Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.     

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.     

Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.     

The mouse homologue of the leukocyte-associated Ig-like receptor-1 is an inhibitory receptor that recruits Src homology region 2-containing protein tyrosine phosphatase (SHP)-2, but not SHP-1

We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.     

Retinal Src homology region 2-containing protein tyrosine phosphatase 2 silencing alleviates diabetic retinopathy via suppressing inflammatory response and oxidative stress by regulating Yes-associated protein 1 activity

Diabetic retinopathy (DR) is the prevalent microvascular complication of diabetes mellitus (DM), and it may lead to permanent blindness. The previous publication has indicated that both inflammatory response and oxidative stress are critical factors involved in DR progression, however, the accurate regulatory mechanism remains to be revealed. Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), a member of the protein tyrosine phosphatase family, was reported to play a role in diabetic nephropathy, whereas its function in DR was unknown and required further exploration. The level of phosphorylated, not the total, SHP2 increased in the retinas of rats with streptozotocin injection-induced DM. Further, the intravitreal injection of SHP2 shRNA lentivirus alleviated retinal pathological changes, and inhibited inflammatory response and oxidative stress, which were accompanied with Yes-associated protein 1 (YAP1) deactivation in DR rats. Additional co-immunoprecipitation results confirmed the interaction of SHP2 and YAP1. Collectively, our data preliminarily show that DR amelioration-induced by SHP2 inhibition in rats may attribute to the deactivation of YAP1 pathway.     

Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions

Inhibitory NK receptors with ligand specificity for MHC class I recruit Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) phosphatase and prevent autocytotoxicity. Activation of SHP-1 depends upon Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of the inhibitory receptor. In this study we demonstrate, by quantitative temporal analysis, that talin, Lck, and SHP-1 are recruited to the synapse within 1 min in both cytolytic and noncytolytic conjugates. Polarization of talin and Lck rapidly disappears in the noncytolytic interactions but persists in cytolytic interactions, where protein kinase C-theta;, Src homology 2 domain-containing leukocyte protein of 76 kDa, and lysosomes are recruited within 5 min. At 1 min SHP-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic synapse. Lck has multifocal distribution in both synapses consistent with the shared requirement for early tyrosine phosphorylation. Our studies indicate that the spatial location of SHP-1 in the synapse distinguishes noncytolytic from cytolytic interactions within the first minute.     

Synthesis and biological evaluation of heterocyclic bis-aryl amides as novel Src homology 2 domain containing protein tyrosine phosphatase-2 (SHP2) inhibitors

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a convergent node for oncogenic cell-signaling cascades including the PD-L1/PD-1 pathway. Consequently, SHP2 has emerged as a compelling target for novel anti-cancer agents. Replacing one of phenyl ring in PTP1B inhibitor 1 with heterocyclic ring led to a series of heterocyclic bis-aryl amide derivatives. The representative compound 7b displayed SHP2 inhibitory activity with IC₅₀ of 2.63 ± 0.08 μM, exhibited about 4-fold selectivity for SHP2 over TCPTP and had no detectable activity against SHP1 and PTP1B. These preliminary results could provide a possible opportunity for the development of novel SHP2 inhibitors with optimal potency and improved pharmacological properties.     

Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation

The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55-60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation.     

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 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.     

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.     

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 region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.     

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.     

Protein kinase C activation inhibits tyrosine phosphorylation of Cbl and its recruitment of Src homology 2 domain-containing proteins.

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCalpha and PKCtheta can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCalpha and PKCtheta in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.