T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.

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Abstract. Previously we have shown that PDGF receptor mutants that do not bind PI-3 kinase internalize after ligand binding, but fail to downregulate and degrade. To define further the role of PI-3 kinase in trafficking processes in mammalian cells, we have investigated the effects of a potent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar concentrations, wortmannin inhibited both the transfer of PDGF receptors from peripheral compartments to juxtanuclear vesicles, and their subsequent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in perinuclear vesicles after 120 min of incubation, was not blocked by wortmannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Thus, the effects of wortmannin on PDGFr trafficking are much more pronounced than its effects on other endocytic events. Unexpectedly, wortmannin also caused a striking effect on the morphology of endosomal compartments, marked by tubulation and enlargement of endosomes containing transferrin or LY. This effect was somewhat similar to that produced by brefeldin A, and was also blocked by pre-treatment of cells with aluminum fluoride (A1F4-). These results suggest two sites in the endocytic pathway where PI-3 kinase activity may be required: (a) to sort PDGF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing lysosomally directed and recycling molecules. This latter function could be mediated through the activation of A1F4--sensitive GTPbinding proteins downstream of PI-3 kinase. p l-3 kinase was first discovered as an activity in immunoprecipitates of receptor and nonreceptor tyrosine kinases that phosphorylated the 3' position of the inositol ring in Pins, Plns(4)P, and Plns(4,5)P2 (37). The association of this lipid kinase activity with receptors that stimulate cellular growth suggested a role for PI-3 kinase in mitogenic signaling (3). More recently, the existence of several biochemically distinct PI-3 kinases in mammalian cells has been reported (32, 33), and PI-3 kinase activity has been implicated in numerous cellular processes including membrane ruffling, chemotaxis, trafficking of membrane proteins, and activation of kinases such as protein kinase C (reviewed in reference 19). Thus, PI-3 kinases in mammalian cells have emerged as a diverse family of proteins that are likely to be involved in multiple essential cellular processes.In yeast, deletions or mutations in the VPS34 gene, which encodes for the only detectable PI-3 kinase activity Address all correspondence to S. Corvera, Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01655. Tel.: (508) 856-6898. Fax: (508) 856-4289. E-mail: Scorvera@bangatel.ummed.edu in these ceUs (31), lead to a dramatic alteration in the sorting of newly synthesized pr...

In Drosophila, the very first steps in neurogenesis appear to be controlled by a small group of zygotically acting genes termed the neurogenic loci. Mutations in any of these genes result in a misrouting of epidermal lineages into the neural pathway. Morphological and molecular studies suggest that the correct ectodermal differentiation is mediated by a cell‐cell interaction mechanism and that at least some of the neurogenic loci are involved in this mechanism. The molecular analyses of the neurogenic loci Notch and Delta revealed that the putative gene products are large transmembrane proteins with homology to mammalian epidermal growth factor. We describe here a molecular analysis of Enhancer of split [E(spl)], a third neurogenic locus, which displays striking genetic interactions with both Notch and Delta, suggesting a close functional relationship of the respective gene products. We provide evidence for a single genetic complementation group corresponding to a single transcription unit which is necessary for wild‐type E(spl) function. P‐element‐mediated transformation indicates that this transcription unit includes functions associated with both the dominant E(spl)D mutation and the recessive visible allele groucho, and is necessary for the correct differentiation of the embryonic nervous system.

Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Here, we demonstrate that antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLC gamma. Furthermore, antibodies specific for the beta isoform of p85 but not antibodies specific for the alpha isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-cbl immunoprecipitates. Thus, pp120 appears to correspond to c-cbl. A direct association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85, and this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus, these results show important functional differences between the alpha and beta isoforms of p85 in vivo and point to c-cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells.