Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly.     

Differential mitogen-activated protein kinase stimulation by Fc gamma receptor IIa and Fc gamma receptor IIIb determines the activation phenotype of human neutrophils

Fc gamma Rs mediate immune complex-induced tissue injury. The hypothesis that Fc gamma RIIa and Fc gamma RIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of Fc gamma RIIa and/or Fc gamma RIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. Fc gamma RIIa and Fc gamma RIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both Fc gamma RIIa and Fc gamma RIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic Fc gamma R cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by Fc gamma RIIIb or heterotypic cross-linking, but had no effect on Fc gamma RIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by Fc gamma R cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either Fc gamma RIIa or Fc gamma RIIIb, but cytochalasin D only enhanced H2O2 production stimulated by Fc gamma RIIIb. Our data indicate that Fc gamma RIIa and Fc gamma RIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of Fc gamma RIIIb in neutrophils.     

High affinity IgG receptor activation of Src family kinases is required for modulation of the Shc-Grb2-Sos complex and the downstream activation of the nicotinamide adenine dinucleotide phosphate (reduced) oxidase

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcgammaRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcgammaRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcgammaRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcgammaRI stimulation. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcgammaR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.     

Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-x(L). Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-x(L) expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-x(L) expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-alpha by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.     

Role of SHPS-1 in the regulation of insulin-like growth factor I-stimulated Shc and mitogen-activated protein kinase activation in vascular smooth muscle cells

Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I-induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I-dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I-stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I-induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I-stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I-dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the beta3 subunit of the alphaVbeta3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I-dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I-stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I-dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.     

Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47(phox) and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47(phox). Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils.     

Activation of several MAP kinases upon stimulation of rat alveolar macrophages: role of the NADPH oxidase.

Zymosan-activated serum (ZAS), a source of C5a, stimulates the rat alveolar macrophages (AM) to release superoxide anion. Here we show that treatment of rat AM with ZAS induced a time-dependent increase in the tyrosine phosphorylation of several proteins (116, 105-110, 82-78, 66-72, 62, 45, 42, and 38 kDa). This increase was sensitive to genistein, a tyrosine kinase inhibitor. ZAS stimulated the tyrosine phosphorylation and activation of three members of a family of serine/threonine kinases known as the mitogen-activated protein kinases (MAPK), i.e., ERK1 and ERK2, as assessed by immunoblotting, immunoprecipitation, and phosphotransferase activity, and p38 MAPK, as determined by immunoblotting with phospho-specific antibodies. In addition, ZAS induced the tyrosine phosphorylation of the SHC proteins and their association with GRB2, suggesting a role for this complex in the activation of the ERK pathway. Addition of extracellular catalase during ZAS stimulation significantly reduced the tyrosine phosphorylation response and the activation of ERK1 and ERK2 and their activator MEK1/2 while it did not affect that of p38 MAPK and MKK3/MKK6. Superoxide dismutase marginally increased the response to ZAS, supporting a role for hydrogen peroxide. In contrast to the results with AM, stimulation of human neutrophils with ZAS in the presence of catalase minimally altered the activation of ERK1 and ERK2. These data show that, in ZAS-stimulated rat AM, activation of the respiratory burst and production of hydrogen peroxide via superoxide dismutation are largely responsible for the activation of the ERK pathway through an upstream target.     

FMLP刺激的HL-60中p38 MAPK对NADPH氧化酶p47~(phox)成分的磷酸化作用

为了解p38促分裂原活化蛋白激酶 (MAPK)参与NADPH氧化酶激活的机理 ,利用p38MAPK抑制剂SB2 0 35 80 ,在甲酰甲硫氨酰 亮氨酰 苯丙氨酸 (FMLP)刺激的分化为中性粒细胞样的HL 6 0细胞中研究p38MAPK对O·2 产生和NADPH氧化酶胞浆成分p4 7phox 的磷酸化作用 .实验发现 ,p38MAPK的激活过程与NADPH氧化酶的激活过程一致 .5 0 μmol LSB2 0 35 80抑制 5 0 % O·2 产生 ,完全抑制p38MAPK激活和部分抑制p4 7phox 体外磷酸化 .结果表明 ,在FMLP刺激的HL 6 0细胞中 ,p38MAPK可以通过磷酸化p4 7phox而参与NADPH氧化酶激活 .     

Hyperosmotic stress activates the insulin receptor in CHO cells

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.     

Mechanism of SHIP-mediated inhibition of insulin- and platelet-derived growth factor-stimulated mitogen-activated protein kinase activity in 3T3-L1 adipocytes

The Src homology 2-containing 5' inositolphosphatases (SHIP and SHIP2) dephosphorylate 3'-phosphorylated PtdIns on the 5' position, decreasing intracellular levels of PtdIns 3,4,5-P3. In the current study, we investigated the role of SHIP in insulin and platelet-derived growth factor (PDGF) signaling by expressing wild-type (WT) and catalytically inactive SHIPDeltaIP in 3T3-L1 adipocytes, utilizing adenoviral infection. Insulin and PDGF both stimulated tyrosine phosphorylation of SHIP-WT and of SHIPDeltaIP, and tyrosine phosphorylation of SHIP-associated proteins increased after ligand stimulation. Tyrosine-phosphorylated PDGFR, IR, and insulin receptor substrate-1 all immunoprecipitated with SHIP. Expression of WT and DeltaIP mutant SHIP did not affect tyrosine phosphorylation of either the insulin or the PDGF receptor, or the expression of insulin receptor substrate-1 and Shc proteins. Both SHIP-WT and SHIPDeltaIP blocked insulin and PDGF-induced MAPK and MAPK kinase phosphorylation as well as, GTP-bound Ras activity, suggesting that the catalytic activity of SHIP is not necessary for these effects. SHIP associated with Shc upon ligand stimulation, indicating that the SHIP-Shc association is phosphorylation dependent. This association was primarily between the SHIP-SH2 domain and the phosphorylated tyrosine residues of Shc because no association was observed when the 3YF-Shc mutant was coexpressed with SHIP. The Shc*Grb2 association was not compromised by SHIP expression, despite complete inhibition of the Ras/MAPK pathway. Interestingly, son-of-sevenless (SOS) protein normally found in Grb2 complexes was markedly reduced in SHIP expressing cells, whereas the displaced SOS was recovered when the post-Grb2-IP supernatants were blotted with anti-SOS antibody. Thus, SHIP competes son-of-sevenless (SOS) away from Shc-Grb2. In summary, 1) SHIP-WT and SHIPDeltaIP expression inhibit insulin and PDGF stimulated Ras, MAPK kinase, and MAPK activities; 2) SHIP associates with tyrosine phosphorylated Shc, and the proline-rich sequences in SHIP associate with Grb2 and titrate out SOS to form Shc*Grb2*SHIP complexes; and 3) dissociation of SOS from the Shc*Grb2 complex inhibits Ras GTP loading, leading to decreased signaling through the MAPK pathway.     

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.     

A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc, contributing to suppress apoptosis by IL-3.

Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains their viability by suppressing apoptosis. IL-3 stimulates tyrosine phosphorylation of the Shc adaptor protein and thereby formation of a complex of Shc with Grb2 at phosphorylated tyrosine (Y) residue 317-Shc. This pathway is implicated in Ras/mitogen-activated protein kinase (MAPK) activation towards c-fos gene expression. We examined the possible involvement of Shc in the antiapoptotic activity of IL-3. Conditional overexpression of the Shc SH2 domain, a dominant-negative mutant of Shc, was found to induce apoptosis of IL-3-dependent Ba/F3 cells along with a reduction of c-myc gene expression. Apoptosis was rescued by the exogenously introduced c-myc gene. Since we identify novel tyrosine phosphorylation sites of Shc: Y239 and Y240, their role on cell survival was tested by mutational analysis. Ba/F3 cells expressing mutant Shc Y317F, which is unable to stimulate efficiently the Ras pathway, still showed resistance to apoptosis. However, cells expressing Shc Y239/240F, which is able to stimulate the Ras pathway, were sensitive to apoptosis. In these cells, induction of the c-myc gene was reduced. These findings suggest that a new signalling pathway for cell survival is generated from Y239/240 of Shc to the nuclei involving c-myc gene expression.     

Hypoxia induces activation and subcellular translocation of focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes.

We previously reported that hypoxia caused rapid activation of RAS/mitogen-activated protein kinase (MAPK) pathway, two other stress-activated MAPK family members, stress-activated protein kinase (SAPK) and p38MAPK, and Src family tyrosine kinases, p60(c-src) and p59(c-fyn) in cultured rat cardiac myocytes. In this study, to elucidate how hypoxia affects adhesive interaction between cardiac myocytes and extracellular matrix (ECM), we investigated the molecular mechanism of the activation of focal adhesion-associated tyrosine kinases p125(FAK) and paxillin. Here, we show that hypoxia induced tyrosine phosphorylation of p125(FAK) and paxillin and that hypoxia-induced activation of p125(FAK) was accompanied by its increased association with adapter proteins Shc and GRB2, and non-receptor type tyrosine kinase p60(c-src). Furthermore, hypoxia caused subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. These results strongly suggest that p125(FAK) is one of the most important components in hypoxia-induced intracellular signaling in cardiac myocytes and may play a pivotal role in adhesive interaction between cardiac myocytes and ECM.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes. Cross-talk via trans-activation of the tyrosine kinase c-kit receptor

We previously reported that activation of mitogen-activated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1). In the present study, we determined signaling mechanisms upstream of MAPK activation that are involved in ET-1 stimulation and their synergism with stem cell factor (SCF). Pretreatment of cultured NHMC with ET(B) receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (), or a protein kinase C inhibitor (calphostine) blocked a transient tyrosine phosphorylation of MAPK induced by ET-1, whereas the addition of a calcium chelator (BAPTA) failed to inhibit that tyrosine phosphorylation of MAPK. Treatment with ET-1 and SCF together synergistically increased DNA synthesis, which was accompanied by synergism for MAPK phosphorylation. The time course of inositol 1,4,5-trisphosphate formation revealed that there is no difference in the level of inositol 1,4,5-trisphosphate stimulated by ET-1 + SCF or by ET-1 alone. Evaluations of the serine phosphorylation of MEK and Raf-1 activity showed a synergistic effect in SCF + ET-1-treated NHMC. Stimulation with SCF + ET-1 induced a more rapid and stronger tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc than did stimulation with SCF only, and this was accompanied by a stronger association of tyrosine-phosphorylated Shc with Grb2. Interestingly, a more rapid and marked tyrosine phosphorylation of c-kit was also detected in NHMC-treated with SCF + ET-1 than NHMC treated with SCF only. These data indicate that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of tyrosine phosphorylation of c-kit, which results in the enhanced formation of the Shc-Grb(2) complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop.     

Shc, Grb2, Sos1, and a 150-kilodalton tyrosine-phosphorylated protein form complexes with Fms in hematopoietic cells.

Fms, the macrophage colony-stimulating factor (M-CSF) receptor, is normally expressed in myeloid cells and initiates signals for both growth and development along the monocyte/macrophage lineage. We have examined Fms signal transduction pathways in the murine myeloid progenitor cell line FDC-P1. M-CSF stimulation of FDC-P1 cells expressing exogenous Fms resulted in tyrosine phosphorylation of a variety of cellular proteins in addition to Fms. M-CSF stimulation also resulted in Fms association with two of these tyrosine-phosphorylated proteins, one of which was identified as the 55-kDa Shc, which is shown in other systems to be involved in growth stimulation, and the other was a previously uncharacterized 150-kDa protein (p150). Fms also formed complexes with Grb2 and Sos1, and neither contained phosphotyrosine. Whereas both Grb2 and Sos1 complexed with Fms only after M-CSF stimulation, the amount of Sos1 complexed with Grb2 was not M-CSF dependent. Shc coimmunoprecipitated Sos1, Grb2, and tyrosine-phosphorylated p150, while Grb2 immunoprecipitates contained mainly phosphorylated p150, Fms, Shc, and Sos1. Shc interacted with tyrosine-phosphorylated p150 via its SH2 domain, and the Grb2 SH2 domain likewise bound tyrosine-phosphorylated Fms and p150. Analysis of Fms mutated at each of four tyrosine autophosphorylation sites indicated that none of these sites dramatically affected p150 phosphorylation or its association with Shc and Grb2. M-CSF stimulation of fibroblast cell lines expressing exogenous murine Fms did not phosphorylate p150, and this protein was not detected either in cell lysates or in Grb2 or Shc immunoprecipitates. The p150 protein is not related to known signal transduction molecules and may be myeloid cell specific. These results suggest that M-CSF stimulation of myeloid cells could activate Ras through the nucleotide exchange factor Sos1 by Grb2 binding to either Fms, Shc, or p150 and that Fms signal transduction in myeloid cells differs from that in fibroblasts.     

A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Nerve growth factor stimulates the tyrosine phosphorylation of a 38-kDa protein that specifically associates with the src homology domain of phospholipase C-gamma 1.

The cellular actions of nerve growth factor (NGF) involve changes in protein phosphorylation, initiated by the binding and subsequent activation of its tyrosine kinase receptor, the trk protooncogene (pp140c-trk). Upon exposure to NGF, a 38-kDa tyrosine-phosphorylated protein (pp38) is identified in both PC-12 pheochromocytoma cells and NIH3T3 cells transfected with the full-length human pp140c-trk cDNA (3T3-c-trk) that is specifically coimmunoprecipitated with pp140c-trk or phosphatidylinositol-phospholipase C (PLC)-gamma 1. In both PC-12 and 3T3-c-trk cells, NGF rapidly stimulates the association of pp140c-trk and pp38 with a fusion protein containing the src homology (SH) domains of PLC gamma 1. This phosphorylation and subsequent association are specific for NGF, since epidermal growth factor, platelet-derived growth factor, and insulin do not stimulate the tyrosine phosphorylation of these proteins or their association with the PLC gamma 1 SH domains, although the receptors for these growth factors do undergo tyrosine phosphorylation and association with the PLC-gamma 1 fusion protein under these conditions. Furthermore, the NGF-dependent pp38-SH binding is specific for the SH2 domains of PLC-gamma 1, since the phosphoprotein does not bind to fusion proteins containing SH domains of ras GTPase-activating protein or the p85 subunit of phosphatidylinositol 3 kinase. Both amino- and carboxyl-terminal SH2 domains of PLC-gamma 1 are necessary for the association of pp38 with PLC-gamma 1, although each SH2 domain is sufficient for the association of pp140c-trk with PLC-gamma 1. In both PC-12 and 3T3-c-trk cells, the phosphorylation and association of pp38 with PLC gamma 1 is rapid, occurring maximally at 1 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over a physiological concentration of the growth factor. The specificity and rapidity of pp38 phosphorylation and its association with PLC-gamma 1 suggest that it may be an important component in signal transduction for NGF.