SH2-B Is Required for Nerve Growth Factor-induced Neuronal Differentiation*

Liangyou RuiDagger , James Herrington, and Christin Carter-Su§

From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PC12 cells, a rat adrenal pheochromocytoma cell line, are a widely used paradigm for studying NGF-induced1 neuronal differentiation. NGF stimulates PC12 cells to differentiate into sympathetic-like neurons by activating the NGF receptor TrkA, a member of the Trk family of receptor tyrosine kinases (1-3). Upon NGF binding, TrkA dimerizes (4) and autophosphorylates multiple tyrosines within its cytoplasmic domain (5). Signaling molecules containing Src homology 2 (SH2) or phosphotyrosine-binding domains, such as phospholipase Cgamma (PLCgamma ), phosphatidylinositol 3-kinase, and Shc proteins, interact with tyrosyl phosphorylated TrkA and transmit NGF signals (5, 6). Phosphatidylinositol 3-kinase is essential for NGF protection of PC12 cells from apoptosis (7) but is not required for NGF-induced neuronal differentiation (8). In contrast, the Shc/Ras/MEK/ERK pathway appears to be essential for NGF-induced neuronal differentiation of PC12 cells (5, 8-10). ERK1/2 can be activated by both NGF and epidermal growth factor (EGF), but the biological responses to NGF and EGF are opposite: NGF promotes differentiation, whereas EGF stimulates proliferation of PC12 cells (11). It was thought that sustained activation of ERK1/2 by NGF compared with transient activation by EGF contributes to the specificity of the NGF differentiation signal (11). Prolonged activation of ERK1/2 by NGF has been shown to be mediated by Rap1 and required for the expression of neuronal specific genes (12). Surprisingly, sustained activation of ERK1/2 is not required for NGF-induced neurite outgrowth (12), demonstrating that morphological differentiation of PC12 cells is mediated by other as yet unidentified signaling proteins/pathway(s).

SH2-Bbeta , a predicted adapter protein with multiple potential protein-protein interaction domains/motifs (e.g. pleckstrin homology, SH2, and proline-rich), has recently been shown to be regulated by a variety of ligands that activate receptor tyrosine kinases or receptor-associated tyrosine kinases (13-15). Three alternatively spliced isoforms of SH2-B (alpha , beta , and gamma ) have been described (13, 16, 17)2 that differ in their C termini downstream of the SH2 domain. No cellular function had been ascribed to any of these isoforms. In this study, we show that NGF stimulates association of SH2-Bbeta with TrkA via the SH2 domain of SH2-Bbeta and tyrosyl phosphorylation of SH2-Bbeta . The mutation of the conserved Arg to Glu within the FLVR motif within the SH2 domain of SH2-Bbeta abolishes the association of the mutant SH2-Bbeta with TrkA and tyrosyl phosphorylation of the mutant SH2-Bbeta in response to NGF. Furthermore, this mutant SH2-Bbeta acts as a dominant negative SH2-Bbeta to block NGF-induced neurite outgrowth when overexpressed in PC12 cells. These results suggest that SH2-Bbeta is an essential component of a signaling pathway(s) that is vital for NGF-induced neurite outgrowth.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Murine NGF and EGF were from Collaborative Biomedical Products. Sodium orthovanadate was from Sigma. Recombinant protein A-agarose was from Repligen. Antibody to rat SH2-Bbeta (alpha SH2-B) was raised against a GST fusion protein containing the C-terminal portion of SH2-Bbeta (13). Monoclonal anti-phosphotyrosine antibody 4G10 (alpha PY) and monoclonal antibody to PLCgamma (alpha PLCgamma ) were from Upstate Biotechnology Inc. Polyclonal antibody to Shc (alpha Shc) was from Transduction Labs. Polyclonal antibody against the extracellular domain of TrkA (alpha TrkA) (18) was kindly provided by Dr. Louis F. Reichardt (University of California, San Francisco). Polyclonal antibody against the cytoplasmic domain of TrkA was purchased from Santa Cruz Biotechnology, Inc. (C-14). Monoclonal antibody to GFP (alpha GFP) was from CLONTECH. Anti-active MAPK (alpha active MAPK) was from Promega.

Plasmids and Transfection-- cDNA encoding rat SH2-Bbeta was subcloned in-frame at BglII/EcoRI sites into pEGFP-C1 (CLONTECH), which encodes a red-shifted variant of GFP. Arg-555 in SH2-Bbeta was mutated to Glu, using QuickChangeTM site-directed mutagenesis kit (Stratagene) with the primer 5'-GTCTTCTTGGTAGAACAGAGTGAGACAAGA-3'. PC12 cells were transfected with pEGFP-C1 encoding GFP, GFP-SH2-Bbeta , or GFP-SH2-Bbeta (R555E), using LipofectAMINE Plus (Life Technologies, Inc.). After 72 h at 37 °C in 5% CO2 in standard medium (Dulbecco's modified Eagle's medium supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, 10% heat-inactivated horse serum, and 5% fetal bovine serum), the transfectants were cultured for 40 additional days in medium supplemented with 1 mg/ml G418. The G418-resistant transfectants were pooled, and the top 2% of cells in terms of expression of GFP fusion proteins were selected by flow cytometry (Elite, ESP).

Immunoprecipitation and Immunoblotting-- Precedures for preparing cell lysates, immunoprecipitation, and immunoblotting were described previously (13). alpha PLCgamma was used at 1:2000, and alpha active-MAPK was used at 1:20,000 for immunoblotting. alpha TrkA and alpha Shc were used at 1:100 for immunoprecipitation.

Cell Imaging-- Cells were plated at a density of 0.2 × 105 and cultured in standard medium supplemented with 1 mg/ml G418 on 60-mm collagen-coated culture dishes (Collaborative Biomedical Products). The next day, NGF was added directly to the medium at the indicated concentrations. Neurite outgrowths were monitored and photographed every 12 h using phase contrast microscopy. NGF was added every 2 days without changing the culture medium.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NGF Stimulates the Association of SH2-Bbeta with TrkA via the SH2 Domain of SH2-Bbeta -- SH2-B in PC12 cells is thought to be the beta  isoform because after dephosphorylation by alkaline phosphatase, it comigrates with dephosphorylated SH2-Bbeta expressed ectopically in COS cells (data not shown). To determine whether SH2-Bbeta plays a role in NGF signaling, we first examined whether SH2-Bbeta interacts with TrkA. Wild type or mutant SH2-Bbeta was fused to GST. PC12 cells overexpressing TrkA (19) were treated with NGF, and the cell extracts were incubated with the indicated GST fusion proteins immobilized on agarose beads. GST fusion proteins containing either wild type (GST-WT) or the C-terminal 20% of SH2-Bbeta with the entire SH2 domain (GST-SH2) bound weakly to TrkA from control cells (Fig. 1a). NGF treatment dramatically increased the binding of TrkA to both GST-SH2-Bbeta and GST-SH2 (Fig. 1a). Densitometric analysis revealed that NGF stimulated the association of TrkA with GST-SH2-Bbeta by more than 5-fold (n = 3) and with GST-SH2 by more than 4.5-fold (n = 3). In contrast, only a residual amount of TrkA bound to GST fusion proteins containing mutant SH2-Bbeta (R555E) in which Glu replaced the critical Arg within the FLVR motif of the SH2 domain of SH2-Bbeta (GST-RE) (Fig. 1a). NGF treatment did not increase the binding of TrkA to GST-RE (Fig. 1a). GST alone did not bind to TrkA, even when cells had been treated with NGF (Fig. 1a). These results suggest that the SH2 domain of SH2-Bbeta is sufficient and necessary for NGF-promoted association of TrkA with SH2-Bbeta . In addition, the weak binding of GST-WT or GST-SH2 to TrkA from control cells suggests that the SH2 domain of SH2-Bbeta may have a low affinity for nontyrosyl phosphorylated, inactive TrkA. Alternatively, there may be other binding site(s) in the C-terminal portion of SH2-Bbeta that bind with a low affinity to nontyrosyl phosphorylated TrkA.


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Fig. 1.   NGF promotes the interaction of SH2-Bbeta with TrkA. a, PC12 cells overexpressing TrkA were stimulated with 100 ng/ml NGF for 10 min. Cell lysates were incubated with GST fusion proteins containing wild type (GST-WT), the SH2 domain of (GST-SH2), or R555E mutant (GST-RE) SH2-Bbeta . Proteins bound to GST fusion proteins were immunoblotted (IB) with alpha TrkA (from Dr. Louis F. Reichardt, 1:500 dilution). b, PC12 cells stably overexpressing GFP-SH2-Bbeta were stimulated with 100 ng/ml NGF for 6 min. Proteins in the cell lysates were immunoprecipitated (IP) with alpha TrkA (from Dr. Louis F. Reichardt, 1:100 dilution) and immunoblotted sequentially with alpha SH2-B and alpha GFP as indicated. The same blot was stripped and reprobed with alpha TrkA.

To determine whether SH2-Bbeta associates with TrkA in cells in response to NGF, PC12 cells overexpressing GFP-tagged SH2-Bbeta were treated with NGF, and proteins in the cell lysates were immunoprecipitated with alpha TrkA. Immunoprecipitated proteins were immunoblotted with either alpha SH2-B or alpha GFP (Fig. 1b). NGF significantly promoted the association of TrkA with GFP-SH2-Bbeta (Fig. 1b). When normalized to the level of TrkA, NGF stimulated the binding of TrkA to GFP-SH2-Bbeta by 4-fold. In contrast, NGF did not stimulate the interaction of TrkA with GFP-SH2-Bbeta (R555E) that has a defective SH2 domain (data not shown). Consistent with activated TrkA binding to SH2-Bbeta , endogenous SH2-Bbeta from NGF-treated cells co-immunoprecipitated with a tyrosyl phosphorylated protein of a size appropriate for TrkA (Fig. 2b, upper panel). Taken together, our results suggest that NGF stimulates the binding of SH2-Bbeta to TrkA via the SH2 domain of SH2-Bbeta .


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Fig. 2.   NGF stimulates tyrosyl phosphorylation of SH2-Bbeta . a, PC12 cells were pretreated with 100 µM Na3VO4 for 60 min prior to 100 ng/ml NGF for 10 min. SH2-Bbeta was immunoprecipitated (IP) with alpha SH2-B and immunoblotted (IB) with alpha PY (upper panel, 1:7,500 dilution). The same blot was reprobed with alpha SH2-B (lower panel). b, PC12 cells overexpressing TrkA were treated with 100 ng/ml NGF for 1 min, and SH2-Bbeta was immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY (upper panel). The same blot was reprobed with alpha SH2-B (lower panel). c, PC12 cells were pretreated with 100 µM Na3VO4 for 60 min prior to 100 ng/ml NGF or 125 ng/ml EGF for 10 min. SH2-Bbeta was immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY.

NGF Stimulates Tyrosyl Phosphorylation of SH2-Bbeta in PC12 Cells-- To examine whether NGF stimulates tyrosyl phosphorylation of SH2-Bbeta , PC12 cells overexpressing either TrkA (Fig. 2b) or SH2-Bbeta tagged with GFP (Fig. 3b) were stimulated with NGF. Proteins in the cell lysates were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY. NGF stimulated tyrosyl phosphorylation of both endogenous SH2-Bbeta (Fig. 2b, upper panel) and GFP-SH2-Bbeta (Fig. 3b, upper panel). In addition, when normal PC12 cells were pretreated for 60 min with 100 µM sodium vanadate (a phosphatase inhibitor) prior to NGF stimulation, significant NGF-stimulated tyrosyl phosphorylation of SH2-Bbeta was detected (Fig. 2a, upper panel). NGF-induced tyrosyl phosphorylation of SH2-Bbeta was difficult to detect in normal PC12 cells without pretreatment with phosphatase inhibitor (data not shown), suggesting that NGF induces phosphorylation of tyrosines within SH2-Bbeta that then undergo rapid dephosphorylation. NGF also stimulated a significant shift in the mobility of SH2-Bbeta (Fig. 2a, lower panel), which is largely due to NGF-promoted phosphorylation of SH2-Bbeta on serines/threonines.3


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Fig. 3.   NGF regulation of stably expressed GFP-tagged wild type (GFP-WT) or R555E mutant (GFP-RE) SH2-Bbeta . a, Equal amounts of lysates of PC12 cells overexpressing GFP, GFP-WT, and GFP-RE were immunoblotted (IB) with alpha SH2-B. b, PC12 cells overexpressing GFP-SH2-Bbeta (GFP-WT) and GFP-SH2-Bbeta (R555E) (GFP-RE) were stimulated with 100 ng/ml NGF for 10 min. Proteins in lysates were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY (upper panel). The same blots were reprobed with alpha SH2-B (lower panel). Because of the difference in the level of expression of GFP-SH2-Bbeta and GFP-SH2-Bbeta (R555E), the exposure time for GFP-SH2-Bbeta (R555E) is approximately three times that for GFP-SH2-Bbeta during ECL.

Interestingly, 1 min of NGF stimulation of PC12 cells overexpressing TrkA caused significant tyrosyl phosphorylation of SH2-Bbeta without a dramatic change in the mobility of SH2-Bbeta (Fig. 2b, lower panel). This is in contrast to the large mobility change observed in endogenous SH2-Bbeta in PC12 cells treated with NGF for 10 min (Fig. 2a, lower panel). This difference in mobility shift in the two figures is likely due to the different times of incubation with NGF, because a large mobility change of SH2-Bbeta was observed when PC12 cells overexpressing TrkA were treated with NGF for 10 min (data not shown). These data support the hypothesis that the shift in the mobility of SH2-Bbeta is caused by phosphorylation of SH2-Bbeta on serines/threonines rather than on tyrosines. The results also suggest that NGF-stimulated tyrosyl phosphorylation of SH2-Bbeta may precede serine/threonine phosphorylation of SH2-Bbeta .

In contrast to NGF, EGF did not stimulate tyrosyl phosphorylation of SH2-Bbeta in PC12 cells even in the presence of phosphatase inhibitor (Fig. 2c). Because EGF stimulates proliferation while NGF promotes neuronal differentiation of PC12 cells, this differential response of SH2-Bbeta to these two growth factors might contribute to the specificity of biological response following the activation of receptors for these two growth factors. Taken together, the results raise the possibility that NGF-stimulated association of SH2-Bbeta with TrkA and/or the subsequent tyrosyl phosphorylation of SH2-Bbeta initiates one or more novel signaling pathways that may be specific for NGF and required for NGF-induced neuronal differentiation.

SH2-Bbeta Is Required for NGF-induced Neurite Outgrowth of PC12 Cells-- To examine the function of SH2-Bbeta in cellular responses to NGF, we generated PC12 cells that stably overexpress GFP, GFP-tagged SH2-Bbeta , or GFP-SH2-Bbeta (R555E) that lacks a functional SH2 domain. To eliminate differences due to clonal variation of PC12 cells, we pooled all G418-resistant clones. The GFP tag enabled us to use flow cytometry to isolate cells expressing high levels of SH2-Bbeta . GFP-SH2-Bbeta and GFP-SH2-Bbeta (R555E) are present at ~60 and 20 times, respectively, the level of endogenous SH2-Bbeta (Fig. 3a).

We first examined whether GFP-SH2-Bbeta , like endogenous SH2-Bbeta , is tyrosyl phosphorylated in response to NGF. PC12 cells overexpressing GFP-SH2-Bbeta (designated as GFP-WT) were stimulated with NGF. Proteins in the cell lysates were immunoprecipitated with alpha SH2-B and then immunoblotted with alpha PY. Significant tyrosyl phosphorylation of GFP-SH2-Bbeta was detected (Fig. 3b, upper panel). Reprobing the same blot with alpha SH2-B showed that NGF also stimulated a shift in the mobility of GFP-SH2-Bbeta (Fig. 3b, lower panel).4 In contrast, NGF did not stimulate tyrosyl phosphorylation of GFP-SH2-Bbeta (R555E) (Fig. 3b, upper panel) as anticipated. These results suggest that like endogenous SH2-Bbeta , GFP-tagged SH2-Bbeta binds to activated and tyrosyl phosphorylated TrkA and is tyrosyl phosphorylated by TrkA in response to NGF.

To study the role of SH2-Bbeta in NGF-induced neuronal differentiation of PC12 cells, we examined whether SH2-Bbeta is required for NGF-induced neurite outgrowth, a hallmark of neuronal differentiation. In the presence of NGF, cells overexpressing GFP alone developed neurite outgrowths in a manner similar to parental PC12 cells (Fig. 4, a and b, and data not shown). Strikingly, overexpression of GFP-SH2-Bbeta (R555E) blocked neurite outgrowth induced by NGF (Fig. 4, c and d). Even treatment of cells with a supramaximal concentration of NGF (100 ng/ml) for 8 days or longer did not induce neurite outgrowth (data not shown). This observation suggests that SH2-Bbeta is required for NGF-induced morphological differentiation of PC12 cells.


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Fig. 4.   Requirement for SH2-Bbeta for NGF-induced neurite outgrowth. PC12 cells overexpressing GFP (a, b, e, and f), GFP-RE (c and d), or GFP-WT (g and h) were plated on 60-mm culture dishes coated with collagen and cultured for 5 days with 100 ng/ml NGF (b and d) or 4 days with 25 ng/ml NGF (f and h). Cells were visualized using phase contrast microscopy. The scale bar in panel h represents 20 µm.

Consistent with SH2-Bbeta playing an essential role in NGF-induced differentiation of PC12 cells, overexpression of GFP-SH2-Bbeta enhanced NGF-induced neurite outgrowth when NGF was tested at a submaximal concentration. For example, 25 ng/ml NGF for 4 days promoted differentiation of the majority of cells expressing GFP-SH2-Bbeta , whereas only a very few of the cells expressing GFP alone differentiated in response to NGF (Fig. 4, e-h). Neurite outgrowth (twice cell diameter) was observed in about 60% of cells expressing GFP-SH2-Bbeta after 2 days of incubation with 15 ng/ml NGF, whereas fewer than 1% of the cells expressing GFP had neurites. By day 5, approximately 95% of the cells expressing GFP-SH2-Bbeta had neurite outgrowth, whereas only about 40% of cells expressing GFP had neurite outgrowth. In the absence of NGF, overexpression of GFP-SH2-Bbeta did not induce neuronal differentiation (Fig. 4g), suggesting that SH2-Bbeta must be activated in some way by NGF, presumably by being phosphorylated, to mediate neurite outgrowth.

NGF-induced Tyrosyl Phosphorylation of TrkA, Shc, and PLCgamma and Activation of ERK1/2 Are Not Affected by Overexpression of SH2-Bbeta (R555E)-- To explore the mechanism of action of SH2-Bbeta , we asked whether overexpression of SH2-Bbeta or SH2-Bbeta (R555E) affects tyrosyl phosphorylation of TrkA, Shc, or PLCgamma , all of which are known to play important roles in NGF signaling (20). Cells stably overexpressing GFP, GFP-SH2-Bbeta , or GFP-SH2-Bbeta (R555E) were treated with NGF, and proteins in the cell lysates were immunoprecipitated with the antibodies against TrkA, Shc, or PLCgamma and immunoblotted with anti-phosphotyrosine antibody. NGF induced tyrosyl phosphorylation of TrkA (Fig. 5a), PLCgamma (Fig. 5c), and all three isoforms of Shc (Fig. 5b). Levels of induction were unchanged in cells overexpressing SH2-Bbeta or SH2-Bbeta (R555E) (Fig. 5), suggesting that TrkA activation and signaling events immediately downstream of TrkA are not compromised by overexpression of SH2-Bbeta (R555E).


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Fig. 5.   Effect of overexpression of GFP-RE and GFP-WT on NGF-induced tyrosyl phosphorylation of TrkA, Shc and PLCgamma . Cells overexpressing GFP alone (GFP), GFP-SH2-Bbeta (GFP-WT), or GFP-SH2-Bbeta (R555E) (GFP-RE) were stimulated with 100 ng/ml NGF for 10 min. a, TrkA was immunoprecipitated (IP) with alpha TrkA (C-14 from Santa Cruz, 1:100 dilution) and immunoblotted (IB) with alpha PY (upper panel). The same blot was reprobed with alpha TrkA (C-14, 1:500 dilution). b, Shc was immunoprecipitated with alpha Shc (1:100 dilution) and immunoblotted with alpha PY (upper panel). The same blot was reprobed with alpha Shc (lower panel, 1:250 dilution). c, PLCgamma was immunoprecipitated with alpha PLCgamma (1:100 dilution) and immunoblotted with alpha PY (upper panel). The same blot was stripped and immunoblotted with alpha PLCgamma (lower panel, 1:2000 dilution).

Because the kinetics of the activation of ERK1/2 have been hypothesized to play an important role in NGF-induced neuronal differentiation of PC12 cells, we examined the time course of activation of ERK1/2 by NGF by immunoblotting cell lysates with antibody recognizing only activated ERK1/2 that is phosphorylated on both tyrosine and threonine. Neither the extent nor the duration of activation of ERK1/2 by NGF was affected by overexpression of SH2-Bbeta or SH2-Bbeta (R555E) (Fig. 6), consistent with the previous observation that sustained activation of ERK1/2 induced by NGF is not sufficient for neuronal differentiation of PC12 cells (21, 22).


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Fig. 6.   Effect of overexpression of GFP-RE and GFP-WT on the activation of ERK1/2 by NGF. Cells overexpressing GFP alone (GFP), GFP-SH2-Bbeta (GFP-WT), or GFP-SH2-Bbeta (R555E) (GFP-RE) were stimulated with 100 ng/ml NGF for the indicated times. An equal amount of protein in the lysates was immunoblotted (IB) with anti-active MAPK antibody (1:20,000 dilution). The same blots were stripped and reprobed with alpha ERK2 that recognizes both ERK1 and ERK2 (1:1000 dilution).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we show that NGF promotes the association of SH2-Bbeta with activated TrkA and stimulates tyrosyl phosphorylation of SH2-Bbeta . Mutating the SH2 domain of SH2-Bbeta abolishes its ability to bind activated TrkA and to be phosphorylated in response to NGF, suggesting that SH2-Bbeta binds via its SH2 domain to tyrosyl phosphorylated TrkA and that this interaction is required for subsequent phosphorylation of SH2-Bbeta . Overexpression of a GFP-tagged SH2-Bbeta (R555E) lacking a functional SH2 domain blocks NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of GFP-tagged wild type SH2-Bbeta enhances NGF-induced neurite outgrowth. SH2-Bbeta represents only the second signaling molecule, the first being Shc, known to bind to activated TrkA, to be phosphorylated, and to be required for NGF-induced neuronal differentiation.

SH2-Bbeta has been shown to associate with receptors for insulin, insulin-like growth factor-1 (14), platelet-derived growth factor (15), and EGF (data not shown), in addition to TrkA. Of the hormones and growth factors that bind to these receptors, only NGF and platelet-derived growth factor have been shown to stimulate tyrosyl phosphorylation of SH2-Bbeta (14, 15). In PC12 cells, both NGF and platelet-derived growth factor (23) promote neurite outgrowth and neuronal differentiation. In contrast, EGF, insulin, and insulin-like growth factor-1 stimulate cell proliferation (11, 24). This correlation raises the possibility that ligand-dependent tyrosyl phosphorylation of SH2-Bbeta plays a critical role in promoting neurite outgrowth of PC12 cells. Consistent with this idea, NGF stimulates robust tyrosyl phosphorylation of GFP-SH2-Bbeta (Fig. 3b), whose expression enhances NGF-induced neurite outgrowth. However, NGF is unable to stimulate the tyrosyl phosphorylation of GFP-SH2-Bbeta (R555E) (Fig. 3b), which acts as a dominant negative SH2-Bbeta to block NGF-induced neurite outgrowth. Because SH2-Bbeta but not SH2-Bbeta (R555E) binds to TrkA (Fig. 1a), it is likely that binding of SH2-Bbeta to TrkA is required for its phosphorylation and that SH2-Bbeta is tyrosyl phosphorylated directly by activated TrkA.

SH2-Bbeta (R555E) neither binds to TrkA nor interferes with phosphorylation of endogenous SH2-Bbeta (data not shown) in response to NGF. Therefore, it seems likely that SH2-Bbeta (R555E) functions as a dominant negative mutant to interfere with the action of endogenous SH2-Bbeta by competing with endogenous SH2-Bbeta for downstream effector(s) and sequestering these putative effector(s) in an inactive state. Phosphorylation of SH2-Bbeta does not appear to be required for sequestration of effectors, because GFP-SH2-Bbeta (R555E) is not significantly phosphorylated in response to NGF (Fig. 2b). However, phosphorylation of SH2-Bbeta may be important for activation of downstream effector(s). Alternatively, the downstream effectors of SH2-Bbeta may be activated when recruited to TrkA-containing complexes by the interaction of SH2-Bbeta with TrkA.

Interestingly, overexpression of neither GFP-SH2-Bbeta (R555E) nor GFP-SH2-Bbeta affect NGF-induced tyrosyl phosphorylation of TrkA, PLCgamma , three isoforms of Shc and activation of ERK1/2, indicating that the signaling events immediately downstream of TrkA and the Shc/Ras/MEK/ERKs cascade are not compromised by overexpression of wild type or dominant negative mutant SH2-Bbeta . This observation demonstrates that the dominant negative effect of SH2-Bbeta (R555E) on NGF-induced neurite outgrowth is not secondary to a defect in the activation of TrkA, the ability of TrkA to tyrosyl phosphorylate its substrates, or the activation of the Shc/Ras/MEK/ERK cascade by NGF. Thus, SH2-Bbeta is likely to initiate a novel pathway required for NGF-induced neurite outgrowth.

In conclusion, we show that the putative adapter protein SH2-Bbeta binds via its SH2 domain to activated TrkA and is tyrosyl phosphorylated in response to NGF. We also demonstrate that overexpression of SH2-Bbeta enhances NGF-induced neuronal differentiation, whereas overexpression of a dominant negative SH2-Bbeta blocks that differentiation. These findings provide the first insight into the cellular function of SH2-B. They also suggest that SH2-Bbeta may be one of the hypothesized "missing links" parallel to or downstream of ERKs that are thought to be required for NGF-induced differentiation.

    ACKNOWLEDGEMENTS

We thank Dr. Louis F. Reichardt for providing us with antibody to TrkA. We thank Drs. L. S. Argetsinger, J. A. VanderKuur, and M. Stofega for discussions, X. Wang for technical assistance, Drs. B. Margolis and D. Meyer for the gift of PC12 cells, and Dr. K. S. O'Shea for help with phase contrast microscopy.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK 34171 (to C. C.-S.) and National Research Service Award F32-DK-09756 (to J. H.). Oligonucleotides were synthesized by the Biomedical Research Core Facilities, University of Michigan, work that was supported in part by National Institutes of Health Grant P60-DK-20572 to the Cancer Center, Michigan Diabetic Research and Training Center and National Institutes of Health Grant P60-AR20557 to University of Michigan Multipurpose Arthritis and Musculoskeletal Diseases Center. This work was also supported in part by National Institutes of Health Grants AR20557 and CA46592 to the University of Michigan Core Flow Cytometry Facility.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Predoctoral Fellowship from the Rackham School of Graduate Studies, University of Michigan.

§ To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622. Tel.: 734-763-2561; Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.

2 K. Nelms, personal communication.

3 Rui, L, J. Herrington and C. Carter-Su, manuscript in preparation.

4 The apparent difference in the amount of GFP-SH2-Bbeta in control and NGF-treated cells in Fig. 3b (lower panel) is an artifact of this mobility shift and the large amount of GFP-SH2-Bbeta . Experiments in which a smaller amount of GFP-SH2-Bbeta was electrophoresed revealed similar amounts of GFP-SH2-Bbeta from control and NGF-stimulated cells.

    ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; EGF, epidermal growth factor; SH, Src homology; PLCgamma , phospholipase Cgamma ; GST, glutathione S-transferase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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