Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.     

Cutting edge: dependence of TCR antagonism on Src homology 2 domain-containing protein tyrosine phosphatase activity

The mechanism by which antagonist peptides inhibit T cell responses is unknown. Mice deficient in Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1) have revealed its importance in the negative regulation of lymphocyte signaling. We investigated a possible role for SHP-1 in T cell antagonism and demonstrate, for the first time, a substantial increase in SHP-1 activity during antagonism of CD4(+) T cells. Furthermore, the removal of functional SHP-1 prevents antagonism in these cells. Our data demonstrate that T cell antagonism occurs via a negative intracellular signal that is mediated by SHP-1.     

Oxidative stress induces lipid-raft-mediated activation of Src homology 2 domain-containing protein-tyrosine phosphatase 2 in astrocytes

Several protein phosphatases are involved in neuroprotection in response to ischemic brain injury. Here, we report that reactive oxygen species (ROS)-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 through lipid rafts in rat primary astrocytes. SHP-2 was transiently phosphorylated during hypoxia/reoxygenation, an effect abrogated by a ROS scavenger and an NADPH oxidase inhibitor. Additionally, exogenous treatment with hydrogen peroxide (H₂O₂) triggered SHP-2 phosphorylation in a time- and dose-dependent manner and led to its translocation into lipid rafts. H₂O₂-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III and methyl-β-cyclodextrin (MCD), lipid-raft-disrupting agents. In the presence of H₂O₂, SHP-2 formed a complex with STAT-3 and reduced the steady-state STAT-3 phosphorylation level. Interestingly, the effect of H₂O₂ on SHP-2 phosphorylation was cell-type specific. Remarkably, SHP-2 phosphorylation was induced strongly by H₂O₂ in astrocytes, but barely detectable in microglia. Our results collectively indicate that SHP-2 is activated by ROS-mediated oxidative stress in astrocytes and functions as a component of the raft-mediated signaling pathway that acts through dephosphorylation and inactivation of other phosphotyrosine proteins, such as STAT-3.     

Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.     

Inappropriate recruitment and activity by the Src homology region 2 domain-containing phosphatase 1 (SHP1) is responsible for receptor dominance in the SHIP-deficient NK cell

We have previously demonstrated that the NKR repertoire is profoundly disrupted by SHIP deficiency. This repertoire disruption is characterized by receptor dominance where inhibitory signals from 2B4 repress killing of complex targets expressing MHC class I and activating ligands. In this study, we examine the molecular basis of receptor dominance in SHIP-/- NK cells. In this study, we show that in SHIP-/- NK cells there is a pronounced bias toward the 2B4 long isoform. We have also characterized signaling molecules recruited to 2B4 in SHIP-/- NK cells. Interestingly, we find that approximately 10- to 16-fold more Src homology region 2 domain-containing phosphatase 1 (SHP1) is recruited to 2B4 in SHIP-/- NK cells when compared with wild type. Consistent with SHP1 overrecruitment, treatment with sodium orthovanadate or a novel inhibitor with micromolar activity against SHP1 restores the ability of SHIP-/- NK cells to kill Rae1+ RMA and M157+ targets. These findings define the molecular basis for hyporesponsiveness by SHIP-deficient NK cells.     

Inhibitory modulation of B cell receptor-mediated Ca2+ mobilization by Src homology 2 domain-containing inositol 5'-phosphatase (SHIP)

Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) mediates inhibitory signals that attenuate intracellular Ca2+ mobilization in B cells upon B cell receptor (BCR) stimulation. To clarify the mechanisms affected by SHIP, we analyzed Ca2+ mobilization in the DT40 B cell line in which the SHIP gene was disrupted. In SHIP-deficient cells, Ca2+ transient elicited by BCR stimulation was more prolonged than that in control cells both in the presence and absence of extracellular Ca2+. Inositol 1,4, 5-trisphosphate production following BCR stimulation was enhanced in SHIP-deficient cells. In SHIP-deficient cells in comparison with the control cells, BCR stimulation in the absence of extracellular Ca2+ induced a greater degree of Ca2+ store depletion and the Ca2+ influx upon re-addition of extracellular Ca2+ was also greater. However, store-operated Ca2+ influx (SOC) elicited by thapsigargin-induced store depletion was not affected by SHIP. These results indicate that the primary target pathway of SHIP is the Ca2+ release from the stores, and that Ca2+ influx by the SOC mechanism is secondarily controlled by the level of Ca2+ in the stores without direct inhibition of SOC. In this way, SHIP may play an important role in ensuring the robust tuning of Ca2+ signaling in B cells.     

Regulation by Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 of alpha-galactosylceramide-induced antimetastatic activity and Th1 and Th2 responses of NKT cells

Interaction of alpha-galactosylceramide (alpha-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by alpha-GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of alpha-GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN-gamma and IL-4 as well as cell proliferation in response to alpha-GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4+ mononuclear cells incubated with alpha-GalCer and CD11c+ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN-gamma and IL-4 than did those incubated with alpha-GalCer and CD11c+ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for alpha-GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4+ T cells by DCs.     

Curcumin suppresses Janus kinase-STAT inflammatory signaling through activation of Src homology 2 domain-containing tyrosine phosphatase 2 in brain microglia

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that the inhibitory action of curcumin on Janus kinase (JAK)-STAT signaling can contribute to its anti-inflammatory activity in the brain. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the ganglioside-, LPS-, or IFN-gamma-stimulated induction of cyclooxygenase-2 and inducible NO synthase, important enzymes that mediate inflammatory processes. These anti-inflammatory effects appear to be due, at least in part, to the suppression of the JAK-STAT inflammatory signaling cascade. Curcumin markedly inhibited the phosphorylation of STAT1 and 3 as well as JAK1 and 2 in microglia activated with gangliosides, LPS, or IFN-gamma. Curcumin consistently suppressed not only NF binding to IFN-gamma-activated sequence/IFN-stimulated regulatory element, but also the expression of inflammation-associated genes, including ICAM-1 and monocyte chemoattractant protein 1, whose promoters contain STAT-binding elements. We further show that activation of Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-2, a negative regulator of JAK activity, is likely to be one of the mechanisms underlying the curcumin-mediated inhibition of JAK-STAT signaling. Treatment of microglial cells with curcumin led to an increase in phosphorylation and association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells.     

Blocking the function of tyrosine phosphatase SHP-2 by targeting its Src homology 2 domains

SHP-2 is an Src homology 2 (SH2) domain-containing tyrosine phosphatase with crucial functions in cell signaling and major pathological implications. It stays inactive in the cytosol and is activated by binding through its SH2 domains to tyrosine-phosphorylated receptors on the cell surface. One such cell surface protein is PZR, which contains two tyrosine-based inhibition motifs responsible for binding of SHP-2. We have generated a glutathione S-transferase fusion protein carrying the tandem tyrosine-based inhibition motifs of PZR, and the protein was tyrosine-phosphorylated by co-expressing c-Src in Escherichia coli cells. The purified phosphoprotein displays a strong binding to SHP-2 and causes its activation in vitro. However, when introduced into NIH 3T3 cells by using a protein delivery reagent, it effectively inhibited the activation of ERK1/2 induced by growth factors and serum but not by phorbol ester, in reminiscence of the effects caused by expression of dominant negative SHP-2 mutants and deletion of functional SHP-2. The data suggest that the exogenously introduced PZR protein specifically binds SHP-2, blocks its translocation, and renders it functionally incompetent. This is further supported by the fact that the phosphorylated PZR protein had no inhibitory effects on fibroblasts derived from mice expressing only a mutant SHP-2 protein lacking most of the N-terminal SH2 domain. This study thus provides a novel and highly specific method to interrupt the function of SHP-2 in cells.     

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the migration of Langerhans cells from the epidermis to draining lymph nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-alpha-induced migration of LCs. The emigration of dendritic cells expressing I-A(b+) from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3beta was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.     

Actin tyrosine dephosphorylation by the Src homology 1-containing protein tyrosine phosphatase is essential for actin depolymerization after membrane IgM cross-linking

Src homology protein 1 (SHP-1) plays an important role in B cell Ag receptor (BCR) differentiation, proliferation, survival, and apoptosis. After BCR stimulation in apoptotic cells, SHP-1 has been shown to be recruited to phosphorylated immunoreceptor tyrosine-based inhibitory motifs present in receptors such as CD22 and CD72. However, the substrates of SHP-1 in the chicken B cell line, DT40, have remained undefined. To identify SHP-1 substrates in DT40, we used a trapping mutant, SHP-1 C/S (a catalytically inactive form). Cross-linking of BCR induced hyperphosphorylation of approximately 44-kDa protein in C/S transfectants. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis revealed that this was actin (cytoplasmic type 5) carrying three immunoreceptor tyrosine-based inhibitory motif-like sequences. SHP-1 was shown to bind to one of these sequences in synthetic peptide binding experiment. Thus, actin is a direct SHP-1 substrate. Furthermore, more SHP-1 molecules translocate into lipid rafts, and their association with actin was increased after BCR stimulation. In C/S transfectants, actin polymerization induced by membrane IgM ligation was sustained to a greater extent for a longer time compared with wild-type transfectants. Therefore, actin dephosphorylation by SHP-1 is essential for actin depolymerization after BCR stimulation. Our data suggest that SHP-1 plays a pivotal role in reorganization of cytoskeletal architecture inducing actin dephosphorylation. These results clearly demonstrate the direct interaction of SHP-1 with actin.     

CD300F blocks both MyD88 and TRIF-mediated TLR signaling through activation of Src homology region 2 domain-containing phosphatase 1

CD300F is known to exhibit inhibitory activity in myeloid cells through its intracellular ITIM. To investigate the effect of CD300F stimulation on TLR signaling, the human acute monocytic leukemia cell line THP-1 was treated with CD300F-specific mAbs or two synthetic peptides that represented the ITIM-like domains of CD300F. Treatment with these agents blocked TLR2-, 3-, 4-, and 9-mediated expression of proinflammatory mediators such as IL-8 and matrix metalloproteinase-9. The luciferase reporter assay in 293T cells and Western blot analysis of THP-1 cells revealed that these inhibitory actions were effective in pathways involving MyD88 and/or TRIF of TLR signaling and associated with marked suppression of IκB kinase activation, phosphorylation/degradation of IκB, and subsequent activation of NF-κB. Use of specific inhibitors and immunoprecipitation analysis further indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatase-1, a protein tyrosine phosphatase with inhibitory activity in hematopoietic cells. These data indicate that CD300F is an active regulator of TLR-mediated macrophage activation through its association with Src homology 2 domain-containing phosphatase-1 and that the synthetic peptides can be applied for the regulation of immune responses that are induced by TLRs.     

Investigating the reason for loss-of-function of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) caused by Y279C mutation through molecular dynamics simulation

Abstract

Noonan syndrome with multiple lentigines (NSML), formerly known as LEOPARD syndrome (LS), is an autosomal dominant inherited multisystemic disorder. Most patients involve mutation in SHP2 encoded by tyrosine-protein phosphatase non-receptor type 11 (PTPN11) gene. Studies have shown that NSML-associated Y279C mutation exhibited the reduced phosphatase activity, leading to loss-of-function (LOF) of SHP2. However, the effect of the Y279C mutation on the SHP2 at the molecular level is unclear. In this study, molecular dynamics simulations of SHP2 wild-type (SHP2^WT) and Y279C mutant (SHP2^Y279C) were performed to investigate the structural differences in proteins after Y279C mutation and to find out the reason for loss-of-function of SHP2. Through a series of post-dynamic analyses, it was found that the protein occupied a smaller phase space after Y279C mutation, showing reduced flexibility. Specifically, due to the mutation of Y279C, the secondary structures of these two regions (residues Lys70-Ala72 and Gly462-Arg465) were significantly transformed from Turn to α-helix and β-strand. Furthermore, by calculating the residue interaction network, hydrogen bond occupancy and binding free energy, it was further revealed that the conformational differences between SHP2^WT and SHP2^Y279C systems were mainly caused by the differences in the interaction between Arg465–Phe469, Ile463–Gly467, Cys279–Lys70, Cys459–Ala72, Gly464–Phe71, Phe71–Ile463, Ile463–Ala505 and Arg465–Glu361. Consequently, this finding is expected to provide a new insight into the reason for loss-of-function of SHP2 caused by Y279C mutation.

Communicated by Ramaswamy H. Sarma     

Targeting Src homology 2 domain-containing tyrosine phosphatase (SHP-1) into lipid rafts inhibits CD3-induced T cell activation

To study the mechanism by which protein tyrosine phosphatases (PTPs) regulate CD3-induced tyrosine phosphorylation, we investigated the distribution of PTPs in subdomains of plasma membrane. We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts, as determined by sucrose density gradient sedimentation. In Jurkat T cells, approximately 5--10% of Src homology 2 domain-containing tyrosine phosphatase (SHP-1) is constitutively associated with plasma membrane, and nearly 50% of SHP-2 is translocated to plasma membrane after vanadate treatment. Similar to transmembrane PTP, CD45, the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts, where other signaling molecules such as Lck, linker for activation of T cells, and CD3 zeta are enriched. We further demonstrated that CD3-induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts, unless PTPs are inhibited. It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane. To test this hypothesis, we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck (residues 1--14). The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3-induced tyrosine phosphorylation of CD3 zeta/epsilon, IL-2 generation, and nuclear mobilization of NF-AT. Collectively, these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR/CD3 activation.     

The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.     

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.