Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.T lymphocyte activation is triggered by interaction between the T cell antigen receptor (TCR) 1 and its cognate antigen. One of the earliest signaling events following TCR stimulation is the rapid increase in tyrosine phosphorylation of a number of proteins (reviewed in Refs. 1 and 2). Unlike growth factor receptors that have intrinsic tyrosine kinase activity (reviewed in Ref. 3), the TCR components transduce their signals through noncovalently associated cytoplasmic tyrosine kinases (1, 2). Three tyrosine kinases, Lck, Fyn, and ZAP-70, have been implicated in the function of the TCR. Identification of substrates for these tyrosine kinases has been an area of intense investigation. Several tyrosine kinase substrates in T cells have been identified in the past few years, including phospholipase C-␥ (4, 5), the guanine nucleotide exchange factor Vav (6 -8), an oligomeric ATPase valosin-containing protein (9, 10), the membrane-cytoskeleton linker protein ezrin (11,12), and the subunit of TCR (13). Recently, c-Cbl, a protooncogene protein of 120 kDa has been identified as another prominent tyrosine kinase substrate in T cells (14). Although it has been demonstrated that c-Cbl becomes rapidly tyrosine-phosphorylated upon TCR stimulation (14) and forms complexes with several signaling molecules (15)(16)(17)(18)(19), the function of c-Cbl in T cells is not clear.T cell activation can be mimicked by the addition of tumorpromoting phorbol esters plus calcium ionophores, suggesting important roles for protein kinase C (PKC) and calcium (20). The role of PKC in T cell activation is further emphasized by studies showing that induction of the transcription factors AP-1, NF-B, and nuclear factor of activated T cell for cytokine gene expression is regulated by PKC (21). PKC is ...