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1.  Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1 
Biochemistry  2013;52(15):2597-2608.
The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals.
doi:10.1021/bi301461t
PMCID: PMC3666314  PMID: 23418871
2.  CD5 Negatively Regulates the T-Cell Antigen Receptor Signal Transduction Pathway: Involvement of SH2-Containing Phosphotyrosine Phosphatase SHP-1 
Molecular and Cellular Biology  1999;19(4):2903-2912.
The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3ζ, ZAP-70, Syk, and phospholipase Cγl but not the Src family tyrosine kinase p56lck. By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.
PMCID: PMC84084  PMID: 10082557
3.  Siglec-9 and SHP-1 are Differentially Expressed in Neonatal and Adult Neutrophils 
Pediatric research  2009;66(3):266-271.
Neonatal PMN exhibit altered inflammatory responsiveness and greater longevity compared to adult PMN; however, the involved mechanisms are incompletely defined. Receptors containing immunoreceptor tyrosine-based inhibitory motif (ITIM) domains promote apoptosis by activating inhibitory phosphatases, such as Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1), that block survival signals. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9, an immune inhibitory receptor with ITIM domains, has been shown to induce cell death in adult PMN in association with SHP-1. To test our hypothesis that neonatal PMN inflammatory function may be modulated by unique Siglec-9 and SHP-1 interactions, we compared expression of these proteins in adult and neonatal PMN. Neonatal PMN exhibited diminished cellular expression of Siglec-9, which was phosphorylated in the basal state. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment decreased Siglec-9 phosphorylation levels in neonatal PMN but promoted its phosphorylation in adult PMN, observations associated with altered survival signaling. While SHP-1 expression was also diminished in neonatal PMN, GM-CSF treatment had minimal effect on phosphorylation status. Further analysis revealed that Siglec-9 and SHP-1 physically interact, as has been observed in other immune cells. Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN.
doi:10.1203/PDR.0b013e3181b1bc19
PMCID: PMC2753195  PMID: 19542910
4.  Phosphorylation and Localization of Protein-Zero Related (PZR) in Cultured Endothelial Cells 
Protein-zero related (PZR) is an IgV-type immunoreceptor with two immunoreceptor tyrosine based inhibitory motifs (ITIMs). PZR interacts with Src homology 2 domain containing tyrosine phosphatase (SHP-2) via its tyrosine phosphorylated ITIMs, for which c-Src is a putative kinase. Towards elucidating PZR function in endothelial cells (ECs), we cloned PZR from bovine aortic endothelial cells (BAECs) and characterized it. Mature bovine PZR had 94.8% and 92.7% sequence identity with canine and human proteins, respectively, and the two ITIM sequences were conserved among higher vertebrates. PZR was expressed in many cell types and was localized to cell contacts and intracellular granules in BAECs and mesothelioma (REN) cells. Co-immunoprecipitation revealed that PZR, Grb-2-associated binder-1 (Gab1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) were three major SHP-2 binding proteins in BAECs. H2O2 enhanced PZR tyrosine phosphorylation and PZR/SHP-2 interaction in ECs in a dose- and time-dependent manner. To see if tyrosine kinases other than Src are also capable of phosphorylating PZR, we co-transfected HEK293 cells with PZR and one of several tyrosine kinases and found that c-Src, c-Fyn, c-Lyn, Csk and c-Abl but not c-Fes phosphorylated PZR and increased PZR/SHP-2 interaction. These results suggest that PZR is a cell adhesion protein which may be involved in SHP-2-dependent signaling at interendothelial cell contacts.
doi:10.1080/10623320802125250
PMCID: PMC2626187  PMID: 18568953
protein zero related (PZR); endothelial cells; tyrosine phosphorylation; ITIM; SHP-2; cell contacts
5.  Paired Immunoglobulin-like Receptor (PIR)-A Is Involved in Activating Mast Cells through Its Association with Fc Receptor γ Chain  
Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain–containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor γ chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated γ chain, whereas PIR-B mediates the inhibitory signal through its ITIMs.
PMCID: PMC2213385  PMID: 9730901
activation signal; Fc receptor γ chain; immunoreceptor tyrosine-based activation motif; mast cell; paired immunoglobulin-like receptor A
6.  Critical Role of Src and SHP-2 in sst2 Somatostatin Receptor-mediated Activation of SHP-1 and Inhibition of Cell Proliferation 
Molecular Biology of the Cell  2003;14(9):3911-3928.
The G protein-coupled sst2 somatostatin receptor acts as a negative cell growth regulator. Sst2 transmits antimitogenic signaling by recruiting and activating the tyrosine phosphatase SHP-1. We now identified Src and SHP-2 as sst2-associated molecules and demonstrated their role in sst2 signaling. Surface plasmon resonance and mutation analyses revealed that SHP-2 directly associated with phosphorylated tyrosine 228 and 312, which are located in sst2 ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This interaction was required for somatostatin-induced SHP-1 recruitment and activation and consequent inhibition of cell proliferation. Src interacted with sst2 and somatostatin promoted a transient Gβγ-dependent Src activation concomitant with sst2 tyrosine hyperphosphorylation and SHP-2 activation. These steps were abrogated with catalytically inactive Src. Both catalytically inactive Src and SHP-2 mutants abolished somatostatin-induced SHP-1 activation and cell growth inhibition. Sst2–Src–SHP-2 complex formation was dynamic. Somatostatin further induced sst2 tyrosine dephosphorylation and complex dissociation accompanied by Src and SHP-2 inhibition. These steps were defective in cells expressing a catalytically inactive Src mutant. All these data suggest that Src acts upstream of SHP-2 in sst2 signaling and provide evidence for a functional role for Src and SHP-2 downstream of an inhibitory G protein-coupled receptor.
doi:10.1091/mbc.E03-02-0069
PMCID: PMC196590  PMID: 12972574
7.  The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells 
Molecular Biology of the Cell  2003;14(9):3519-3528.
Growth factor signaling is usually analyzed in isolation without considering the effect of ligand occupancy of transmembrane proteins other than the growth factor receptors themselves. In smooth muscle cells, the transmembrane protein Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) has been shown to be an important regulator of insulin-like growth factor-I (IGF-I) signaling. SHPS-1 is phosphorylated in response to IGF-I, leading to recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2). Subsequently, SHP-2 is transferred to IGF-I receptor and regulates the duration of IGF-I receptor phosphorylation. Whether ligand occupancy of SHPS-1 influences SHPS-1 phosphorylation or SHP-2 recruitment, thereby altering growth factor signaling, is unknown. Previous studies have shown that integrin associated protein (IAP) associates with SHPS-1. We undertook these studies to determine whether this interaction controlled SHPS-1 phosphorylation and/or SHP-2 recruitment and thereby regulated IGF-I signaling. Disruption of IAP-SHPS-1 binding, by using an IAP monoclonal antibody or cells expressing mutant forms of IAP that did not bind to SHPS-1, inhibited IGF-I–stimulated SHPS-1 phosphorylation and SHP-2 recruitment. This was associated with a lack of SHP-2 transfer to IGF-I receptor and sustained receptor phosphorylation. This resulted in an inability of IGF-I to stimulate sustained mitogen-activated protein kinase activation, cell proliferation, and cell migration. The effect was specific for IGF-I because disruption of the IAP–SHPS-1 interaction had no effect on platelet-derived growth factor-stimulated SHPS-1 phosphorylation or cell migration. In summary, our results show that 1) ligand occupancy of SHPS-1 is a key determinant of its ability to be phosphorylated after IGF-I stimulation, and 2) the interaction between IAP and SHPS-1 is an important regulator of IGF-I signaling because disruption of the results in impaired SHP-2 recruitment and subsequent inhibition of IGF-I–stimulated cell proliferation and migration.
doi:10.1091/mbc.E03-04-0239
PMCID: PMC196546  PMID: 12972543
8.  Regulation of hERG and hEAG Channels by Src and by SHP-1 Tyrosine Phosphatase via an ITIM Region in the Cyclic Nucleotide Binding Domain 
PLoS ONE  2014;9(2):e90024.
Members of the EAG K+ channel superfamily (EAG/Kv10.x, ERG/Kv11.x, ELK/Kv12.x subfamilies) are expressed in many cells and tissues. In particular, two prototypes, EAG1/Kv10.1/KCNH1 and ERG1/Kv11.1/KCNH2 contribute to both normal and pathological functions. Proliferation of numerous cancer cells depends on hEAG1, and in some cases, hERG. hERG is best known for contributing to the cardiac action potential, and for numerous channel mutations that underlie ‘long-QT syndrome’. Many cells, particularly cancer cells, express Src-family tyrosine kinases and SHP tyrosine phosphatases; and an imbalance in tyrosine phosphorylation can lead to malignancies, autoimmune diseases, and inflammatory disorders. Ion channel contributions to cell functions are governed, to a large degree, by post-translational modulation, especially phosphorylation. However, almost nothing is known about roles of specific tyrosine kinases and phosphatases in regulating K+ channels in the EAG superfamily. First, we show that tyrosine kinase inhibitor, PP1, and the selective Src inhibitory peptide, Src40-58, reduce the hERG current amplitude, without altering its voltage dependence or kinetics. PP1 similarly reduces the hEAG1 current. Surprisingly, an ‘immuno-receptor tyrosine inhibitory motif’ (ITIM) is present within the cyclic nucleotide binding domain of all EAG-superfamily members, and is conserved in the human, rat and mouse sequences. When tyrosine phosphorylated, this ITIM directly bound to and activated SHP-1 tyrosine phosphatase (PTP-1C/PTPN6/HCP); the first report that a portion of an ion channel is a binding site and activator of a tyrosine phosphatase. Both hERG and hEAG1 currents were decreased by applying active recombinant SHP-1, and increased by the inhibitory substrate-trapping SHP-1 mutant. Thus, hERG and hEAG1 currents are regulated by activated SHP-1, in a manner opposite to their regulation by Src. Given the widespread distribution of these channels, Src and SHP-1, this work has broad implications in cell signaling that controls survival, proliferation, differentiation, and other ERG1 and EAG1 functions in many cell types.
doi:10.1371/journal.pone.0090024
PMCID: PMC3938566  PMID: 24587194
9.  Leishmania-Induced IRAK-1 Inactivation Is Mediated by SHP-1 Interacting with an Evolutionarily Conserved KTIM Motif 
Parasites of the Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases (e.g. Erk1/2, IKKα/β) involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.
Author Summary
Leishmania developed several methods to seize control of macrophage signalling pathways in an effort to inactivate their killing abilities. One effective method utilized by the parasite is the activation of host protein tyrosine phosphatases, specifically SHP-1. This increased phosphatase activity contributes to the inactivation of signalling molecules involved in critical macrophage functions such as NO and cytokine production. Interestingly, the absence of SHP-1 results in stronger macrophage inflammatory responses to a bacterial cell wall component known as LPS, a molecule detected by macrophages through Toll-like receptors (TLRs). This observation suggested a role for SHP-1 in the regulation of TLR signalling. Our study reveals that upon Leishmania infection, SHP-1 is able to rapidly bind to and inactivate a critical kinase (IRAK-1) in this pathway. This regulatory binding was shown to be mediated by an evolutionarily conserved motif identified in the kinase. This motif was also present in other kinases involved in Toll signalling and therefore could represent a regulatory mechanism of relevance to many kinases. This work not only reports a unique mechanism by which Leishmania can avoid harmful TLR signalling, but also provides a platform on which extensive investigation on host evasion mechanisms and regulation of cellular kinases can be gained.
doi:10.1371/journal.pntd.0000305
PMCID: PMC2596967  PMID: 19104650
10.  The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells  
The Journal of Experimental Medicine  1998;187(9):1417-1426.
Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.
PMCID: PMC2212277  PMID: 9565634
11.  Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine-based inhibition motif peptide motif. 
Molecular and Cellular Biology  1997;17(8):4305-4311.
Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.
PMCID: PMC232283  PMID: 9234687
12.  Negative Signaling Pathways of the Killer Cell Inhibitory Receptor and FcγRIIb1 Require Distinct Phosphatases  
Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (FcγRIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of FcγRIIb1 with BCR and with FcεRI on mast cells leads to recruitment of the inositol 5′ phosphatase SHIP and to inhibition of mast cells from SHP-1–deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of FcγRIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by FcγRIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the FcγRIIb1 tail, providing functional evidence that SHIP plays a role in the FcγRIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by FcγRIIb1 requires the inositol phosphatase SHIP.
PMCID: PMC2199004  PMID: 9236201
13.  C-Reactive Protein Inhibits Insulin Activation of Endothelial Nitric Oxide Synthase via the Immunoreceptor Tyrosine-Based Inhibition Motif of FcγRIIB and SHIP-1 
Circulation research  2009;104(11):1275-1282.
Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcγRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcγRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain–containing inositol 5′-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcγRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.
doi:10.1161/CIRCRESAHA.108.192906
PMCID: PMC2733870  PMID: 19423845
C-reactive protein; insulin; eNOS; FcγRIIB; SHIP1
14.  Role of SHPS-1 in the Regulation of Insulin-like Growth Factor I–stimulated Shc and Mitogen-activated Protein Kinase Activation in Vascular Smooth Muscle Cells 
Molecular Biology of the Cell  2005;16(7):3353-3364.
Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I–induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain–containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I–dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I–stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I–induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I–stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I–dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the β3 subunit of the αVβ3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I–dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I–stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I–dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.
doi:10.1091/mbc.E04-10-0918
PMCID: PMC1165417  PMID: 15888547
15.  An Investigation of Hierachical Protein Recruitment to the Inhibitory Platelet Receptor, G6B-b 
PLoS ONE  2012;7(11):e49543.
Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen.
doi:10.1371/journal.pone.0049543
PMCID: PMC3501490  PMID: 23185356
16.  SHP-1 Phosphatase Is a Critical Regulator in Preventing Natural Killer Cell Self-Killing 
PLoS ONE  2012;7(8):e44244.
Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these “de-regulated” SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.
doi:10.1371/journal.pone.0044244
PMCID: PMC3432062  PMID: 22952938
17.  SHP-2 expression negatively regulates NK cell function1,2 
Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)4 is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human natural killer (NK) cells upon recruitment to killer cell Ig-like receptors (KIR)4. To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-γ production, whereas cells overexpressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center (MTOC)4 polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts MTOC-based cytoskeletal rearrangement and granule release.
doi:10.4049/jimmunol.0900088
PMCID: PMC2783243  PMID: 19915046
Natural Killer Cells; Protein Kinases/Phosphatases; Cell Surface Molecules; Cell Activation; Cytotoxicity
18.  G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2*S⃞ 
The Journal of Biological Chemistry  2008;283(51):35419-35427.
Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR γ-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cγ (PLCγ). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5′-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR γ-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation.
doi:10.1074/jbc.M806895200
PMCID: PMC2602894  PMID: 18955485
19.  The Lectin-like Receptor KLRE1 Inhibits Natural Killer Cell Cytotoxicity 
The Journal of Experimental Medicine  2003;197(11):1551-1561.
We report the cloning and functional characterization in the mouse and the rat of a novel natural killer (NK) cell receptor termed KLRE1. The receptor is a type II transmembrane protein with a COOH-terminal lectin-like domain, and constitutes a novel KLR family. Rat Klre1 was mapped to the NK gene complex. By Northern blot and flow cytometry using newly generated monoclonal antibodies, KLRE1 was shown to be expressed by NK cells and a subpopulation of CD3+ cells, with pronounced interstrain variation. Western blot analysis indicated that KLRE1 can be expressed on the NK cell surface as a disulphide-linked dimer. The predicted proteins do not contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or a positively charged amino acid in the transmembrane domain. However, in a redirected lysis assay, the presence of whole IgG, but not of F(ab′)2 fragments of a monoclonal anti-KLRE1 antibody inhibited lysis of Fc-receptor bearing tumor target cells. Moreover, the tyrosine phosphatase SHP-1 was coimmunoprecipitated with KLRE1 from pervanadate-treated interleukin 2–activated NK cells. Together, our results indicate that KLRE1 may form a functional heterodimer with an as yet unidentified ITIM-bearing partner that recruits SHP-1 to generate an inhibitory receptor complex.
doi:10.1084/jem.20021253
PMCID: PMC2193914  PMID: 12782717
natural immunity; lymphocytes; immunological receptors; molecular sequence data; protein-tyrosine-phosphatase
20.  Hematopoietic cell phosphatase, SHP-1, is constitutively associated with the SH2 domain-containing leukocyte protein, SLP-76, in B cells 
Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1; previously named HCP, PTP1C, SH-PTP1, and SHP) is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. Recent data have demonstrated that the gene encoding SHP-1 is mutated in motheaten (mc) and viable motheaten (mc') mice resulting in autoimmune disease. More recently, SHP-1 has been shown to negatively regulate B cell antigen receptor (BCR)-initiated signaling. To elucidate potential mechanisms of SHP-1 action in BCR signal transduction, we studied proteins that interact with SHP-1 in B cells. Both anti-SHP-1 antibody and the two SH2 domains of SHP-1 expressed as glutathione S-transferase fusion proteins precipitated at least three phosphoproteins of approximately 75, 110, and 150 kD upon anti-immunoglobulin M stimulation of the WEHI- 231 immature B cell line. Binding of SHP-1 to the 75- and 110-kD proteins appeared to be mediated mainly by the NH2-terminal SH2 domain of SHP-1, whereas both the NH2- and COOH-terminal SH2 domains are required for maximal binding to the 150-kD protein. Immunoprecipitation and Western blot analysis revealed that the SHP-1-associated 75-kD protein is the hematopoietic cell-specific, SH2-containing protein SLP- 76. Further, this protein-protein association was constitutively observed and stable during the early phase of BCR signaling. However, significant tyrosine phosphorylation of SLP-76 as well as of SHP-1 was observed after BCR ligation. Constitutive association of SHP-1 with SLP- 76 could also be detected in normal splenic B cells. Collectively, these results suggest possible mechanisms by which SHP-1 may modulate signals delivered by BCR engagement.
PMCID: PMC2192711  PMID: 8760799
21.  Inhibitory signaling potential of a T cell receptor-like molecule in lamprey 
European journal of immunology  2009;39(2):571-579.
Summary
A T-cell receptor-like molecule (TCRL) with two canonical ITIM motifs has been identified in the sea lamprey. We show here that TCRL is preferentially expressed by lymphocytes bearing variable lymphocyte receptors. To examine the potential of the TCRL inhibitory motifs, chimeric proteins comprised by the FcγRIIb extracellular and transmembrane domains and the TCRL intracellular domain were expressed in a mouse B cell line. BCR co-ligation with the wild type version of the FcγRIIb/TCRL chimeric protein resulted in its tyrosine phosphorylation and the inhibition of BCR-induced calcium mobilization, whole cell protein tyrosine phosphorylation and Erk/Akt/JNK activation. Tyrosine to phenylalanine mutations in either or both ITIM compromised the inhibitory capacity of this receptor chimera. Analysis of receptor associated proteins indicated that the inhibition is mediated by recruitment of the protein tyrosine kinases, SHP1 and SHP2. These findings demonstrate the inhibitory potential of TCRL and its expression by clonally-diverse lymphocytes bearing the variable lymphocyte receptors, thereby implying an immunomodulatory role for this ancestral TCR relative in a jawless vertebrate.
doi:10.1002/eji.200838846
PMCID: PMC3711646  PMID: 19130486
IgSF; immune signaling; ITIM; lamprey
22.  Negative Regulation of Phagocytosis in Murine Macrophages by the Src Kinase Family Member, Fgr 
Ingestion of opsonized pathogens by professional phagocytes results in the generation and release of microbicidal products that are essential for normal host defense. Because these products can result in significant tissue injury, phagocytosis must be regulated to limit damage to the host while allowing for optimal clearance and destruction of opsonized pathogens. To pursue negative regulation of phagocytosis, we assessed the effect of the Src kinase family member, Fgr, on opsonin-dependent phagocytosis by mouse macrophages. We chose Fgr because it is present in high concentrations in circulating phagocytes but is not essential for Fcγ receptor–mediated ingestion by mouse macrophages. Although expression of Fgr both in a macrophage cell line and in primary macrophages significantly attenuates ingestion mediated by Fcγ receptors and CR3, it does not affect macropinocytosis or receptor-mediated endocytosis. This selective effect of Fgr is independent of its tyrosine kinase function. After Fcγ receptor cross-linking, Fgr becomes associated with the immunoreceptor tyrosine-based inhibition motif (ITIM)–containing receptor, SIRPα (a member of the signal-regulatory protein family, also known as Src homology 2 domain–containing protein tyrosine phosphatase [SHP] substrate 1 [SHPS-1], brain immunoglobulin-like molecule with tyrosine-based activation motifs [BIT], and P84) and potentiates the association of the phosphatase SHP-1 with SIRPα. This association is responsible, at least in part, for decreasing positive signaling essential for optimal phagocytosis. These data demonstrate an important negative regulatory role for this Src kinase family member and suggest that this homeostatic function must be overcome for optimal uptake and clearance of opsonized pathogens.
PMCID: PMC2195814  PMID: 10662797
phagocytes; Src family kinases; protein tyrosine phosphatase
23.  The SHP-1 protein tyrosine phosphatase negatively modulates Akt signaling in the ghrelin/GHSR1a system 
Molecular Biology of the Cell  2011;22(21):4182-4191.
The SHP-1 tyrosine phosphatase is a negative regulator of ghrelin activity, being a critical signaling component for proper regulation of Akt-dependent processes. Based on the SHP-1 expression pattern in white adipose tissue (WAT) and its regulation in a positive energy balance situation, it is possible to speculate about its role in the enlargement of WAT in obesity.
The aim of the present study was to identify the signaling mechanism(s) responsible for the modulation of growth hormone secretagogue receptor type 1a (GHSR1a)-associated Akt activity. Ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early Gi/o protein-dependent pathway and a late pathway mediated by β-arrestins. We found that the Src homology 2–containing protein tyrosine phosphatase (SHP-1) was an essential molecule in both Gi/o protein–dependent and β-arrestin–mediated pathways. More specifically, the role of SHP-1 in the Gi/o protein–dependent pathway was demonstrated by the fact that the overexpression of a catalytically defective SHP-1 augments tyrosine phosphorylation of the PI3K regulatory subunit p85, leading to an increase in the phosphorylation of cSrc and phosphoinositide-dependent protein kinase 1, and finally activating Akt. The presence of SHP-1 in the β-arrestin–scaffolded complex and its attenuating effect on the cSrc and Akt activities verified that SHP-1 regulates not only the Gi/o protein–dependent pathway but also the β-arrestin–mediated pathway. Assays performed in preadipocyte and adipocyte 3T3-L1 cells showed SHP-1 expression. According to our results in HEK-GHSR1a cells, ghrelin stimulated SHP-1 phosphorylation in 3T3-L1 cells. The increase in ghrelin-induced Akt activity was enhanced by small interfering RNA of SHP-1 in preadipocyte 3T3-L1 cells. These results were reproduced in white adipose tissue obtained from mice, in which SHP-1 exhibited higher expression in omental than in subcutaneous tissue. Furthermore, this pattern of expression was inverted in mice fed a high-fat diet, suggesting a role for SHP-1 in controlling ghrelin sensitivity in adipose tissue. Indeed, SHP-1 deficiency was associated with augmented ghrelin-evoked Akt phosphorylation in omental tissue, as well as decreased phosphorylation under overexpression of SHP-1 in subcutaneous tissue. These findings showed a novel role for SHP-1 in the regulation of Akt activity through the modulation of the ghrelin/GHSR1a system signaling.
doi:10.1091/mbc.E11-04-0373
PMCID: PMC3204078  PMID: 21900501
24.  PECAM-1 is involved in BCR/ABL signaling and may downregulate imatinib-induced apoptosis of Philadelphia chromosome-positive leukemia cells 
International Journal of Oncology  2012;42(2):419-428.
PECAM-1 (CD31) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing surface glycoprotein expressed on various hematopoietic cells as well as on endothelial cells. PECAM-1 has been shown to play roles in regulation of adhesion, migration and apoptosis. The BCR/ABL fusion tyrosine kinase is expressed in chronic myeloid leukemia and Philadelphia-positive (Ph+) acute lymphoblastic leukemia cells, and its inhibition by the clinically used tyrosine kinase inhibitors imatinib or dasatinib induces apoptosis of these cells. In the present study, we demonstrate that PECAM-1 is tyrosine phospho rylated in its ITIM motifs in various BCR/ABL-expressing cells including primary leukemia cells. Studies using imatinib and dasatinib as well as transient expression experiments in 293T cells revealed that PECAM-1 was phosphorylated directly by BCR/ABL, which was enhanced by the imatinib-resistant E255K and T315I mutations, or partly by the Src family tyrosine kinases, including Lyn, which were activated dependently or independently on BCR/ABL. We also demonstrate by using a substrate trapping mutant of SHP2 that tyrosine phosphorylated PECAM-1 binds SHP2 and is a major substrate for this tyrosine phosphatase in BCR/ABL-expressing cells. Overexpression of PECAM-1 in BCR/ABL-expressing cells, including K562 human leukemia cells, enhanced cell adhesion and partially inhibited imatinib-induced apoptosis involving mitochondria depolarization and caspase-3 cleavage, at least partly, in an ITIM-independent manner. These data suggest that PECAM-1 may play a role in regulation of apoptosis as well as adhesion of BCR/ABL-expressing cells to modulate their imatinib sensitivity and would be a possible candidate for therapeutic target in Ph+ leukemias.
doi:10.3892/ijo.2012.1729
PMCID: PMC3583636  PMID: 23233201
PECAM-1; BCR/ABL; imatinib; apoptosis; leukemia
25.  SHP2-interacting Transmembrane Adaptor Protein (SIT), A Novel Disulfide-linked Dimer Regulating Human T Cell Activation  
The Journal of Experimental Medicine  1999;189(8):1181-1194.
T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.
PMCID: PMC2193021  PMID: 10209036
T lymphocytes; T cell receptor; transmembrane adaptor proteins; signal transduction

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