FORMATION OF C-CBL-CENTER-DOT-PHOSPHATIDYLINOSITOL 3-KINASE COMPLEXESON LYMPHOCYTE MEMBRANES BY A P56(LCK)-INDEPENDENT MECHANISM

The proto-oncogene c-Cbl was originally identified as a cellular homol ogue of the transforming protein expressed by the murine Cas NS-1 retr ovirus. The full-length c-Cbl protein is a predominantly cytoplasmic p rotein, abundant in lymphoid cells, and potentially involved in signal transduction in several cell types, The specific signal transduction pathways in which c-Cbl participates, and its precise role in these pa thways, are unclear. Previous studies from our laboratory have shown t hat c-Cbl is the predominant tyrosine-phosphorylated protein bound to the p85 subunit of phosphatidylinositol (PI) 3-kinase on T lymphocyte and B lymphocyte activation, To further understand the properties of c -Cbl and the significance of its interactions with PI 3-kinase, we hav e further studied the cellular biological and biochemical responses of c-Cbl to stimulation in lymphoid cells, We show that stimulation indu ces the association of a highly tyrosine-phosphorylated pool of c-Cbl with lymphocyte membranes and with a detergent-insoluble particulate f raction, Immunoprecipitation of c-Cbl from subcellular fractions revea ls that p85 is predominantly associated with the c-Cbl pool recovered from the membrane fraction, despite the fact that this pool represents a small amount of total cellular c-Cbl. The formation of c-Cbl . PI 3 -kinase complexes on lymphocyte membranes did not depend on the cataly tic activity of PI 3-kinase since it was unaltered by the treatment of cells with wortmannin prior to stimulation, Interestingly, c-Cbl tyro sine phosphorylation and the formation of c-Cbl PI 3-kinase complexes were also observed in a mutant Jurkat cell line, JCaM1.6, lacking p56( lck) expression. Because p56(lck) is critical for mitogenic signal tra nsduction in response to T cell receptor activation, our results sugge st that the activation of c-Cbl and the formation of c-Cbl . PI 3-kina se complexes occur upstream or independently of mitogenic signal trans duction pathways in T cells.