Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.     

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.     

Regulation of phospholipase C-gamma activity by glycosphingolipids

Glycosphingolipid-enriched domains are hot spots for cell signaling within plasma membranes and are characterized by the enrichment of glycosphingolipids. A role for glucosylceramide-based glycosphingolipids in phospholipase C-mediated inositol 1,4,5-trisphosphate formation has been previously documented. These earlier studies utilized a first generation glucosylceramide synthase inhibitor to deplete cells of their glycosphingolipids. Recently, more active and specific glucosylceramide synthase inhibitors, including d-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol (d-t-EtDO-P4), have been designed. d-t-EtDO-P4 has the advantage of blocking glucosylceramide synthase at low nanomolar concentrations but does not cause secondary elevations in cell ceramide levels. In the present study, d-t-EtDO-P4 depleted cellular glucosylceramide and lactosylceramide in cultured ECV304 cells at nanomolar concentrations without obvious cellular toxicity. The expression of several signaling proteins was evaluated in glycosphingolipid-depleted ECV304 cells to study the role of glycosphingolipids in phospholipase C-mediated signaling. No difference was observed in the cellular expression of phospholipase C-gamma between controls and glycolipid-depleted cells. Western blot analysis, however, revealed that depletion of endogenous glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 induced tyrosine phosphorylation of phospholipase C-gamma in a concentration-dependent manner with maximum induction at 100 nm. The phosphorylation of phospholipase C-gamma induced by d-t-EtDO-P4 was abolished by exogenously added glucosylceramide, consistent with a specific glycosphingolipid-phospholipase C-gamma interaction. The phospholipase C-gamma phosphorylation was maximally enhanced by bradykinin when cells were exposed to 100 nm d-t-EtDO-P4. The measurement of cellular activity of phospholipase C-gamma, by myo-inositol 1,4,5-trisphosphate radioreceptor assay, demonstrated that depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 resulted in significantly increased formation of inositol 1,4,5-trisphosphate above base line, and an increased sensitivity of phospholipase C-gamma to bradykinin stimulation. Thus, the activation of phospholipase C-gamma is negatively regulated by membrane glycosphingolipids in ECV304 cells.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

IL-7 receptor mediates tyrosine phosphorylation but does not activate the phosphatidylinositol-phospholipase C-gamma 1 pathway.

IL-7 is a glycoprotein involved in the regulation of lymphocyte precursor growth. In addition, it has a comitogenic effect on mature T cells but not on mature B cells. The exact mechanism whereby IL-7R mediates these cell growth properties remains unknown. Because many growth factor receptor systems on various cell types transduce signals by activating a tyrosine kinase, we have studied here the effect of IL-7R ligation on protein tyrosine phosphorylation. We found that human rIL-7 consistently induced tyrosine phosphorylation of five major proteins, of 175, 155, 135, 110, and 85 kDa, and five minor proteins. The effect of human rIL-7 on tyrosine phosphorylation of these substrates was concentration and time dependent. One of the known substrates that is phosphorylated on tyrosine residues after binding of growth factors to their receptors is the phosphoinositide-specific phospholipase C. Several phospholipase C isozymes have been recently recognized; one isozyme, phospholipase C-gamma 1, was demonstrated to be phosphorylated rapidly after ligand binding to the platelet-derived growth factor receptor and the T cell Ag receptor. We show here that, in contrast to Ag receptor ligation, activation of IL-7R does not induce tyrosine phosphorylation on phospholipase C-gamma 1. Consistent with these results, human rIL-7 failed to increase phosphatidylinositol turnover and did not induce a rise in cytosolic free Ca2+ in the thymocytes, mature T cells, or pre-pre-B cells. The results indicate that the IL-7R mediates the activation of the tyrosine phosphorylation pathway but does not induce the phosphatidylinositol-phospholipase C pathway.     

Src kinase mediates the regulation of phospholipase C-gamma activity by glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.     

CCK causes PKD1 activation in pancreatic acini by signaling through PKC-delta and PKC-independent pathways

Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependent increase in serine phosphorylation by activation of high- and low-affinity CCK(A) receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-zeta pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-delta, but not PKC-epsilon, or treatment with PKC-delta translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCK(A) receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.     

Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.     

Inositol phospholipids-specific phospholipase C: interaction of the gamma 1 isoform with tyrosine kinase.

The generation of second messengers from inositol phospholipids is catalysed by enzymes from the phospholipase C family. Activation of phospholipase C-gamma 1 through tyrosine phosphorylation provides a link between mitogenic and inositol phospholipid signaling.     

Overexpression of phospholipase C-gamma in NIH 3T3 fibroblasts results in increased phosphatidylinositol hydrolysis in response to platelet-derived growth factor and basic fibroblast growth factor.

Overexpression of phospholipase C-gamma in fibroblasts led to increased tyrosine phosphorylation of this enzyme in response to platelet-derived growth factor and basic fibroblast growth factor. This correlated with increased phosphoinositide release but not with enhanced mitogenicity. Thus, phospholipase C-gamma-mediated phosphoinositide metabolism may not be limiting in the signaling pathways initiated by these growth factors.     

Autophosphorylation of the intracellular domain of the epidermal growth factor receptor results in different effects on its tyrosine kinase activity with various peptide substrates. Phosphorylation of peptides representing Tyr(P) sites of phospholipase C-gamma.

The effect of autophosphorylation on the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is not well understood. We previously demonstrated that phospholipase C-gamma physically associates with the EGF-activated EGFR, but not with a kinase-negative mutant of the EGFR, and, moreover, that only the tyrosine-phosphorylated EGFR is able to associate with phospholipase C-gamma. We have now investigated the effect of autophosphorylation on the tyrosine kinase activity of the EGFR by employing the purified kinase-active intracellular domain of the EGFR (EGFR-IC) produced by a baculovirus expression system. Synthetic peptides, including ones which contain the individual major tyrosine phosphorylation sites of phospholipase C-gamma, were used as substrates. We found that the extensively prephosphorylated EGFR-IC exhibited similar reaction kinetics to the unphosphorylated EGFR-IC when angiotensin II was used as a nonspecific substrate. In contrast there was a clear stimulation of kinase activity due to autophosphorylation of the EGFR-IC when peptides representing either the major autophosphorylation site of the EGFR or the EGFR phosphorylation sites of phospholipase C-gamma were used as substrates. However, the modes of stimulation for these peptides differed. The binding affinity (Km) for the unphosphorylated EGFR-IC for the peptide containing Tyr-771 of phospholipase C-gamma was relatively poor compared with other peptides, but improved 5-6-fold when the EGFR-IC was prephosphorylated. On the other hand, autophosphorylation improved the reaction velocity (Vm) of the phosphorylation of other peptides by 2-3-fold, with little or no increase in affinity. These results suggest that autophosphorylation of the EGFR may induce a conformational change of its kinase domain which enhances its kinase activity with exogenous substrates and may induce association with phospholipase C-gamma by increasing its affinity to a domain containing Tyr-771.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

Oxidative stress-induced phospholipase C-gamma 1 activation enhances cell survival

Phospholipase C-gamma1 (PLC-gamma1) is rapidly activated in response to growth factor stimulation and plays an important role in regulating cell proliferation and differentiation through the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C (PKC) and increased levels of intracellular calcium, respectively. Given the existing overlap between signaling pathways that are activated in response to oxidant injury and those involved in responding to proliferative stimuli, we investigated the role of PLC-gamma1 during the cellular response to oxidative stress. Treatment of normal mouse embryonic fibroblasts (MEF) with H2O2 resulted in time- and concentration-dependent tyrosine phosphorylation of PLC-gamma1. Phosphorylation could be blocked by pharmacological inhibitors of Src family tyrosine kinases or the epidermal growth factor receptor tyrosine kinase, but not by inhibitors of the platelet-derived growth factor receptor or phosphatidylinositol 3-kinase. To investigate the physiologic relevance of H2O2-induced tyrosine phosphorylation of PLC-gamma1, we compared survival of normal MEF and PLC-gamma1-deficient MEF following exposure to H2O2. Treatment of PLC-gamma1-deficient MEF with H2O2 resulted in rapid cell death, whereas normal MEF were resistant to the stress. Pretreatment of normal MEF with a selective pharmacological inhibitor of PLC-gamma1, or inhibitors of inositol trisphosphate receptors and PKC, increased their sensitivity to H2O2, whereas treatment of PLC-gamma1-deficient MEF with agents capable of directly activating PKC and enhancing calcium mobilization significantly improved their survival. Finally, reconstitution of PLC-gamma1 protein expression in PLC-gamma1-deficient MEF restored cell survival following H2O2 treatment. These findings suggest an important protective function for PLC-gamma1 activation during the cellular response to oxidative stress.     

Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in WEHI-231 B lymphoma cells did not increase the amount of PtdIns 3-kinase activity which could be precipitated with anti-Tyr(P) antibodies. Similarly, inhibition of the phosphoinositide pathway did not abrogate the ability of mIg cross-linking to stimulate this response. Thus, mIg-induced tyrosine phosphorylation regulates PtdIns 3-kinase, an important mediator of growth control in fibroblasts and potentially an important regulatory component in B cells as well.     

CD3-zeta surface expression is required for CD4-p56lck-mediated upregulation of T cell antigen receptor-CD3 signaling in T cells.

It has been proposed that during T cell receptor antigen recognition, CD4- or CD8-p56lck molecules interact with the T cell antigen receptor-CD3 complex (TCR-CD3) to phosphorylate various undefined substrates, which then initiate signal transduction through the TCR-CD3 complex. The ability of CD4 to modulate the TCR-CD3-induced increase in intracellular Ca2+, [Ca2+]i, and substrate tyrosine phosphorylation was studied in mutants of the human leukemic T cell line HPB-ALL characterized by their low expression of the TCR-CD3 complex on the cell surface. In TCR-CD3low cells, in which CD3-zeta was found to be associated with the TCR-CD3 complex, cross-linking CD3 with CD4 resulted in a profile of calcium mobilization, CD3-zeta, and phospholipase C-gamma 1 tyrosine phosphorylation similar to that observed in HPB-ALL cells, although the magnitude of generalized substrate tyrosine phosphorylation appeared to be smaller, as compared with wild-type cells. Responses were weak or absent when CD3 was cross-linked alone. In contrast, in a mutant in which association of CD3-zeta 2 with the TCR-CD3 was defective, cross-linking of CD3 with CD4 had a weaker effect on any of the activation parameters tested. These experiments showed that the presence of CD3-zeta 2 in the TCR-CD3 complex is of critical importance for the ability of CD4 to enhance early transducing signals inside the cell. The data also suggest that CD4-associated protein tyrosine kinase p56lck could up-regulate defective CD3-mediated induction of phospholipase C activity by increasing tyrosine phosphorylation of phospholipase C-gamma 1.     

Phospho-LAT-independent activation of the ras-mitogen-activated protein kinase pathway: a differential recruitment model of TCR partial agonist signaling.

Stimulation of mature T cells with agonist ligands of the Ag receptor (TCR) causes rapid phosphorylation of tyrosine-based activation motifs in the intracellular portion of TCR-zeta and CD3 and activation of several intracellular signaling cascades. Coordinate activation of these pathways is dependent on Lck- and ZAP-70-mediated tyrosine phosphorylation of a 36-kDa linker for activation of T cells and subsequent recruitment of phospholipase C-gamma1, Grb2-SOS, and SLP-76-vav. Here, we show that TCR partial agonist ligands can selectively activate one of these pathways, the Ras-mitogen-activated protein kinase pathway, by inducing recruitment of Grb2-SOS complexes to incompletely phosphorylated p21 phospho-TCR-zeta. This bypasses the need for activation of Lck and ZAP-70, and for phosphorylation of the linker for activation of T cells to activate Ras. We propose a general model in which differential recruitment of activating complexes away from transmembrane linker proteins may determine selective activation of a given signaling pathway.