von Willebrand M, Baier G, Couture C, Burn P, Mustelin T
Division of Cell Biology, Institute for Allergy and Immunology, La Jolla, CA 92037.
Eur J Immunol. 1994 Jan;24(1):234-8. doi: 10.1002/eji.1830240137.
Activation of resting T lymphocytes by ligands to the T cell receptor (TcR)/CD3 complex is initiated by phosphorylation of a number of key regulatory proteins on specific tyrosine residues. One such protein is the heterodimeric enzyme phosphatidylinositol-3-kinase (PI3K). We recently found that this enzyme is also rapidly activated following TcR/CD3 triggering and that immunoprecipitated PI3K was activated in vitro by direct tyrosine phosphorylation. Here we show that TcR/CD3-induced tyrosine phosphorylation and activation of PI3K in Jurkat T leukemia cells depend on the presence of the p56lck tyrosine kinase: in a variant of the Jurkat T cell line lacking p56lck, JCaM1, these responses were absent. We also show that p56lck directly activates PI3K purified from transfected COS-1 cells, indicating that other T cell-specific proteins are not required for the process. Finally, tryptic peptide maps show that p56lck phosphorylates three tyrosine residues in the p85 alpha subunit of PI3K and two in p110 of PI3K. Our results suggest that p56lck is required for activation of PI3K in Jurkat T cells and can itself directly activate it by phosphorylating one or several stimulatory sites.
通过T细胞受体（TcR）/CD3复合物的配体激活静息T淋巴细胞，是由一些关键调节蛋白在特定酪氨酸残基上的磷酸化引发的。其中一种蛋白是异二聚体酶磷脂酰肌醇-3激酶（PI3K）。我们最近发现，该酶在TcR/CD3触发后也会迅速被激活，并且免疫沉淀的PI3K在体外通过直接酪氨酸磷酸化而被激活。在此我们表明，在Jurkat T白血病细胞中，TcR/CD3诱导的PI3K酪氨酸磷酸化和激活依赖于p56lck酪氨酸激酶的存在：在缺乏p56lck的Jurkat T细胞系变体JCaM1中，这些反应不存在。我们还表明，p56lck直接激活从转染的COS-1细胞中纯化的PI3K，这表明该过程不需要其他T细胞特异性蛋白。最后，胰蛋白酶肽图显示，p56lck使PI3K的p85α亚基中的三个酪氨酸残基和PI3K的p110中的两个酪氨酸残基磷酸化。我们的结果表明，p56lck是Jurkat T细胞中PI3K激活所必需的，并且它本身可以通过磷酸化一个或几个刺激位点直接激活PI3K。
