein C3G, and a fraction of the C3G protein was coimmunoprecipitated with Cbl in activated Jurkat T cells. These results suggest the possibility that Cbl may participate in a signaling pathway that regulates guanine nucleotide exchange on small G-proteins in T cells.
我们和其他研究人员最近鉴定出c-cbl原癌基因的蛋白产物Cbl是T细胞活化后早期的酪氨酸激酶底物，并表明Cbl在体内与Src家族酪氨酸激酶、Grb2衔接蛋白以及PI-3激酶的p85亚基形成复合物。在此我们表明，在Jurkat T细胞的T细胞受体激活后，Cbl与人类Crk蛋白的三种形式均相关联，主要是CrkL。在正常人外周血来源的T细胞中证实了Cbl与Crk蛋白之间的关联。在体外，Cbl能够与Crk的SH2结构域相互作用，但不能与SH3结构域相互作用。对应于Cbl中潜在的Crk SH2结构域结合基序（pYDVP）的磷酸肽特异性抑制Cbl与Crk SH2结构域之间的结合。抗Cbl抗体完全免疫沉淀了与CrkL相关的120kDa磷酸酪氨酸多肽，这表明最近描述的T细胞中与p130cas相关的Crk相关p116可能是Cbl。与此可能性一致，用于鉴定p116多肽的4F4抗体在一维或二维凝胶上分离时与Cbl蛋白发生交叉反应。CrkL与大量鸟嘌呤核苷酸交换蛋白C3G组成性相关，并且在活化的Jurkat T细胞中有一部分C3G蛋白与Cbl共免疫沉淀。这些结果提示Cbl可能参与调节T细胞中小G蛋白上鸟嘌呤核苷酸交换的信号通路。
