T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.