The Trkb Receptor Tyrosine Kinase Regulates Cellular Proliferation Via Signal Transduction Pathways Involving Shc,Plcγ, and Cbl

Abstract

The TrkB protein tyrosine kinase is a high affinity receptor for brain derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). TrkB autophosphorylation occurs on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Using site directed mutagenesis, we have assessed the importance of TrkB tyrosines 484 and 785 in affecting TrkB-mediated signaling events leading to NIH 3T3 cell mitogenesis and survival. Mutation of TrkB tyrosine 484, while having no affect on BDNF-inducible PLCγ and Cbl tyrosine phosphorylation, is essential for the phosphorylation of Shc, the complete activation of extracellular regulated kinase 1/2 (ERK1/2) and the induction of c-fos protein synthesis. In contrast, mutation of Y785 does not significantly affect BDNF-inducible Shc phosphorylation, ERK1/2 activation, or c-fos protein synthesis, but completely inhibits the tyrosine phosphorylation of PLCγ and Cbl. These data indicate that both ERK-dependent and ERK-independent signaling pathways lead to BDNF-inducible mitogenesis and survival.     

Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses

Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase Cgamma1 (PLCgamma1). Cytoplasmic loading with inhibitory antibodies to PLCgamma1, Gbeta, and Galpha(i/o/t/z) inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Galpha(i/o) inhibitor, pertussis toxin, reduced o-HA-induced PLCgamma1 tyrosine phosphorylation, protein kinase C (PKC) alpha and beta1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCgamma1, and PKC. In particular, we demonstrated a possible role for PKCalpha in mitogenesis and PKCbeta1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited Gbeta subunit co-precipitation with PLCgamma1, suggesting a possible role for Src in activation of PLCgamma1 and interaction between two distinct o-HA-induced signaling pathways.     

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.     

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.     

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.     

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Pertussis toxin-sensitive and -insensitive thrombin stimulation of Shc phosphorylation and mitogenesis are mediated through distinct pathways

Activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) result in phosphorylation of the adaptor protein Shc, providing sites of interaction for proteins in downstream signal transduction cascades. The mechanism of Shc phosphorylation and its function in G protein signaling pathways is still unclear. By examining Shc phosphorylation in response to thrombin in two cell lines, we have defined distinct pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the tyrosines in Shc, we show that the three sites of tyrosine phosphorylation, Y239, Y240, and Y317, are necessary for thrombin signaling in both systems. The SH2 (src homology 2) domain of Shc is also critical for signaling, but not required for phosphorylation of Shc. In both cell types, inhibition of src family member kinases by chemical inhibitors or microinjection block Shc phosphorylation and bromodeoxyuridine (BrdU) incorporation in response to thrombin. However, in the PTX-sensitive thrombin pathway, both betagamma function and the epidermal growth factor receptor (EGFR) are necessary for Shc phosphorylation and BrdU incorporation. In contrast, signaling in the PTX-insensitive pathway is not mediated through betagamma or the EGFR. Thus, while phosphorylation and function of Shc appear to be the same in both thrombin pathways, the mechanism of tyrosine kinase activation proximal to Shc is different. The differences in signaling between the two thrombin pathways may be representative of mechanisms used by other PTX-sensitive and -insensitive GPCRs to mediate specific responses. In addition, transactivation of RTKs may be a manner by which GPCRs can amplify their signal.     

The protein tyrosine phosphatase, Shp2, is required for the complete activation of the RAS/MAPK pathway by brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) and other neurotrophins induce a unique prolonged activation of mitogen-activated protein kinase (MAPK) compared with growth factors. Characterization and kinetic and spatial modeling of the signaling pathways underlying this prolonged MAPK activation by BDNF will be important in understanding the physiological role of BDNF in many complex systems in the nervous system. In addition to Shc, fibroblast growth factor receptor substrate 2 (FRS2) is required for the BDNF-induced activation of MAPK. BDNF induces phosphorylation of FRS2. However, BDNF does not induce phosphorylation of FRS2 in cells expressing a deletion mutant of TrkB (TrkBDeltaPTB) missing the juxtamembrane NPXY motif. This motif is the binding site for SHC. NPXY is the consensus sequence for phosphotyrosine binding (PTB) domains, and notably, FRS2 and SHC contain PTB domains. This NPXY motif, which contains tyrosine 484 of TrkB, is therefore the binding site for both FRS2 and SHC. Moreover, the proline containing region (VIENP) of the NPXY motif is also required for FRS2 and SHC phosphorylation, which indicates this region is an important component of FRS2 and SHC recognition by TrkB. Previously, we had found that the phosphorylation of FRS2 induces association of FRS2 and growth factor receptor binding protein 2 (Grb2). Now, we have intriguing data that indicates BDNF induces association of the SH2 domain containing protein tyrosine phosphatase, Shp2, with FRS2. Moreover, the PTB association motif of TrkB containing tyrosine 484 is required for the BDNF-induced association of Shp2 with FRS2 and the phosphorylation of Shp2. These results imply that FRS2 and Shp2 are in a BDNF signaling pathway. Shp2 is required for complete MAPK activation by BDNF, as expression of a dominant negative Shp2 in cells attenuates BDNF-induced activation of MAPK. Moreover, expression of a dominant negative Shp2 attenuates Ras activation showing that the protein tyrosine phosphatase is required for complete activation of MAPKs by BDNF. In conclusion, Shp2 regulates BDNF signaling through the MAPK pathway by regulating either Ras directly or alternatively, by signaling components upstream of Ras. Characterization of MAPK signaling controlled by BDNF is likely to be required to understand the complex physiological role of BDNF in neuronal systems ranging from the regulation of neuronal growth and survival to the regulation of synapses.     

EphB1 recruits c-Src and p52Shc to activate MAPK/ERK and promote chemotaxis

Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal-regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.     

Phosphorylation of the linker for activation of T-cells by Itk promotes recruitment of Vav

The linker for activation of T-cells (LAT) is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and contains nine consensus putative tyrosine phosphorylation sites, several of which have been shown to serve as SH2 binding sites. Upon T-cell antigen receptor (TCR/CD3) engagement, LAT is phosphorylated by protein tyrosine kinases (PTK) and binds to the adaptors Gads and Grb2, as well as to phospholipase Cgamma1 (PLCgamma1), thereby facilitating the recruitment of key signal transduction components to drive T-cell activation. The LAT tyrosine residues Y(132), Y(171), Y(191), and Y(226) have been shown previously to be critical for binding to Gads, Grb2, and PLCgamma1. In this report, we show by generation of LAT truncation mutants that the Syk-family kinase ZAP-70 and the Tec-family kinase Itk favor phosphorylation of carboxy-terminal tyrosines in LAT. By direct binding studies using purified recombinant proteins or phosphopeptides and by mutagenesis of individual tyrosines in LAT to phenylalanine residues, we demonstrate that Y(171) and potentially Y(226) are docking sites for the Vav guanine nucleotide exchange factor. Further, overexpression of a kinase-deficient mutant of Itk in T-cells reduced both the tyrosine phosphorylation of endogenous LAT and the recruitment of Vav to LAT complexes. These data indicate that kinases from distinct PTK families are likely responsible for LAT phosphorylation following T-cell activation and that Itk kinase activity promotes recruitment of Vav to LAT.     

Role of protein kinase C in signal attenuation following T cell receptor engagement.

T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.     

Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.     

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed.     

Inhibition of insulin-like growth factor-1 receptor and IRS-2 signaling by ethanol in SH-SY5Y neuroblastoma cells

The effect of ethanol on insulin-like growth factor-1 (IGF-I)-mediated signal transduction and functional activation in neuronal cells was examined. In human SH-SY5Y neuroblastoma cells, ethanol inhibited tyrosine autophosphorylation of the IGF-I receptor. This corresponded to the inhibition of IGF-I-induced phosphorylation of p42/p44 mitogen-activated/extracellular signal-regulated protein kinase (MAPK) by ethanol. Insulin-related substrate-2 (IRS-2) and focal adhesion kinase phosphorylation were reduced in the presence of ethanol, which corresponded to the prevention of lamellipodia formation (30 min). By contrast, ethanol had no effect on Shc phosphorylation when measured up to 1 h, and did not affect the association of Grb-2 with Shc. Neurite formation at 24 h was similarly unaffected by ethanol. The data indicate that the IGF-I receptor is a target for ethanol in SH-SY5Y cells However, there is diversity in the sensitivity of signaling elements within the IGF-I receptor tyrosine kinase signaling cascades to ethanol, which can be related to the inhibition of specific functional events in neuronal activation.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Growth hormone-induced phosphorylation of epidermal growth factor (EGF) receptor in 3T3-F442A cells. Modulation of EGF-induced trafficking and signaling

Growth hormone (GH) promotes signaling by causing activation of the non-receptor tyrosine kinase, JAK2, which associates with the GH receptor. GH causes phosphorylation of epidermal growth factor receptor (EGFR; ErbB-1) and its family member, ErbB-2. For EGFR, JAK2-mediated GH-induced tyrosine phosphorylation may allow EGFR to serve as a scaffold for GH signaling. For ErbB-2, GH induces serine/threonine phosphorylation that dampens basal and EGF-induced ErbB-2 kinase activation. We now further explore GH-induced EGFR phosphorylation in 3T3-F442A, a preadipocytic fibroblast cell line that expresses endogenous GH receptor, EGFR, and ErbB-2. Using a monoclonal antibody that recognizes ERK consensus site phosphorylation (PTP101), we found that GH caused PTP101-reactive phosphorylation of EGFR. This GH-induced EGFR phosphorylation was prevented by MEK1 inhibitors but not by a protein kinase C inhibitor. Although GH did not discernibly affect EGF-induced EGFR tyrosine phosphorylation, we observed by immunoblotting a substantial decrease of EGF-induced EGFR degradation in the presence of GH. Fluorescence microscopy studies indicated that EGF-induced intracellular redistribution of an EGFR-cyan fluorescent protein chimera was markedly reduced by GH cotreatment, in support of the immunoblotting results. Notably, protection from EGF-induced degradation and inhibition of EGF-induced intracellular redistribution afforded by GH were both prevented by a MEK1 inhibitor, suggesting a role for GH-induced ERK activation in regulating the trafficking itinerary of the EGF-stimulated EGFR. Finally, we observed augmentation of early aspects of EGF signaling (EGF-induced ERK2 activation and EGF-induced Cbl tyrosine phosphorylation) by GH cotreatment; the GH effect on EGF-induced Cbl tyrosine phosphorylation was also prevented by MEK1 inhibition. These data indicate that GH, by activating ERKs, can modulate EGF-induced EGFR trafficking and signaling and expand our understanding of mechanisms of cross-talk between the GH and EGF signaling systems.     

LAR protein tyrosine phosphatase receptor associates with TrkB and modulates neurotrophic signaling pathways

The identities of receptor protein tyrosine phosphatases (PTPs) that associate with Trk protein tyrosine kinase (PTK) receptors and modulate neurotrophic signaling are unknown. The leukocyte common antigen-related (LAR) receptor PTP is present in neurons expressing TrkB, and like TrkB is associated with caveolae and regulates survival and neurite outgrowth. We tested the hypothesis that LAR associates with TrkB and regulates neurotrophic signaling in embryonic hippocampal neurons. Coimmunoprecipitation and coimmunostaining demonstrated LAR interaction with TrkB that is increased by BDNF exposure. BDNF neurotrophic activity was reduced in LAR-/- and LAR siRNA-treated LAR+/+ neurons and was augmented in LAR-transfected neurons. In LAR-/- neurons, BDNF-induced activation of TrkB, Shc, AKT, ERK, and CREB was significantly decreased; while in LAR-transfected neurons, BDNF-induced CREB activation was augmented. Similarly, LAR+/+ neurons treated with LAR siRNA demonstrated decreased activation of Trk and AKT. LAR is known to activate the Src PTK by dephosphorylation of its negative regulatory domain and Src transactivates Trk. In LAR-/- neurons, or neurons treated with LAR siRNA, phosphorylation of the Src regulatory domain was increased (indicating Src inactivation), consistent with a role for Src in mediating LAR's ability to up-regulate neurotrophic signaling. Interactions between LAR, TrkB, and Src were further confirmed by the findings that Src coimmunoprecipitated with LAR, that the Src inhibitor PP2 blocked the ability of LAR to augment TrkB signaling, and that siRNA-induced depletion of Src decreased LAR interaction with TrkB. These studies demonstrate that receptor PTPs can associate with Trk complexes and promote neurotrophic signaling and point to receptor PTP-based strategies as a novel approach for modulating neurotrophin function.     

Brain-derived neurotrophic factor induces phosphorylation of fibroblast growth factor receptor substrate 2

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival. Gaining an understanding of how BDNF, via the tropomyosin-related kinase B (TRKB) receptor, elicits specific cellular responses is of contemporary interest. Expression of mutant TrkB in fibroblasts, where tyrosine 484 was changed to phenylalanine, abrogated Shc association with TrkB, but only attenuated and did not block BDNF-induced phosphorylation of mitogen-activated protein kinase (MAPK). This suggests there is another BDNF-induced signaling mechanism for activating MAPK, which compelled a search for other TrkB substrates. BDNF induces phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2) in both fibroblasts engineered to express TrkB and human neuroblastoma (NB) cells that naturally express TrkB. Additionally, BDNF induces phosphorylation of FRS2 in primary cultures of cortical neurons, thus showing that FRS2 is a physiologically relevant substrate of TrkB. Data are presented demonstrating that BDNF induces association of FRS2 with growth factor receptor-binding protein 2 (GRB2) in cortical neurons, fibroblasts, and NB cells, which in turn could activate the RAS/MAPK pathway. This is not dependent on Shc, since BDNF does not induce association of Shc and FRS2. Finally, the experiments suggest that FRS2 and suc-associated neurotrophic factor-induced tyrosine-phosphorylated target are the same protein.