SH2-containing Phosphatase Research Articles

LAIR1 drives glioma progression by nuclear focal adhesion kinase dependent expressions of cyclin D1 and immunosuppressive chemokines/cytokines

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR1), an immune receptor containing immunoreceptor tyrosine-based inhibiory motifs (ITIMs), has emerged as an attractive target for cancer therapy. However, the intrinsic function of LAIR1 in gliomas remains unclear. In this study, the poor prognosis of glioma patients and the malignant proliferation of glioma cells in vitro and in vivo were found to be closely correlated with LAIR1. LAIR1 facilitates focal adhesion kinase (FAK) nuclear localization, resulting in increased transcription of cyclin D1 and chemokines/cytokines (CCL5, TGFβ2, and IL33). LAIR1 specifically supports in the immunosuppressive glioma microenvironment via CCL5-mediated microglia/macrophage polarization. SHP2Q510E (PTP domain mutant) or FAKNLM (non-nuclear localizing mutant) significantly reversed the LAIR1-induced growth enhancement in glioma cells. In addition, LAIR1Y251/281F (ITIMs mutant) and SHP2Q510E mutants significantly reduced FAK nuclear localization, as well as CCL5 and cyclin D1 expression. Further experiments revealed that the ITIMs of LAIR1 recruited SH2-containing phosphatase 2 (SHP2), which then interacted with FAK and induced FAK nuclear localization. This study uncovered a critical role for intrinsic LAIR1 in facilitating glioma malignant progression and demonstrated a requirement for LAIR1 and SHP2 to enhance FAK nuclear localization.

ES28.01 Biology of KRAS Targeting Agents

ES28.01 Biology of KRAS Targeting Agents

Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling

IntroductionSeverely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART).MethodsPhenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR.ResultsThe ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase(SHP)-1 and a reduced expression of positive regulators like Janus kinase(JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells.ConclusionsFunctionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs further investigation. The information may be useful in design of novel therapeutic targets for better management of disease.

Model-driven experimental analysis of the function of SHP-2 in IL-6-induced Jak/STAT signaling

Misregulated interleukin-6 (IL-6)-induced Jak/STAT signaling contributes to many diseases. Under non-pathological conditions Jak/STAT signaling is tightly regulated by a complex network of regulators. One of these regulators is the protein tyrosine phosphatase SH2-containing phosphatase 2 (SHP2). Although SHP2 is known to be a negative regulator of IL-6-induced Jak/STAT signaling, its exact molecular function is not entirely understood. To elucidate the function of SHP2 in IL-6 signaling we followed a systems biology approach, in which modeling, stepwise model refinement, and experimental analysis are closely linked. We come up with an identifiable model of early Jak/STAT signaling that describes the data and proves to be predictive. The model-based analysis implies that (1) the stepwise association of IL-6 with gp80 and gp130 and (2) STAT3 dimerization at the receptor are essential for the dynamics of early pathway activation, and (3) phosphorylation of SHP2 is nonlinear. Furthermore, the modeling results indicate that SHP2 does not act as a feedback inhibitor in an early phase of IL-6-induced Jak/STAT signaling. However, experimental data reveal that SHP2 exhibits a basal repressory function.

Suppression of Normal and Malignant Kit Signaling by a Bispecific Antibody Linking Kit with CD300a

Through its receptor Kit (CD117), stem cell factor (SCF) critically regulates human mast cell (MC) differentiation, survival, priming, and activation. The dominance of SCF in setting these parameters compels stringent contra-regulation to maintain a balanced MC phenotype. We have synthesized a library of bispecific Ab fragments to examine the effect of linking Kit with CD300a. In this study, we report that CD300a exerts a strong inhibitory effect on Kit-mediated SCF-induced signaling, consequently impairing MC differentiation, survival, and activation in vitro. This effect derives from Kit-mediated tyrosine phosphorylation of CD300a and recruitment of the SHIP-1 but not of SH2-containing protein phosphatase 1. CD300a inhibits the constitutive activation of the human leukemic HMC-1 cells but not their survival. Finally, CD300a abrogates the allergic reaction induced by SCF in a murine model of cutaneous anaphylaxis. Our findings highlight CD300a as a novel regulator of Kit in human MC and suggest roles for this receptor as a suppressor of Kit signaling in MC-related disorders.

Down-Modulation of Shp1 and Hsp70 Provides a Survival Advantage to the Ph+ Cells of CML Patients Additive to That Related to the Oncogenic BCR/ABL Protein and May Account for Resistance to Ima Treatment.

Some chronic myeloid leukemia (CML) patients in chronic-phase and even more in advanced-phase, demonstrate primary resistance to imatinib or develop secondary resistance during treatment. In this study we investigate the mechanisms of resistance in a couple of Ima-sensitive and -resistant Kcl22 cell lines (Kcl22-s and Kcl22-r) combining a gene expression profile and proteomics approach. This couple of cells is interesting for a series of reasons: none of the known mechanisms of Ima resistance, i.e. Bcr-Abl point mutations or its overexpression or MDR up-regulation, has been described in these cells; in addition, differently respect to the various Ph+ cell lines and more similarly to a Ph+ stem cells, the original parental Kcl22-s show a high level of resistance to the Ima-induced apoptosis. Microarray indicated that 153 genes are up-regulated and 103 are down-regulated in the Kcl22-r respect to the Kcl22-s cells. Up-regulated genes comprise genes encoding for some membrane transporters, cell signalling proteins including MAPK kinase pathway molecules, cancer testis antigens, apoptosis negative regulators and DNA repair proteins. Among the down-regulated genes, that comprised also Annexin-1 and the HSP-70 and HSP27 proteins, we found a very low levels of the non-receptor, SH2 domain-containing protein tyrosin phosphatase-1 (Shp1). This protein is widely expressed in hematopoietic cells, and is directly involved in the negative regulation of cytokine/protein tyrosine kinase-mediated signalling. Methylation-specific PCR analysis of specific regions of SHP-1 and HSP70 promoters showed that the down-regulation of these genes is related to the hypermethylation of their promoters. Treatment of Kcl22-r with the 5-Azacytidine (5-AC), a demethylant agent, resulted in induction of Shp1 thus providing a further confirmation for the role of promoter methylation in the down regulation of this gene. Interestingly, the 5-AC-induced expression of Shp1 correlated, in Kcl22-r, with a significant reduction of phosphorylation of both pSTAT3 and pERK1/2, as well as of proliferation capacity of these cells. The phosphorylation of these latter signalling proteins is rather low in Kcl22-s cells independently by the Bcr-Abl p210 activity and it is not changed by the 5-AC treatment. We also show that Shp1 interacts in these cells with the Shp2, another SH2-containing phosphatase with different biological properties. Indeed, it has been shown that Shp2 is able to positively regulate signalling pathways, including the Ras/MAPK pathway. Moreover, the Shp2-dependent activation of the Ras/MAPK pathway in Kcl22-r cells may be also due to Hsp70 protein down-regulation. Indeed, this chaperone protein has been recently shown to stabilize the inactive form of the oncogenic Shp2 phosphatase. Taken together, our results indicate that a complex pathway of proliferation and survival in Ph+ cells based on the balance between the levels of Shp1 and 2 phosphatases and Hsp70 may account for the Bcr-Abl independent activation of Ras/MAPK pathway and for the resistance to Ima.

Interleukin-4 Receptor Alpha Chain ITIM Motif Mutant Mice Potentiate Stat6 Activation and IgE Production

RATIONALE: Signaling via the IL-4 receptor alpha chain (IL-4Rα) plays a critical role in the evolution of Th2 responses and the pathology of allergic diseases such as asthma. The IL-4Rα is endowed with an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the fifth tyrosine residue in the cytoplasmic domain of IL-4Rα (Y709). This motif binds a number of SH2-containing phosphatases, including SHP-1, SHP-2, and SHIP. Its role in vivo remains unclear. METHODS: The in vivo function of this ITIM motif in IL-4R signaling was analyzed in mutant mice in which the Y709 residue was mutated into phenylalanine (Y709F) by knockin mutagenesis. RESULTS: B cells from the Y709F mutation demonstrated enhanced Stat6 but not Jak1 phosphorylation following IL-4 stimulation. In vivo, the Y709F mutant mice exhibited increased baseline levels of IgE in the serum and augmented total and antigen-specific IgE responses following immunization. T lymphocytes from this mutation also exhibit significantly increased production of IL-4 in a Th2 polarized milieu. CONCLUSIONS: These results point to a negative regulatory role for the Y709 ITIM motif in signaling via IL-4Rα in vivo. Mutations or polymorphisms that affect ITIM function may play an important role in disease pathogenesis of allergic inflammation such as asthma.

Aberrant Methylation of SHP-1 (SH2-Containing Phosphatases 1) and SOCS-1 (Suppressor of Cytokine Signalling 1) Genes in Immunodeficiency-Related Lymphomas.

SHP-1 and SOCS-1 are members of a family of negative regulators of signaling that act downstream of cytokine receptors, receptor tyrosine kinases and receptor complexes of the immune and hemopoietic system. SHP-1 and SOCS-1 act through the inhibition of the JAK2/STAT pathway and their inactivation is considered to be a relevant mechanism in JAK2/STAT pathway induction. Several observations point to induction of the JAK/STAT pathway as an important mechanism of lymphomagenesis. Since DNA methylation is a mechanism of gene silencing involved in the pathogenesis of human cancers, including lymphoma, here we tested the involvement of SHP-1 and SOCS-1 methylation in immunodeficiency-related lymphoma. Tumor samples from 34 HIV-related non-Hodgkin lymphoma (HIV-NHL) and 25 post-transplant lymphoproliferative disorders (PTLD) were analyzed for SOCS-1 and SHP-1 methylation by methylation-specific polymerase chain reaction. The tumor panel included 15 HIV-diffuse large B cell lymphoma (HIV-DLBCL), 10 HIV-Burkitt lymphoma (HIV-BL), 9 HIV-primary effusion lymphoma (HIV-PEL), 10 PTLD-immunoblastic (PTLD-IB), 7 PTLD-centroblastic (PTLD-CB), 4 polymorphic PTLD (P-PTLD), 3 PTLD-BL and 1 PTLD multiple myeloma (MM). Among HIV-NHL, SHP-1 methylation occurred in 23/34 (67%) cases, including 12/15 (80%) HIV-DLBCL, 4/10 (40%) HIV-BL, and 7/9 (78%) HIV-PEL. SOCS-1 methylation occurred in 6/34 (17%) HIV-NHL, including 5/9 (55%) HIV-PEL and 1/15 (7%) HIV-DLBCL. When considering EBV status, SHP-1 was methylated in 10/11 (91%) EBV-negative HIV-NHL and in 12/22 (54%) EBV-positive HIV-NHL. Among PTLD, SHP-1 methylation was detected in 20/25 (80%) cases, including 7/10 (70%) PTLD-IB, 6/7 (86%) PTLD-CB, 3/4 (75%) P-PTLD, 2/3 (66%) PTLD-BL and 1 PTLD MM. SOCS-1 methylation was detected in 3/25 (12%) PTLD, including 1/10 (10%) PTLD-IB and 2/7 (28%) PTLD-CB. When considering EBV status, SHP-1 was methylated in 12/13 (92%) EBV-negative PTLD and in 6/11 (54%) EBV-positive PTLD. The implications of our data are threefold. First, SHP-1 methylation is involved in the majority of HIV-NHL and PTLD. Second, it is remarkable that virtually all EBV-negative HIV-NHL and PTLD carry SHP-1 methylation. In EBV-positive B-cells, EBV infection activates the STAT pathway. It is conceivable that, in EBV-negative HIV-NHL and in EBV-negative PTLD, SHP-1 inactivation through aberrant methylation may surrogate EBV infection for STAT activation. Third, similar to observations in NHL of the immunocompetent host, SOCS-1 methylation is rarely implicated in the pathogenesis of immunodeficiency-related NHL. This notion is supported by the phenotype of SOCS1-deficient mice, that die of a myeloproliferative disease but do not develop a lymphoproliferative disease.

Ubiquitous Activation of Ras and Jak/Stat Pathways in Human HCC

Although the natural history and pathologic characteristics of human hepatocellular carcinoma (HCC) are well documented, the molecular pathogenesis of HCC remains poorly understood. Here, we define the role for Ras and Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathways in human HCC. Promoter and genomic status of Ras and Jak/Stat inhibitors were assessed in 80 HCCs by methylation-specific polymerase chain reaction and microsatellite analysis. Activation of Ras and Jak/Stat signaling pathways was determined by DNA sequencing, Western blot, and immunoprecipitation analysis. Suppression of Ras and Jak/Stat pathways in HCC cell lines was evaluated by viability and apoptosis assays. Activation of Ras and Jak/Stat pathways was enhanced in all HCCs when compared with nonneoplastic surrounding and normal livers coincidently with the suppression of at least 1 Ras (RASSF1A and/or NORE1A) and 2 Jak/Stat inhibitors (cytokine-inducible SH2-protein [CIS]; suppressor of cytokine signaling [SOCS]1, 2, 3; and SH2-containing phosphatases [SHP1]). HCC associated with cirrhosis showed significantly higher frequency of RASSF1A, CIS, and SOCS1 promoter methylation than HCC without cirrhosis (P < .002, P < .02, and P < .02, respectively). Furthermore, aberrant methylation of NORE1A and SOCS3 promoters was observed only in a subclass of HCC with poor survival, suggesting that inactivation of these 2 genes might be involved in HCC progression. Combined treatment of HCC cell lines with Ras and Jak/Stat inhibitors as well as with the demethylating agent zebularine induced a strong apoptotic response. These data demonstrate the ubiquitous activation of Ras and Jak/Stat pathways in HCC and suggest the potential use of Ras and Jak/Stat inhibitors and demethylating agents as therapeutic modality for human liver cancer.

An alternative exon 1 of the CD5 gene regulates CD5 expression in human B lymphocytes

AbstractT lymphocytes and a subpopulation of B lymphocytes express the CD5 coreceptor. Its functional importance is evident from the multiple levels and developmental stages of the regulation of its expression. We here report the discovery of a novel regulatory exon upstream of the noncoding region of the CD5 gene in humans. This alternate exon 1 is designated E1B (with the conventional exon 1 renamed E1A) and was shown to regulate the expression of CD5. E1B-containing transcripts existed exclusively in B lymphocytes and encoded a protein that was truncated and retained intracellularly. As a consequence, the amount of E1A-containing transcripts was down-regulated and the membrane CD5 expression was diminished in the presence of E1B-containing transcripts. High levels of E1A transcripts were found in chronic lymphocytic leukemia, and there were no E1A transcripts in 697 pre-B cells, which have no membrane CD5. Introduction of E1B into Jurkat cells reduced their membrane expression of CD5, and sequence analysis revealed that the E1B motif is a defective human endogenous retrovirus. A balance between the 2 alternative exons 1 might be central to the regulation of membrane CD5 in human B cells, and, through CD5-associated SH2-containing phosphatase 1, to the modulation of B-cell antigen receptor-transduced signals.

Controlling cytokine signaling by constitutive inhibitors

Cytokines are secreted proteins that regulate diverse biological functions by binding to receptors at the cell surface to activate complex signal transduction pathways including the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Stringent mechanisms of signal attenuation are essential for ensuring an appropriate, controlled cellular response. Three families of proteins, the SH2-containing phosphatases (SHP), the protein inhibitors of activated STATs (PIAS), and the suppressors of cytokine signaling (SOCS), inhibit specific and distinct aspects of cytokine signal transduction. The analysis of mice lacking genes for members of the SHP has shed much light on the roles of these proteins in vivo. In recent in vitro studies, the protein modifiers ubiquitin and small ubiquitin-like modifier (SUMO) have emerged as key players in the strategies employed by SOCS and PIAS to repress signaling. This review throws light on the mechanisms of action of these regulators as being evolved by the latest researches.

The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells

AbstractThe CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways. (Blood. 2004;104:4063-4070)

Suppression of Growth Hormone (GH) Janus Tyrosine Kinase 2/Signal Transducer and Activator of Transcription 5 Signaling Pathway in Transgenic Mice Overexpressing Bovine GH

High continuous GH levels in vivo produce desensitization of the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway of GH signaling in the liver. To evaluate the mechanisms involved in this desensitization, transgenic mice overexpressing bovine GH were used. In these animals, GH receptor and membrane-associated JAK2 kinase are increased 4.5- and 6-fold, respectively. However, JAK2. STAT5a and -5b do not become tyrosine phosphorylated in response to GH stimulus, nor are these STAT proteins recruited to membranes, suggesting that they cannot bind to the receptor. The content of the suppressor cytokine-inducible src homology 2 (SH2)-containing protein (CIS), both total and membrane-associated, is markedly increased in the liver of GH transgenic mice. This could account for the inhibition of STAT5 activation, because CIS competes with STAT5 for GH receptor docking sites. Existence of an alternative mechanism of negative regulation of this signaling pathway by chronically elevated GH levels is suggested by the low level of JAK2 phosphorylation that transgenic mice exhibit. Whereas total SH2-containing phosphatase 2 (SHP-2) content is the same in both kinds of mice, membrane-associated SHP-2 protein levels increase 4.5-fold in GH transgenic animals. This could explain the dramatic inhibition of JAK2 phosphotyrosine level, thus contributing to the suppression of GH signaling observed in these transgenic mice.

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and beta-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA.

Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcgammaRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) in FcgammaRIIB-mediated inhibition of FcgammaR signaling. Coexpression of SHP-1 enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA phagocytosis in COS-1 cells. SHP-1 also enhances the reduction in FcgammaRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP-1. Furthermore, the activation of SHP-1 tyrosine phosphorylation is observed following stimulation of FcgammaRII in COS-1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP-1 also enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA, indicating that FcgammaRIIB can use more than one pathway for its inhibitory action. In addition, SHP-1 and SHIP-1 can inhibit FcgammaRIIA phagocytosis and signal transduction in the absence of FcgammaRIIB. The data support emerging evidence that SH2-containing phosphatases, such as SHP-1 and SHIP-1, can modulate signaling by "activating" receptors.

B cell inhibitory receptors and autoimmunity.

The immune system of any organism must maintain a fine balance between activation and inhibition. It must possess adequate reactivity to raise an effective immune response to target non-self molecules while not harming the organism itself. Essential to this process is the ability to control the timing and place of activation and to limit the extent of, and eventually terminate, activation. Failure to maintain this balance will result in either immunodeficiency or autoimmunity. Inhibitory receptors are involved in this regulation, a number having been shown to be critical in controlling the B cell immune response. There are two broad classes of inhibitory receptor − most are of the immunoglobulin (Ig) superfamily while the remainder are lectin-like molecules. They share a number of structural and functional similarities. Each inhibitory receptor contains one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain essential for generation and transduction of inhibitory signals. The ITIM consists of a six amino acid consensus sequence (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val).1 Ligation of the inhibitory receptor to an immunoreceptor tyrosine-based activatory motif (ITAM)-containing activatory molecule results in tyrosine kinase phosphorylation of the tyrosine residue within the ITIM2 by lyn.3 Tyrosine phosphorylation of the ITIM allows it to bind and activate phosphatases containing an src homology 2 (SH2) domain. Two classes of SH2-containing inhibitory phosphatases have been identified: the protein tyrosine phosphatases SHP-1 and SHP-2, and the phosphoinositol phosphatases SHIP and SHIP2. These classes have separate downstream signalling pathways through which they modulate cellular inhibition. In general, each class of phosphatase interacts with the ITIMs of different inhibitory receptors but each inhibitory receptor appears to act predominantly through only one class of phosphatase.4 A number of inhibitory receptors have been described on B cells, the details of which are summarized in Fig. 1 and Table 1. We will concentrate on three of these, FcγRII, CD22 and PD−1, and in addition will discuss lyn, SHP-1 and SHIP, which are crucial elements in the signalling pathways of the inhibitory receptors. We will describe their probable physiological roles in immune regulation, and then review the evidence from knockout mice, spontaneous mouse models of autoimmunity and human disease that defective regulation by B cell inhibitory receptors can lead to autoimmunity. Open in a separate window Figure 1 B cell inhibitory receptors.

Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.

SHP2 and cbl participate in α-chemokine receptor CXCR4–mediated signaling pathways

AbstractStromal cell–derived factor (SDF)-1α and its receptor, CXCR4, play an important role in cell migration, embryonic development, and human immunodeficiency virus infection. However, the cellular signaling pathways that mediate these processes are not fully elucidated. We and others have shown that the binding of SDF-1α to CXCR4 activates phosphatidylinositol-3 kinase (PI-3 kinase), p44/42 mitogen-associated protein kinase, and the transcription factor nuclear factor–κB, and it also enhances the tyrosine phosphorylation and association of proteins involved in the formation of focal adhesions. In this study, we examined the role of phosphatases in CXCR4-mediated signaling pathways. We observed significant inhibition of SDF-1α–induced migration by phosphatase inhibitors in CXCR4-transfected pre-B lymphoma L1.2 cells, Jurkat T cells, and peripheral blood lymphocytes. Further studies revealed that SDF-1α stimulation induced robust tyrosine phosphorylation in the SH2-containing phosphatase SHP2. SHP2 associated with the CXCR4 receptor and the signaling molecules SHIP, cbl, and fyn. Overexpression of wild-type SHP2 increased SDF-1α–induced chemotaxis. Enhanced activation of fyn and lyn kinases and the tyrosine phosphorylation of cbl were also observed. In addition, SDF-1α stimulation enhanced the association of cbl with PI-3 kinase, Crk-L, and 14-3-3β proteins. Our results suggest that CXCR4-mediated signaling is regulated by SHP2 and cbl, which collectively participate in the formation of a multimeric signaling complex.

Negative regulation of FcϵRI signaling by FcγRII costimulation in human blood basophils

Background: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcϵRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcγRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine–based inhibitory motifs (ITIMs) in the FcγRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. Objective: In human basophils and mast cells FcϵRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcϵRI signaling is inhibited by FcγRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. Methods: FcγR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcγRII/FcϵRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcγRIIB ITIM sequence were used for adsorption assays. Results: We report that peripheral blood basophils express FcγRII (in both the ITIM-containing FcγRIIB and the immunoreceptor tyrosine–based activation motif–containing FcγRIIA forms) and that costimulating FcγRII and FcϵRI inhibits basophil FcϵRI-mediated histamine release, IL-4 production, and Ca2+ mobilization. The inhibition of basophil FcϵRI signaling by FcγRII/FcϵRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. Conclusions: Evidence that FcγRII/FcϵRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcγRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcϵRI signaling by costimulated FcγRII. (J Allergy Clin Immunol 2000;106:337-48.)

Effects of Src Homology Domain 2 (SH2)-Containing Inositol Phosphatase (SHIP), SH2-Containing Phosphotyrosine Phosphatase (SHP)-1, and SHP-2 SH2 Decoy Proteins on FcγRIIB1-Effector Interactions and Inhibitory Functions

Coaggregation of Fc gamma RIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate Fc gamma RIIB1 effects suggest that the receptor uses SH2-containing inositol phosphatase (SHIP) and SH2-containing phosphotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant Fc gamma RIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants' dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on Fc gamma RIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated Fc gamma RIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind Fc gamma RIIB1 and block Fc receptor function. The SHIP SH2, while not affecting Fc gamma RIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, Fc gamma RIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of Fc gamma RIIB1-mediated inhibitory signaling.