A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (Fc⑀RI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P 3 production. Using immune complex phospholipase assays, we show that Fc⑀RI cross-linking activates both PLC␥1 and PLC␥2. Activation is accompanied by the increased phosphorylation of both PLC␥ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLC␥ isoforms have distinct subcellular localizations. PLC␥1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLC␥1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLC␥2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after Fc⑀RI cross-linking. The activation of PLC␥1, but not of PLC␥2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P 3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLC␥1 with little inhibition of PLC␥2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLC␥1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLC␥1. Although less abundant than PLC␥2, activated PLC␥1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P 3 production in RBL-2H3 cells. INTRODUCTIONIn mast cells and basophils, cross-linking the high affinity cell surface receptors for IgE (Fc⑀RI) 1 activates the tyrosine kinases Lyn and Syk (Eiseman and Bolen, 1992;Hutchcroft et al., 1992) and initiates a signaling cascade that leads to the secretion of histamine and other granule constituents, to changes in adhesive properties, cell shape, and surface topography and to the de novo synthesis of lipid mediators and cytokines (reviewed in Beaven & Metzger, 1993;Oliver et al., 1997). Tyrosine kinase activation by the Fc⑀RI and related members of the multisubunit immunoreceptor family, which includes the T cell antigen receptor, the B cell antigen receptor, and several Fc␥ receptors, depends on immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of individual receptor subunits (Cambier, 1995). The heterotrimeric (␣␤␥ 2 ) Fc⑀RI of RBL-2H3 mast cells contains ITAM motifs in both the ␥ and ␤ subunit cytoplasmic domains (Metzger, 1992 to ITAM phosphorylation and Syk activation by its association with the ␥ subunit phospho-ITAM. Syk activation, resulting in the phosphorylation of multiple protein substrates, in turn initiates the signaling cascade Oliver et al., 1994;Wilson et al., 1995;Rivera and Brugge, 1995). Pharmacological studies have established that Sykdependent tyrosine phosphorylation is required for the antigen-stimulated synthesis of inositol (1,4,5) trisphosphate (I...

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (FcER1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, FcER1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27-28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30 -40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short halflife. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.G proteins transduce signals across the plasma membrane by sequential interactions with cell surface receptors and appropriate second messenger-producing effectors (e.g. enzymes and ion channels). These interactions are modulated by nucleotide-driven conformational changes in the ␣ subunits of heterotrimeric G proteins (G␣).1 A ligand-bound receptor catalyzes the exchange of GDP for GTP on its cognate G␣ and the dissociation of G␣ from the complex of G protein ␤ and ␥ subunits (G␤␥). These dissociated subunits are competent to modulate the activity of effectors. The duration of G protein-mediated responses are dependent on the intrinsic GTPase rate of G␣ and on extrinsic factors, such as regulators of G protein signaling (RGS proteins).RGS proteins serve to regulate G protein signaling by functioning as GTPase-activating proteins (GAPs). GAP activity can sharpen the termination of a signal upon removal of a stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network ...

Cross-linking the IgE-bound Fc⑀RI with polyvalent antigen leads to Ca 2؉ -dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of Fc⑀RI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLC␥ activity and is demonstrated by a dramatic inhibition of Fc⑀RI-induced Ins(1,4,5)P 3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P 3 production in at least two ways: by promoting translocation and phosphorylation of PLC␥1 and by direct stimulation of both PLC␥ isoforms. In vitro stimulation of PLC␥ activity by PtdIns(3,4,5)P 3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca 2؉ influx. Compared with control cells, Ca 2؉ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca 2؉ after antigen and an inhibition of the sustained Ca 2؉ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca 2؉ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P 3 is synthesized in wortmannintreated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca 2؉ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca 2؉ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase. J. Leukoc. Biol. 65: 321-329; 1999.