The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

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Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.

SummaryTyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (FceR1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of FcetLIinduced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Sykdependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.

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The synthetic lactyl anhydride isobutylcarbonyl lactyl anhydride (iBCLA), a selective and potent inhibitor of L-(+)-lactate transport in rabbit erythrocytes, reduces the chemical labeling of a 40-50-kdalton polypeptide by tritiated 4,4'-diisothiocyanato-2,2'-dihydrostilbenedisulfonate ([3H]H2DIDS). iBCLA does so in a dose-dependent manner at concentrations that strongly inhibit lactate lactate exchange but not chloride-phosphate exchange. These labeling experiments and inhibition reversal studies using iBCLA, p-(chloro-mercuri)benzenesulfonic acid (pCMBS), and dithiothreitol (DDT) suggest that iBCLA does not act at sulfhydryl groups but at or near an amino group that is near a disulfide linkage in the polypeptide which catalyzes lactate transport. These experiments support the association between specific monocarboxylate transport and a 40-50-kdalton membrane-bound polypeptide of the rabbit erythrocyte.

CD45 is a major transmembrane glycoprotein expressed on all nucleated hematopoietic cells. Eight isoforms of CD45 are distributed through the immune system according to cell type and degree of cellular differentiation. Heterogeneity among the isoforms is found entirely in the extracellular domain, arising from the differential splicing of up to four exons of a single gene. The control of isoform expression suggests that the extracellular domain may participate in protein-protein interactions with isoform-specific ligands. The intracellular domain of CD45 is large (approximately 700 amino acids), identical for all isoforms, and highly conserved across species. Two nonidentical intracellular sequences of about 240 amino acids that are homologous with a tyrosine phosphatase consensus sequence have been identified. Studies with purified CD45 have shown that all isoforms possess enzymatic activity in in vitro assays. In several T and B cell lines and in natural killer cells, it appears that CD45 is required for optimal signal transduction after stimulation through a number of surface receptors. Although an in vivo substrate has not been identified conclusively, one model suggests that CD45 functions to dephosphorylate a negative-regulatory tyrosine residue on one or more protein tyrosine kinases involved in receptor-mediated second messenger formation. In T cells, the src family kinases, lck and fyn, are candidates for this regulated kinase. In this review, some of the structural and functional aspects of CD45 and its role in signal transduction in the immune system are discussed.

Esters of N-hydroxysulfosuccinimide strongly inhibit L-(+)-lactate transport in rabbit erythrocytes, probably by acylating amino groups on the transport protein. Lactate transport studies using bis(sulfosuccinimido) suberate (BS3), bis(sulfosuccinimido) adipate (BS2A), bis(sulfosuccinimido) dithiobis(propionate), and a variety of monocarboxylate esters suggest that an exofacial amino group of the lactate transport protein is essential for lactate transport. Also, reductive methylation studies show that even when positive charge is preserved in modified amino groups, the transport is strongly inhibited. At pH less than 6, band 3 mediated inorganic anion transport is enhanced in BS3-treated cells, while at pH greater than 6, it is inhibited. BS3-induced inhibition of L-(+)-lactate transport does not have this pH dependence. BS3 reduces the labeling of a 40-50-kDa membrane polypeptide (band R) by tritiated 4,4'-diisothiocyanato-2,2-dihydrostilbenedisulfonate ([3H]H2DIDS) and by tritiated bis(sulfosuccinimido) adipate ([3H]BS2A). Tritiated sulfosuccinimido acetate (S2[3H]acetate) also labels band R, over a range of concentrations where lactate transport is inhibited in a dose-dependent manner by S2 acetate. BS3 is a known impermeant protein cross-linker. S2 acetate permeates rabbit red cell membranes by an H2DIDS-inhibitable mechanism. BS3 cross-links the proteolytic fragments of rabbit band 3 produced by extracellular chymotrypsin. These labeling experiments support an association between band R and specific monocarboxylate transport.