Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT

Chemoattractants are thought to be the first mediators generated at sites of bacterial infection. We hypothesized that signaling through G protein-coupled chemoattractant receptors may stimulate cytokine production. To test this hypothesis, a human mast cell line (HMC-1) that normally expresses receptors for complement components C3a and C5a at low levels was stably transfected to express physiologic levels of fMLP receptors. We found that fMLP, but not C3a or C5a, induced macrophage inflammatory protein (MIP)-1ss (CCL4) and monocyte chemoattractant protein-1 (CCL2) mRNA and protein. Although fMLP stimulated both sustained Ca(2+) mobilization and phosphorylation of extracellular signal-regulated kinase (ERK), these responses to C3a or C5a were transient. However, transient expression of C3a receptors in HMC-1 cells rendered the cells responsive to C3a for sustained Ca(2+) mobilization and MIP-1ss production. The fMLP-induced chemokine production was blocked by pertussis toxin, PD98059, and cyclosporin A, which respectively inhibit G(i)alpha activation, mitgen-activated protein kinase kinase-mediated ERK phosphorylation, and calcineurin-mediated activation of NFAT. Furthermore, fMLP, but not C5a, stimulated NFAT activation in HMC-1 cells. These data indicate that chemoattractant receptors induce chemokine production in HMC-1 cells with a selectivity that depends on the level of receptor expression, the length of their signaling time, and the synergistic interaction of multiple signaling pathways, including extracellular signal-regulated kinase phosphorylation, sustained Ca(2+) mobilization and NFAT activation.     

TrkA signalling pathways in human airway smooth muscle cell proliferation

NGF may play a role in airway inflammation and hyperresponsiveness. We studied its possible involvement in airway remodelling and report here its proliferative effect and its receptor and signalling pathways in human airway smooth muscle cells in culture (HASMC). Proliferation of HASMC induced by NGF (0.1-10 pM) was assessed by the XTT and BrdU techniques with and without kinase inhibitors. Immunoprecipitation and Western blotting were used to study phosphorylation of TrkA and MAPK. NGF caused dose-dependent proliferation of HASMC and induced TrkA phosphorylation, both abolished by the tyrosine-kinase inhibitor K252a. PI3K and JNK inhibitors had no effect. PKC inhibitors partially inhibited NGF-induced proliferation and totally abolished p38 phosphorylation but did not affect ERK1/2 phosphorylation. The rafK inhibitor decreased NGF-induced proliferation, and totally abolished ERK1/2 phosphorylation, but did not affect p38 phosphorylation. This finding was confirmed by the decrease of NGF-induced proliferation after treatment with inhibitors of the p38 or of ERK1/2 pathways. In conclusion, NGF activation of the TrkA receptor involves two distinct signalling pathways: PKC selectively activates p38, and the ras/raf pathway selectively activates ERK1/2. Both are necessary to induce HASMC proliferation.     

Crosstalks between the receptors tyrosine kinase EGFR and TrkA and the GPCR,FPR, in human monocytes are essential for receptors-mediated cell activation

The G-protein coupled receptor (GPCR) fMLP receptor (FPR) and the two receptors tyrosine kinase (RTK), the nerve growth factor (NGF) receptor TrkA and the epidermal growth factor (EGF) receptor (EGFR) are involved in reactive oxygen species (ROS), matrix metalloproteinase-9 (MMP-9) production and CD11b membrane integrin upregulation. We show that in monocytes the three receptors crosstalk each other to modulate these pro-inflammatory mediators. Tyrphostin AG1478, the EGFR inhibitor, inhibits fMLP and NGF-associated ROS production, fMLP-associated CD11b upregulation and NGF-induced TrkA phosphorylation; K252a, the NGF receptor inhibitor, inhibits fMLP or EGF-associated ROS production, CD11b expression and EGF-induced EGFR phosphorylation; cyclosporine H, the FPR inhibitor inhibits EGF or NGF-associated ROS production, EGF-associated CD11b upregulation and prevents EGFR and TrkA phosphorylation by their respective ligand EGF and NGF. In response to fMLP, TrkA phosphorylation is inhibited by the EGFR inhibitor while EGFR phosphorylation is inhibited by the TrkA inhibitor. Receptor crosstalks are Src and ERK dependent. Down-regulation of each receptor by specific siRNA suppresses the ability of the two other receptors to promote ligand-mediated ERK phosphorylation and pro-inflammatory activities including ROS, MMP-9 production and CD11b upregulation. Thus, in monocytes GPCR ligands' activity involves activation of RTK while RTK-ligands activity engages GPCR-signalling molecules.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

Activation of Ras, Raf-1 and protein kinase C in differentiating human neuroblastoma cells after treatment with phorbolester and NGF

The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The results identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms.     

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

Platelet-activating factor-induced chemokine gene expression requires NF-kappaB activation and Ca2+/calcineurin signaling pathways. Inhibition by receptor phosphorylation and beta-arrestin recruitment

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.     

NGF activates the phosphorylation of MAP1B by GSK3beta through the TrkA receptor and not the p75(NTR) receptor

We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75NTR but not TrkA receptors, does not stimulate GSK3beta phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3beta phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75NTR and also fail to show NGF-dependent GSK3beta phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3beta phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3beta phosphorylation of MAP1B. These results suggest that the activation of GSK3beta by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75NTR receptors.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Dual signaling by the alpha(v)beta(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells

Vitronectin, which ligates the alpha(v)beta(3)-integrin, increases both lung capillary permeability and lung endothelial Ca(2+). In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located alpha(v)beta(3)-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha(v)beta(3) monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha(v)beta(5). Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA(2) inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca(2+) or cPLA(2) translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca(2+) chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical alpha(v)beta(3) in BPAECs caused ERK2 activation and an increase of intracellular Ca(2+), both conjointly required for cPLA(2) activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated "two-hit" signaling pathway that regulates an integrin-induced proinflammatory response.     

A novel apoptotic pathway induced by nerve growth factor-mediated TrkA activation in medulloblastoma

Nerve growth factor (NGF) induces apoptosis in a human medulloblastoma cell line (MED283) engineered to express TrkA (MED283-TrkA) (Muragaki, Y., Chou, T. T., Kaplan, D. R., Trojanowski, J. Q., and Lee, V. M. (1997) J. Neurosci. 17, 530-542). To dissect the molecular signaling pathway that mediates this novel effect, specific receptor mutations in Trk have been employed. We showed that phosphorylation of tyrosine 490 is required for activation of phosphoinositide 3-OH kinase, whereas phosphorylation of tyrosine 785 is required for activation of phospholipase C-gamma. TrkA-mediated apoptosis was abolished when either the ATP-binding site or both tyrosines 490 and 785 were mutated. Because tyrosines 490 and 785 mediate redundant signaling through the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, we examined the role of Ras-ERK signaling in NGF-induced apoptosis. We found that MED283-TrkA cells expressing a dominant negative Ras inhibitor (N17Ras) failed to undergo ERK activation and apoptosis following NGF treatment, whereas the ERK kinase (mitogen-activated protein kinase kinase) inhibitors PD98059 and U0126 eliminated ERK activation but had no effect on apoptosis. We infer from these data that NGF-induced apoptosis is mediated by a novel Ras and/or Raf signaling pathway.     

Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular-free Ca(2+) mobilization and induces mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in MCF-7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca(2+)-free medium did not alter Cch-stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12-myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch-evoked MAPK/ERK activation was unaffected by down-regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC-zeta pseudosubstrate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3-kinase (PI3K), diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pretreated with genistein, a non-selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC-zeta, PI3K, and Src family of tyrosine kinases, but not intracellular-free Ca(2+) mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells.     

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.