CD22 is a functional ligand for SH2 domain-containing protein-tyrosine phosphatase-1 in primary T cells

The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells.     

Negative regulation of monocyte adhesion to arterial elastic laminae by signal regulatory protein alpha and Src homology 2 domain-containing protein-tyrosine phosphatase-1

Elastic laminae are extracellular matrix constituents that not only contribute to the stability and elasticity of arteries but also play a role in regulating arterial morphogenesis and pathogenesis. We demonstrate here that an important function of arterial elastic laminae is to prevent monocyte adhesion, which is mediated by the inhibitory receptor signal regulatory protein (SIRP) alpha and Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1. In a matrix-based arterial reconstruction model in vivo, elastic laminae were resistant to leukocyte adhesion and transmigration compared with the collagen-dominant arterial adventitia. The density of leukocytes within the elastic lamina-dominant media was about 58-70-fold lower than that within the adventitia from 1 to 30 days. An in vitro assay confirmed the inhibitory effect of elastic laminae on monocyte adhesion. The exposure of monocytes to elastic laminae induced activation of SIRP alpha, which in turn activated SHP-1. Elastic lamina degradation peptides extracted from arterial specimens could also activate SIRP alpha and SHP-1. The knockdown of SIRP alpha and SHP-1 by specific small interfering RNA diminished the inhibitory effect of elastic laminae, resulting in a significant increase in monocyte adhesion. These observations suggest that SIRP alpha and SHP-1 potentially mediate the inhibitory effect of elastic laminae on monocyte adhesion.     

Identification of a novel lipid raft-targeting motif in Src homology 2-containing phosphatase 1

The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 DeltaC) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 DeltaC was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.     

Src homology domain 2-containing tyrosine phosphatase 2 associates with intercellular adhesion molecule 1 to regulate cell survival

Intercellular adhesion molecule-1 (ICAM-1) binds to the plasma protein fibrinogen (Fg) to mediate leukocyte/endothelial cell interactions. In our studies, the ligation of Fg to ICAM-1 on tumor necrosis factor-alpha-stimulated endothelial cells resulted in the tyrosine phosphorylation of Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). The ICAM-1 cytoplasmic sequence IKKYRLQ conforms poorly to the concensus immunoreceptor tyrosine-based inhibition motifs found in receptors that bind SHP-2. Nevertheless, the tyrosine phosphorylated sequence (IKKpYRLQ) bound specifically to the SH2 domain proximal to the NH(2)-terminal of SHP-2 (SHP-2-N) but not to the SH2 domain proximal on the COOH-terminal side (SHP-2-C). Phosphorylated ICAM-1 bound SHP-2-N. In immunoprecipitation experiments, SHP-2 associated with phosphorylated ICAM-1. Cells expressing truncated ICAM-1 that lacked the cytoplasmic sequence (ICAM-1(TR)) failed to associate with SHP-2. ICAM-1 containing the tyrosine to alanine substitution at position 485 (ICAM-1(Y485A)) associated weakly with SHP-2. Cells expressing ICAM-1(TR) and ICAM-1(Y485A) underwent apoptosis upon adhesion to Fg, whereas the wild type ICAM-1 maintained cell survival. These results indicate that ICAM-1 interactions with SHP-2 allow better cellular survival mediated through Fg-ICAM-1 ligation.     

The Src homology 2-containing inositol 5-phosphatase 1 (SHIP1) is involved in CD32a signaling in human neutrophils

Phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P(3)) plays important signaling roles in immune cells, particularly in the control of activating pathways and of survival. It is formed by a family of phosphatidylinositol 3'-kinases (PI3Ks) which phosphorylate PtdIns(4,5)P(2) in vivo. In human neutrophils, the levels of PtdIns(3,4,5)P(3) increase rapidly at the leading edge of locomoting cells and at the base of the phagocytic cup during FcgammaR-mediated particle ingestion. Even though these, and other, data indicate that PtdIns(3,4,5)P(3) is involved in the control of chemotaxis and phagocytosis in human neutrophils, the mechanisms that regulate its levels have yet to be fully elucidated in these cells. We evaluated the potential implication of SHIP1 and PTEN, two lipid phosphatases that utilize PtdIns(3,4,5)P(3) as substrate, in the signaling pathways called upon in response to CD32a cross-linking. We observed that the cross-linking of CD32a resulted in a transient accumulation of PtdIns(3,4,5)P(3). CD32a cross-linking also induced the tyrosine phosphorylation of SHIP1, its translocation to the plasma membrane and its co-immunoprecipitation with CD32a. CD32a cross-linking had no effect on the level of serine/threonine phosphorylation of PTEN and did not stimulate its translocation to the plasma membrane. PP2, a Src kinase inhibitor, inhibited the tyrosine phosphorylation of SHIP1 as well as its translocation to the plasma membrane. Wortmannin, a PI3K inhibitor, had no effect on either of these two indices of activation of SHIP1. Our results indicate that SHIP1 is involved, in a Src kinase-dependent manner, in the early signaling events observed upon the cross-linking of CD32a in human neutrophils.     

CD22 ligand binding regulates normal and malignant B lymphocyte survival in vivo

The CD22 extracellular domain regulates B lymphocyte function by interacting with alpha2,6-linked sialic acid-bearing ligands. To understand how CD22 ligand interactions affect B cell function in vivo, mouse anti-mouse CD22 mAbs were generated that inhibit CD22 ligand binding to varying degrees. Remarkably, mAbs which blocked CD22 ligand binding accelerated mature B cell turnover by 2- to 4-fold in blood, spleen, and lymph nodes. CD22 ligand-blocking mAbs also inhibited the survival of adoptively transferred normal (73-88%) and malignant (90%) B cells in vivo. Moreover, mAbs that bound CD22 ligand binding domains induced significant CD22 internalization, depleted marginal zone B cells (82-99%), and reduced mature recirculating B cell numbers by 75-85%. The CD22 mAb effects were independent of complement and FcRs, and the CD22 mAbs had minimal effects in CD22AA mice that express mutated CD22 that is not capable of ligand binding. These data demonstrate that inhibition of CD22 ligand binding can disrupt normal and malignant B cell survival in vivo and suggest a novel mechanism of action for therapeutics targeting CD22 ligand binding domains.     

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.     

Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.     

Downstream of tyrosine kinases-1 and Src homology 2-containing inositol 5'-phosphatase are required for regulation of CD4+CD25+ T cell development

The adaptor protein, downstream of tyrosine kinases-1 (Dok-1), and the phosphatase SHIP are both tyrosine phosphorylated in response to T cell stimulation. However, a function for these molecules in T cell development has not been defined. To clarify the role of Dok-1 and SHIP in T cell development in vivo, we compared the T cell phenotype of wild-type, Dok-1 knockout (KO), SHIP KO, and Dok-1/SHIP double-knockout (DKO) mice. Dok-1/SHIP DKO mice were runted and had a shorter life span compared with either Dok-1 KO or SHIP KO mice. Thymocyte numbers from Dok-1/SHIP DKO mice were reduced by 90%. Surface expression of both CD25 and CD69 was elevated on freshly isolated splenic CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO, suggesting these cells were constitutively activated. However, these T cells did not proliferate or produce IL-2 after stimulation. Interestingly, the CD4(+) T cells from SHIP KO and Dok-1/SHIP DKO mice produced higher levels of TGF-beta, expressed Foxp3, and inhibited IL-2 production by CD3-stimulated CD4(+)CD25(-) T cells in vitro. These findings suggest Dok-1 and SHIP function in pathways that influence regulatory T cell development.     

Regulation of the Src homology 2-containing inositol 5-phosphatase SHIP1 in HIP1/PDGFbeta R-transformed cells

It has been shown previously that the Huntingtin interacting protein 1 gene (HIP1) was fused to the platelet-derived growth factor beta receptor gene (PDGFbetaR) in leukemic cells of a patient with chronic myelomonocytic leukemia. This resulted in the expression of the chimeric HIP1/PDGFbetaR protein, which oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the Ba/F3 murine hematopoietic cell line to interleukin-3-independent growth. Tyrosine phosphorylation of a 130-kDa protein (p130) correlates with transformation by HIP1/PDGFbetaR and related transforming mutants. We report here that the p130 band is immunologically related to the 125-kDa isoform of the Src homology 2-containing inositol 5-phosphatase, SHIP1. We have found that SHIP1 associates and colocalizes with the HIP1/PDGFbetaR fusion protein and related transforming mutants. These mutants include a mutant that has eight Src homology 2-binding phosphotyrosines mutated to phenylalanine. In contrast, SHIP1 does not associate with H/P(KI), the kinase-dead form of HIP1/PDGFbetaR. We also report that phosphorylation of SHIP1 by HIP1/PDGFbetaR does not change its 5-phosphatase-specific activity. This suggests that phosphorylation and possible PDGFbetaR-mediated sequestration of SHIP1 from its substrates (PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4)) might alter the levels of these inositol-containing signal transduction molecules, resulting in activation of downstream effectors of cellular proliferation and/or survival.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

Src homology 2-containing inositol 5-phosphatase 1 binds to the multifunctional docking site of c-Met and potentiates hepatocyte growth factor-induced branching tubulogenesis

Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathione S-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis.     

Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation.

SH-PTP2 is a nontransmembrane human protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains and binds to insulin receptor substrate 1 (IRS-1) via these domains in response to insulin. The expression of a catalytically inactive mutant of SH-PTP2 (containing the mutation Cys-459-->Ser) in Chinese hamster ovary cells that overexpress human insulin receptors (CHO-IR cells) markedly attenuated insulin-stimulated Ras activation. Expression of mutant SH-PTP2 also inhibited MAP kinase activation in response to insulin but not in response to 12-O-tetradecanoyl phorbol-13-acetate. In contrast, the insulin-induced association of phosphoinositide 3-kinase activity with IRS-1 was not affected by the expression of inactive SH-PTP2. Furthermore, the expression of mutant SH-PTP2 had no effect on the binding of Grb2 to IRS-1, on the tyrosine phosphorylation of Shc, or on the formation of the complex between Shc and Grb2 in response to insulin. However, the amount of SH-PTP2 bound to IRS-1 in insulin-treated CHO-IR cells expressing mutant SH-PTP2 was greater than that observed in CHO-IR cells overexpressing wild-type SH-PTP2. Recombinant SH-PTP2 specifically dephosphorylated a synthetic phosphopeptide corresponding to the sequence surrounding Tyr-1172 of IRS-1, a putative binding site for SH-PTP2. Additionally, phenylarsine oxide, an inhibitor of protein-tyrosine phosphatases, inactivated SH-PTP2 in vitro and increased the insulin-induced association of SH-PTP2 with IRS-1. These results suggest that SH-PTP2 may regulate an upstream element necessary for Ras activation in response to insulin and that this upstream element may be required for the Grb2- or Shc-dependent pathway. Furthermore, these results are consistent with the notion that SH-PTP2 may bind to IRS-1 through its SH2 domains in response to insulin and dephosphorylate the phosphotyrosine residue to which it binds, thereby regulating its association with IRS-1.     

Loss of Src homology region 2 domain-containing protein tyrosine phosphatase-1 increases CD8+ T cell-APC conjugate formation and is associated with enhanced in vivo CTL function

Extensive evidence has been accumulated to implicate the intracellular protein tyrosine phosphatase, Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), as a negative regulator of TCR-signaling thresholds. Specifically, T cells from the SHP-1-deficient mouse, motheaten, exhibit a hyperproliferative phenotype when activated by cognate peptide-pulsed APCs. However, the cellular basis for this phenotype has not been fully explained. Using the intracellular fluorescent dye, CFSE, we show that a greater proportion of motheaten vs control naive CD8(+) T cells undergo cell division when activated by peptide-pulsed APCs. Furthermore, there is a greater likelihood of TCRs on SHP-1-deficient vs control T cells binding to peptide/MHC ligands on APCs when using TCR down-regulation as an indirect measure of TCR engagement. In addition, T cell-APC conjugate assays provide direct evidence that a greater proportion of SHP-1-deficient T cells are capable of forming stable conjugates with APCs and this may explain, at least in part, their hyperproliferative response to TCR-triggered stimulation. The physiological relevance of the combined in vitro observations is demonstrated by the significantly enhanced in vivo expansion and CTL capacity generated in mice receiving adoptively transferred SHP-1-deficient naive CD8(+) T cells when compared with control T cells.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

Deficiency of Src homology 2-containing phosphatase 1 results in abnormalities in murine neutrophil function: studies in motheaten mice

Neutrophils, an essential component of the innate immune system, are regulated in part by signaling pathways involving protein tyrosine phosphorylation. While protein tyrosine kinase functions in regulating neutrophil behavior have been extensively investigated, little is known about the role for specific protein tyrosine phosphatases (PTP) in modulating neutrophil signaling cascades. A key role for Src homology 2 domain-containing phosphatase 1 (SHP-1), a PTP, in neutrophil physiology is, however, implied by the overexpansion and inappropriate activation of granulocyte populations in SHP-1-deficient motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. To directly investigate the importance of SHP-1 to phagocytic cell function, bone marrow neutrophils were isolated from both me/me and me(v)/me(v) mice and examined with respect to their responses to various stimuli. The results of these studies revealed that both quiescent and activated neutrophils from motheaten mice manifested enhanced tyrosine phosphorylation of cellular proteins in the 60- to 80-kDa range relative to that detected in wild-type congenic control neutrophils. MOTHEATEN: neutrophils also demonstrated increased oxidant production, surface expression of CD18, and adhesion to protein-coated plastic. Chemotaxis, however, was severely diminished in the SHP-deficient neutrophils relative to control neutrophils, which was possibly attributable to a combination of defective deadhesion and altered actin assembly. Taken together, these results indicate a significant role for SHP-1 in modulating the tyrosine phosphorylation-dependent signaling pathways that regulate neutrophil microbicidal functions.     

A filarial nematode-secreted phosphorylcholine-containing glycoprotein uncouples the B cell antigen receptor from extracellular signal-regulated kinase-mitogen-activated protein kinase by promoting the surface Ig-mediated recruitment of Src homology 2 domain-containing tyrosine phosphatase-1 and Pac-1 mitogen-activated kinase-phosphatase

Unraveling the molecular mechanisms by which filarial nematodes, major human pathogens in the tropics, evade the host immune system remains an elusive goal. We have previously shown that excretory-secretory product-62 (ES-62), a homologue of phosphorylcholine-containing molecules that are secreted by human parasites and which is active in rodent models of filarial infection, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal transduction elements in B lymphocytes. Such activation mediates desensitization of subsequent B cell Ag receptor (BCR) ligation-induced activation of extracellular signal-regulated kinase-mitogen-activated protein (ErkMAP) kinase and ultimately B cell proliferation. We now show that the desensitization is due to ES-62 targeting two major regulatory sites of B cell activation. Firstly, pre-exposure to ES-62 primes subsequent BCR-mediated recruitment of SHP-1 tyrosine phosphatase to abolish recruitment of the RasErkMAP kinase cascade via the Igalphabeta-ShcGrb2Sos adaptor complex interactions. Secondly, any ongoing ErkMAP kinase signaling in ES-62-primed B cells is terminated by the MAP kinase phosphatase, Pac-1 that is activated consequently to challenge via the BCR.     

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.