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PubMed1997

Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS-1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase.

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.

Animals↗
PubMed1999

The Shp-2 tyrosine phosphatase activates the Src tyrosine kinase by a non-enzymatic mechanism.

Previously, we demonstrated that the Src tyrosine kinase interacts with the Shp-2 tyrosine phosphatase. To determine whether Shp-2 regulates Src kinase activity, we measured Src activity in cells overexpressing wild-type or catalytically-inactive C463S Shp-2. We observed a 2-3-fold increase in the specific activity of Src in both cell types and the increase did not appear to be due to dephosphorylation of Tyr 527 or phosphorylation of Tyr 416 on Src. Conversely, we observed a 2-3-fold decrease in the specific activity of Src when Shp-2 expression was inhibited. Using glutathione S-transferase-fusion proteins, we demonstrated that Shp-2 binds to the SH3 domain of Src. Our findings reveal that the Shp-2 tyrosine phosphatase can regulate the Src tyrosine kinase by a non-enzymatic mechanism. We also found that the phosphatase activity of Shp-2 immunoprecipitates is downregulated in cells transformed by Src or other proteins, and that Shp-2 preferentially associates with the membrane fraction of transformed cells. We suggest that membrane-association of Shp-2 is important for regulating Shp-2 activity.

3T3 Cells↗
PubMed2001

Protein tyrosine phosphatase phi regulates paxillin tyrosine phosphorylation and mediates colony-stimulating factor 1-induced morphological changes in macrophages.

Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase phi (PTP phi). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP phi and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTP phi induces cell rounding and ruffle formation, while C325S PTP phi has no effect. In contrast, in CSF-1-starved cells, C325S PTP phi behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP phi increases adhesion in cycling cells, while WT PTP phi enhances motility. In WT PTP phi-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTP phi-expressing cells. Moreover, a complex containing PTP phi, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTP phi selectively binds paxillin and Pyk2 in vitro. Although PTP phi and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTP phi in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.

Blotting, Western↗
PubMed2003

TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex.

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.

3T3 Cells↗
PubMed2001

Tyrosine phosphorylation-dependent activation of NF-kappa B. Requirement for p56 LCK and ZAP-70 protein tyrosine kinases.

Phosphorylation of the N-terminal domain of I kappa B inhibitory subunits induces activation of the transcription factor NF-kappa B. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome-mediated degradation of I kappa B-alpha, little is known about the mechanisms that lead to release of active NF-kappa B in T cells as a consequence of tyrosine phosphorylation of I kappa B-alpha [Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell 86, 787--798]. The involvement of the tyrosine kinases p56(lck) and ZAP-70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate-induced phosphorylation of I kappa B-alpha on Tyr42 and NF-kappa B activation, we observed that, in p56(lck)-deficient Jurkat mutants, NF-kappa B could still associate with I kappa B-alpha despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56(lck) appeared to be required for pervanadate-induced NF-kappa B activation but not for Tyr42 phosphorylation. These results show that p56(lck) and ZAP-70 are key components of the signaling pathway that leads to phosphotyrosine-dependent NF-kappa B activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF-kappa B. Finally, we show that H(2)O(2), which stimulates p56(lck) and ZAP-70 in T cells, is an activator of NF-kappa B through tyrosine phosphorylation of I kappa B-alpha.

DNA↗
PubMed2005

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases.

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.

Animals↗
PubMed2003

Evidence for protein tyrosine phosphatase, tyrosine kinase, and G-protein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons.

The slow EPSP (sEPSP) or slow EPSC (sEPSC) at parallel fiber to Purkinje neuron synapses is attributable to a nonselective cation channel coupled to activation of metabotropic type 1 glutamate receptors (mGluR1s). Photorelease of L-glutamate in 1 msec from 4-methoxy-7-nitroindolinyl-or 7-nitroindolinyl-caged glutamate in cerebellar slices was used to isolate and study postsynaptic mechanisms coupling mGluR1 to the cation channel. L-Glutamate immediately activated a glutamate transporter current, followed by the slow mGluR1-activated conductance. Inhibitors of kinases, phosphatases, and G-proteins were tested on the peak glutamate-evoked currents. No effects of the inhibitors were seen on the initial glutamate transporter currents. In contrast, the later mGluR1 currents were either unaffected or enhanced by the protein tyrosine kinase (PTK) inhibitors PP1, K252a, and staurosporine were diminished or blocked by phosphatase inhibitors but were unaffected by inhibitors of serine-threonine kinases PKA, PKC, or PKG. The selective src-PTK inhibitor PP1 (10 microm intracellularly) potentiated submaximal mGluR1 currents evoked by low L-glutamate concentrations but had no effect on maximal responses (80 or 160 microm L-glutamate). L-Glutamate-evoked mGluR1 currents and parallel fiber sEPSCs were reversibly and completely inhibited by protein tyrosine phosphatase (PTP) inhibitor bpV(phen) (50-200 microm) and by nonselective phosphatase inhibitor orthovanadate (0.5 or 1 mm). mGluR1 currents were completely inhibited by GDPbetaS applied intracellularly (5 mm). The results confirm a role for a GTPase postsynaptically, show that tyrosine phosphorylation inhibits mGluR1 coupling to the channel, and show that PTPs increase activation by tyrosine dephosphorylation most likely upstream of the sEPSP cation channel.

Animals↗
PubMed1993

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.

Amino Acid Sequence↗
PubMed1991

Conversion of p-tyrosine to p-tyramine in the isolated perfused rat kidney: modulation by perfusate concentrations of p-tyrosine.

We used the isolated perfused rat kidney to evaluate the role of renal decarboxylation of p-tyrosine as the source of urinary p-tyramine. Kidneys were perfused with concentrations of p-tyrosine ranging from 0.02 mM to 2.0 mM. p-Tyramine was measured by a sensitive and specific radioenzymatic assay. An increase in the perfusate concentration of p-tyrosine resulted in a significant increase in p-tyramine production that was blocked by the addition of NSD-1015, an inhibitor of aromatic-1-amino decarboxylase (AADC). We conclude p-tyrosine is the precursor for the renal production of p-tyramine, renal AADC catalyzes the formation of urinary p-tyramine, synthesized p-tyramine is predominantly excreted in the urine, and p-tyramine synthesis is modulated by the arterial delivery of p-tyrosine to the kidney.

Animals↗
PubMed1995

The tyrosine activation motif as a target of protein tyrosine kinases and SH2 domains.

The signaling subunits of antigen receptor and Fc receptor complexes carry a tyrosin-based activation motif (ITAM). Work of the recent years showed that this motif is required for the activation of protein tyrosine kinases (PTK) via these receptors. We discuss here two models of how ITAM either in its phosphorylated or unphosphorylated state may interact with PTKs. After receptor cross-linking the activated PTKs will also phosphorylate the tyrosines of ITAM. We have found that different members of the src-family of kinases can phosphorylate either both tyrosines or only the first tyrosine of ITAM. We further discuss how this alternative phosphorylation of ITAM can result in the interaction of the BCR with different SH2-containing proteins and thus influence the signal transduction from the receptor.

Amino Acid Sequence↗
PubMed2005

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z.

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

Amino Acid Sequence↗
PubMed2002

Simultaneous determination of 3-nitrotyrosine and tyrosine in plasma proteins of rats and assessment of artifactual tyrosine nitration.

A sensitive and specific isotope dilution liquid chromatography-electrospray tandem mass spectrometry method was developed for the determination of the 3-nitrotyrosine residue levels in rat plasma proteins. The assay is based on the cleavage of proteins with concentrated hydrochloric acid to release both 3-nitrotyrosine and tyrosine. To control the potential artifactual nitration of tyrosine residues during the proteolysis, samples are spiked with (13)C(9)-labeled tyrosine and the level of (13)C(9)-labeled 3-nitrotyrosine is measured. The clean-up process entails hydrolysate fortification with 2,5,6-d(3)-3-nitrotyrosine, followed by solid-phase extraction on octadecylsilyl (to isolate tyrosine) and aminopropylsilyl (to isolate 3-nitrotyrosine) cartridges. Tyrosine and 3-nitrotyrosine fractions are mixed in an appropriate ratio prior to the analysis. The method was applied to animals exposed to ferric nitrilotriacetate to induce oxidative stress.

Animals↗
PubMed2004

Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages.

Loss-of-function mutations in the murine dominant white spotting/c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.

Animals↗
PubMed1996

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor.

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.

Animals↗
PubMed1996

Growth and survival signals transmitted via two distinct NPXY motifs within leukocyte tyrosine kinase, an insulin receptor-related tyrosine kinase.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase, which belongs to the insulin receptor family and is mainly expressed in pre-B cells and brain. In this study, we show that LTK utilizes insulin receptor substrate-1 (IRS-1) and Shc as major two substrates and possesses two NPXY motifs for them separately, tyrosine 485 of one NPXY motif at the juxtamembrane domain for IRS-1 and tyrosine 862 of another NPXY motif at the carboxyl-terminal domain for Shc. By using Ba/F3 cells expressing epidermal growth factor receptor-LTK chimeric receptors containing a mutation at each NPXY site, we showed that while both NPXY motifs equally contribute to activation of the Ras pathway and generation of mitogenic signals, only tyrosine 485 of LTK transmits cell survival signals. These data suggest that IRS-1 possesses anti-apoptotic function at least in LTK signaling. Moreover, our data indicate that the survival signaling pathway of LTK is distinct from the Ras pathway and the p70(S6) kinase pathway. Our results provide a useful insight in understanding the distinctive roles of Shc and IRS-1 in the signal transduction system of the insulin receptor family, and this anti-apoptotic function of IRS-1 may explain the survival effects of insulin, IGF-1, and interleukin 4.

Amino Acid Sequence↗
PubMed1996

p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase.

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling.

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca(2+) flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr(171), and Tyr(191), but not Tyr(226), are necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma1 binding, suggesting there might be multiple binding sites for PLC-gamma1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA.

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.

Adaptor Proteins, Signal Transducing↗