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PubMed2005

Phosphatidylinositol phosphate kinase type Igamma directly associates with and regulates Shp-1 tyrosine phosphatase.

Tyrosine phosphorylation plays a critical role in many regulatory aspects of cellular signaling, and dephosphorylation of phosphotyrosine residues is crucial for termination of signals initiated by tyrosine kinases. Previous work has shown that the tyrosine kinase Src phosphorylates Tyr644 on phosphatidylinositol phosphate kinase type I (PIPKI) gamma661 in a focal adhesion kinase-dependent manner. Phosphorylation of this residue is essential for high affinity binding of PIPKI gamma661 to the focal adhesion protein talin and for targeting of PIPKI gamma661 to focal adhesions. A yeast two-hybrid screen performed with the C-terminal 178-amino acid tail of PIPKI gamma661 identified an interaction with the phosphatase domain of the tyrosine phosphatase Shp-1. The interaction between PIPKI gamma661 and Shp-1 was confirmed via co-immunoprecipitation from HEK293 cell lysates. In addition, Src-phosphorylated PIPKI gamma661 is a substrate for Shp-1, and Shp-1 modulates both the association between PIPKI gamma661 and talin and the targeting of PIPKI gamma661 to focal adhesions in mammalian cells. Finally, we showed that Shp-1 phosphatase activity is inhibited by the product of PIPKI gamma661, phosphatidylinositol 4,5-bisphosphate, in vitro. These combined results suggest a model in which the reciprocal actions of Src tyrosine kinase and Shp-1 tyrosine phosphatase dynamically regulate the association between PIPKI gamma661 and talin.

Animals↗
PubMed2006

The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast cell proliferation by activating the Rac/JNK pathway.

The scaffolding adapter Gab2 mediates cell signaling and responses evoked by various extracellular stimuli including several growth factors. Kit, the receptor for stem cell factor (SCF), plays a critical role in the proliferation and differentiation of a variety of cell types, including mast cells. Kit, via Tyr(567) and Tyr(719), activates Src family kinases (SFK) and PI3K respectively, which converge on the activation of a Rac/JNK pathway required for mast cell proliferation. However, how Kit Tyr(567) signals to Rac/JNK is not well understood. By analyzing Gab2(-/-) mast cells, we find that Gab2 is required for SCF-evoked proliferation, activation of Rac/JNK, and Ras. Upon Kit activation in wild-type mast cells, Gab2 becomes tyrosyl-phosphorylated and associates with Kit and Shp-2. Tyr(567), an SFK binding site in Kit, and SFK activity were required for Gab2 tyrosyl phosphorylation and association with Shp-2. By re-expressing Gab2 or a Gab2 mutant that cannot bind Shp-2 in Gab2(-/-) mast cells or acutely by deleting Shp-2 in mast cells, we found that Gab2 requires Shp-2 for SCF-evoked Rac/JNK, Ras activation, and mast cell proliferation. Lastly, by analyzing mast cells from mice with compound Gab2 and Kit Y719F mutations (i.e., Gab2(-/-): KitY719F/Y719F mice), we find that Gab2, acting in a parallel pathway to PI3K from Kit Tyr(719), regulates mast cell proliferation and development in specific tissues. Our data show that Gab2 via Shp-2 is critical for transmitting signals from Kit Tyr(567) to activate the Rac/JNK pathway controlling mast cell proliferation, which likely contributes to mast cell development in specific tissues.

Adaptor Proteins, Signal Transducing↗
PubMed2006

SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth.

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle-specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]-SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK-SHP-2-null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.

Animals↗
PubMed2002

A transcriptional inhibitor targeted by the atypical orphan nuclear receptor SHP.

SHP (short heterodimer partner, NROB2) is an atypical orphan member of the mammalian nuclear receptor family that consists only of a putative ligand-binding domain and thus cannot bind DNA. Instead, SHP acts as a transcriptional coregulator by inhibiting the activity of various nuclear receptors (downstream targets) via occupation of the coactivator-binding surface and active repression. However, repression mechanisms have remained elusive and may involve coinhibitory factors (upstream targets) distinct from known nuclear receptor corepressors. Here, we describe the isolation of mouse E1A-like inhibitor of differentiation 1 (EID1) as a candidate coinhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action and propose that SHP inhibition of transcription involves EID1 antagonism of CBP/p300-dependent coactivator functions.

Acetyltransferases↗
PubMed2002

Expression of SHP-1 phosphatase indicates post-germinal center cell derivation of B-cell posttransplant lymphoproliferative disorders.

SHP-1 tyrosine phosphatase acts as a negative regulator of signaling by receptors for growth factors, cytokines, and chemokines and by receptors involved in immune response. Our recent study showed that SHP-1 is tightly regulated at various stages of B-cell differentiation and is expressed in the mantle and marginal zones, interfollicular B cells, and plasma cells, whereas it is nondetectable in germinal center cells. In this study we evaluated expression of SHP-1 in vitro and in vivo in nine cell lines representing three different types of EBV+ B-cell populations closely resembling or derived from posttransplant lymphoproliferative disorders (PTLDs). Furthermore, we examined tissue samples from 58 patients with B-cell PTLDs, both EBV+ (85% of the cases analyzed) and EBV- (15%). SHP-1 protein was strongly expressed in all cell lines and PTLD cases. In addition, the PTLD cases were essentially negative for germinal center B-cell markers: none expressed CD10 and only one expressed BCL-6. More than 40% expressed a late post-germinal B-cell marker, CD138. The universal expression of SHP-1, lack of expression of CD10 and BCL-6, and frequent expression of CD138 suggest that PTLDs are derived from post-germinal center B cells regardless of the EBV cell infection status. Based on the immunophenotype, B-cell PTLDs could be divided into two broad categories corresponding to the early (CD10-/BCL-6-/SHP-1+/CD138-) and late (CD10-/BCL-6-/SHP-1+/CD138+) post-germinal center cells. By being expressed earlier, SHP-1 is a more sensitive marker of post-germinal center B cells than CD138, which is seen on the terminally differentiated immunoblasts and plasma cells.

B-Lymphocytes↗
PubMed1995

Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

Amino Acid Sequence↗
PubMed2006

Role of host protein tyrosine phosphatase SHP-1 in Leishmania donovani-induced inhibition of nitric oxide production.

In order to survive within the macrophages of its host organism, the protozoan parasite Leishmania inhibits a number of critical, gamma interferon (IFN-gamma)-inducible, macrophage functions, including the generation of nitric oxide. We have previously shown that the protein tyrosine phosphatase SHP-1 (Src-homology 2 domain containing phosphatase-1) is activated during Leishmania infection and plays an important role in both the survival of Leishmania within cultured macrophages and disease progression in vivo by inhibiting nitric oxide production. Here we use a SHP-1-/- macrophage cell line derived from motheaten mice to address the mechanisms by which SHP-1 prevents IFN-gamma-dependent nitric oxide production during Leishmania donovani infection. We show that Leishmania inhibits nitric oxide production in response to IFN-gamma poorly in SHP-1-deficient macrophages. This correlates with the inability of Leishmania to alter JAK2 and mitogen-activated protein kinase extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and to prevent nuclear translocation of transcription factors NF-kappaB and AP-1, although the latter two to a lesser extent. Surprisingly, Leishmania inactivated the transcription factor STAT1 to a similar extent in SHP-1-deficient and wild-type macrophages, so STAT1 is not necessary for nitric oxide production by infected macrophages. Overall, this study demonstrates that induction of SHP-1 by Leishmania is vital for inhibition of nitric oxide generation and that this inhibition occurs through the inactivation of JAK2 and ERK1/2, and transcription factors NF-kappaB and AP-1.

Animals↗
PubMed2000

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase.

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

Animals↗
PubMed2003

Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity.

Here, we present data suggesting a novel mechanism for regulation of natural killer (NK) cell cytotoxicity through inhibitory receptors. Interaction of activation receptors with their ligands on target cells induces cytotoxicity by NK cells. This activation is under negative control by inhibitory receptors that recruit tyrosine phosphatase SHP-1 upon binding major histocompatibility class I on target cells. How SHP-1 blocks the activation pathway is not known. To identify SHP-1 substrates, an HLA-C-specific inhibitory receptor fused to a substrate-trapping mutant of SHP-1 was expressed in NK cells. Phosphorylated Vav1, a regulator of actin cytoskeleton, was the only protein detectably associated with the catalytic site of SHP-1 during NK cell contact with target cells expressing HLA-C. Vav1 trapping was independent of actin polymerization, suggesting that inhibition of cellular cytotoxicity occurs through an early dephosphorylation of Vav1 by SHP-1, which blocks actin-dependent activation signals. Such a mechanism explains how inhibitory receptors can block activating signals induced by different receptors.

Actins↗
PubMed2003

Inhibition of tumor necrosis factor-[alpha]-induced SHP-2 phosphatase activity by shear stress: a mechanism to reduce endothelial inflammation.

OBJECTIVE: Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity. METHODS AND RESULTS: Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation. CONCLUSIONS: These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Angiotensin II type 2 receptor inhibits epidermal growth factor receptor transactivation by increasing association of SHP-1 tyrosine phosphatase.

Angiotensin (Ang) II has 2 major receptor isoforms, Ang type 1 (AT(1)) and Ang type (AT(2)). AT(1) transphosphorylates epidermal growth factor receptor (EGFR) to activate extracellular signal-regulated kinase (ERK). Although AT(2) was shown to inactivate ERK, the action of AT(2) on EGFR activation remains undefined. Using AT(2)-overexpressing vascular smooth muscle cells from AT(2) transgenic mice, we studied these undefined actions of AT(2). Maximal ERK activity induced by Ang II was increased 1.9- and 2.2-fold by AT(2) inhibition, which was abolished by orthovanadate but not okadaic acid or pertussis toxin. AT(2) inhibited AT(1)-mediated EGFR tyrosine phosphorylation by 63%. The activity of SHP-1 tyrosine phosphatase was significantly upregulated 1 minute after AT(2) stimulation and association of SHP-1 with EGFR was increased, whereas AT(2) failed to tyrosine phosphorylate SHP-1. Stable overexpression of SHP-1-dominant negative mutant completely abolished AT(2)-mediated inhibition of EGFR and ERK activation. AT(1)-mediated c-fos mRNA accumulation was attenuated by 48% by AT(2) stimulation. Induction of fibronectin gene containing an AP-1 responsive element in its 5'-flanking region was decreased by 37% after AT(2) stimulation, corresponding to the results of gel mobility assay with the AP-1 sequence of fibronectin as a probe. These findings suggested that AT(2) inhibits ERK activity by inducing SHP-1 activity, leading to decreases in AP-1 activity and AP-1-regulated gene expression, in which EGFR dephosphorylation plays an important role via association of SHP-1.

Angiotensin II↗
PubMed2005

Evaluation of normal and neoplastic human mast cells for expression of CD172a (SIRPalpha), CD47, and SHP-1.

Signal regulatory proteins (SIRPs) and tyrosine phosphatases have recently been implicated in the control of receptor tyrosine kinase (RTK)-dependent cell growth. In systemic mastocytosis (SM), neoplastic cells are driven by the RTK KIT, which is mutated at codon 816 in most patients. We examined expression of SIRPalpha, SIRPalpha ligand CD47, and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), a tyrosine phosphatase-type, negative regulator of KIT-dependent signaling, in normal human lung mast cells (HLMC) and neoplastic MC obtained from nine patients with SM. As assessed by multicolor flow cytometry, normal LMC expressed SIRPalpha, CD47, and SHP-1. In patients with SM, MC also reacted with antibodies against SIRPalpha and CD47. By contrast, the levels of SHP-1 were low or undetectable in MC in most cases. Corresponding data were obtained from mRNA analysis. In fact, whereas SIRPalpha mRNA and CD47 mRNA were detected in all samples, the levels of SHP-1 mRNA varied among donors. To demonstrate adhesive functions for SIRPalpha and CD47 on neoplastic MC, an adhesion assay was applied using the MC leukemia cell line HMC-1, which was found to bind to immobilized extracellular domains of SIRPalpha1 (SIRPalpha1ex) and CD47 (CD47ex), and binding of these cells to CD47ex was inhibited by the CD172 antibody SE5A5. In summary, our data show that MC express functional SIRPalpha and CD47 in SM, whereas expression of SHP-1 varies among donors and is low compared with LMC. It is hypothesized that CD172 and CD47 contribute to MC clustering and that the "lack" of SHP-1 in MC may facilitate KIT-dependent signaling in a subgroup of patients.

Antigens, CD↗
PubMed2000

Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B.

Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning to be understood, the signaling events that terminate GH signaling, such as dephosphorylation of tyrosyl-phosphorylated signaling molecules, are poorly understood. In this report, we examine the role of the SH2 (Src homology-2) domain-containing protein tyrosine phosphatase SHP-2 in GH signaling. We demonstrate that the SH2 domains of SHP-2 bind directly to tyrosyl phosphorylated GHR from GH-treated cells. Tyrosine-to-phenylalanine mutation of tyrosine 595 of rat GHR greatly diminishes association of the SH2 domains of SHP-2 with GHR, and tyrosine-to-phenylalanine mutation of tyrosine 487 partially reduces association of the SH2 domains of SHP-2 with GHR. Mutation of tyrosine 595 dramatically prolongs the duration of tyrosyl phosphorylation of the signal transducer and activator of transcription STAT5B in response to GH, while mutation of tyrosine 487 moderately prolongs the duration of STAT5B tyrosyl phosphorylation. Consistent with the effects on STAT5B phosphorylation, tyrosine-to-phenylalanine mutation of tyrosine 595 prolongs the duration of tyrosyl phosphorylation of GHR and JAK2. These data suggest that tyrosine 595 is a major site of interaction of GHR with SHP-2, and that GHR-bound SHP-2 negatively regulates GHR/JAK2 and STAT5B signaling.

3T3 Cells↗
PubMed2002

Deregulation of SHP-2 tyrosine phosphatase by the Helicobacter pylori virulence factor CagA.

Helicobacter pylori (H. pylori) is estimated to infect about half of the world population. It causes gastric diseases ranging from gastritis to cancer and has been classified as a class I carcinogen by WHO. However, little is known about the molecular mechanisms by which H. pylori induces pathogenesis. CagA is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe clinical outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. We found that wild-type, but not phosphorylation-resistant CagA, is capable of inducing a growth factor-like morphological change, termed the hummingbird phenotype, in cells. Furthermore, CagA specifically binds the SH2-containing protein tyrosine phosphatase SHP-2 in a tyrosine phosphorylation-dependent manner and stimulates phosphatase activity. Disruption of the CagA-SHP-2 complex abolishes the CagA-dependent morphological change. Conversely, constitutively active SHP-2 is capable of inducing a CagA-like morphological change when it is plasma membrane-targeted. Our results show that CagA perturbs cellular functions by deregulating SHP-2 phosphatase after translocation from H. pylori into gastric epithelial cells. Given the positive regulatory roles of SHP-2 in both cell proliferation and cell movement, the CagA-SHP-2 interaction may play an important role in the oncogenic transformation that is a hallmark of cagA+ H. pylori infection.

Antigens, Bacterial↗
PubMed2002

SHP-1- and phosphotyrosine-independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells.

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-gamma production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.

Amino Acid Motifs↗
PubMed2004

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2).

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.

Amino Acid Sequence↗
PubMed1996

Multiple in vivo phosphorylated tyrosine phosphatase SHP-2 engages binding to Grb2 via tyrosine 584.

SHP-2 (also named PTP1D, syp, or SH-PTP2) has been identified as a phosphotyrosine phosphatase comprising two src-homology-2 (SH2) domains. Upon growth factor stimulation, SHP-2 becomes tyrosine phosphorylated, thereby increasing its catalytic activity. Here, we identified SHP-2 to be phosphorylated on multiple tyrosine residues in response to different stimuli and unmasked the carboxyl-terminal tyrosine 584 as a major phosphorylation site in human cell lines. Tyrosine 584 shares, together with tyrosine 546, the consensus sequence pY-X-N-X, a characteristic of potential binding sites for the SH2 domain of growth factor receptor-bound protein 2 (Grb2). We show here that mutation of tyrosine 584, but not tyrosine 546, to phenylalanine totally abolished the binding of Grb2 to SHP-2. By using a systematic mutagenesis approach, phosphorylation of additional tyrosines in each of the SH2 domains of SHP-2 was detected after coexpression of epidermal growth factor receptor, but not after coexpression of platelet-derived growth factor receptor, whereas tyrosine 263 located in the interspace between SH2 and catalytic domain appears to be exclusively recognized by platelet-derived growth factor receptor. Immunoprecipitation of SHP-2 from a panel of mammary carcinoma cell lines copurifies several tyrosine phosphorylated proteins; the most prominent band has an apparent molecular weight of M(r) 115,000.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Protein tyrosine phosphatase SHP-1 fails to associate with cytoskeleton but is normally phosphorylated upon thrombin stimulation of thrombasthenic platelets.

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.

Blood Platelets↗