Oxidation sensitivity of the catalytic cysteine of the protein-tyrosine phosphatases SHP-1 and SHP-2

Reversible oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTPs) has emerged as a putative mechanism of activity regulation by physiological cell stimulation with growth factors, and by cell treatments with adverse agents such as UV irradiation. We compared SHP-1 and SHP-2, two structurally related cytoplasmic protein-tyrosine phosphatases with different cellular functions and cell-specific expression patterns, for their intrinsic susceptibility to oxidation by H(2)O(2). The extent of oxidation was monitored by detecting the modification of the PTP catalytic cysteine by three different methods, including a modified in-gel PTP assay, alkylation with a biotinylated iodoacetic acid derivative, and an antibody against oxidized PTPs. Dose-response curves for oxidation of the catalytic domains of SHP-1 and SHP-2 were similar. SHP-1 and -2 require relatively high H(2)O(2) concentrations for oxidation (half-maximal oxidation at 0.1-0.5 mM). For SHP-1, the SH2 domains had a significant protective function with respect to oxidation. In EOL-1 cells, SHP oxidation by exogenous H(2)O(2) in general and SHP-2 oxidation in particular was strongly diminished compared to HEK293 cells, at least partially related to a generally lower oxidant sensitivity of the EOL-1 cells. The data suggest that the differential cell functions of SHP-1 and SHP-2 are not related to differences in oxidation sensitivity. The modulating effects of SH2 domains for oxidation of these PTPs are in support of an enhanced oxidation susceptibility of activated SHPs.     

Molecular cloning of MIS, a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and SHP-2

We describe the molecular cloning and characterization of a novel myeloid inhibitory siglec, MIS, that belongs to the family of sialic acid-binding immunoglobulin-like lectins. A full-length MIS cDNA was obtained from murine bone marrow cells. MIS is predicted to contain an extracellular region comprising three immunoglobulin-like domains (V-set amino-terminal domain followed by two C-set domains), a transmembrane domain and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. The closest relative of MIS in the siglec family is human siglec 8. Extracellular regions of these two siglecs share 47% identity at the amino acid level. Southern blot analysis suggests the presence of one MIS gene. MIS is expressed in the spleen, liver, heart, kidney, lung and testis tissues. Several isoforms of MIS protein exist due to the alternative splicing. In a human promonocyte cell line, MIS was able to bind Src homology 2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2. This binding was mediated by the membrane-proximal ITIM of MIS. Moreover, MIS exerted an inhibitory effect on FcgammaRI receptor-induced calcium mobilization. These data suggest that MIS can play an inhibitory role through its ITIM sequences.     

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.     

Tyrosine phosphatases SHP-1 and SHP-2 are associated with distinct tyrosine-phosphorylated proteins.

SHP-1 and SHP-2 are two SH2 domain-containing tyrosine phosphatases. They share significant overall sequence identity but their functions are often opposite. The mechanism underlying this is not well understood. In this study, we have investigated the association of SHP-1 and SHP-2 with tyrosine-phosphorylated proteins in mouse tissues and in cultured cells treated with a potent tyrosine phosphatase inhibitor, pervanadate. Pervanadate was introduced into mice by intravenous injection. It induced robust tyrosine phosphorylation of cellular proteins in a variety of tissues. Both SHP-1 and SHP-2 were phosphorylated on tyrosyl residues upon pervanadate treatment, and they became associated with distinct tyrosine-phosphorylated proteins in different tissues and cells. Among these proteins, PZR and PECAM were identified as major SHP-2-binding proteins while LAIR-1 was shown to be a major SHP-1-binding protein. A number of other proteins are to be identified. We believe that the different binding proteins may determine the distinct physiological functions of SHP-1 and SHP-2. The present study also provides a general method to induce tyrosine phosphorylation of cellular proteins and to study protein-protein interactions involving tyrosine phosphorylation in vivo and in vitro.     

Redox regulation of dimerization of the receptor protein-tyrosine phosphatases RPTPalpha, LAR, RPTPmu and CD45

Whether dimerization is a general regulatory mechanism of receptor protein-tyrosine phosphatases (RPTPs) is a subject of debate. Biochemical evidence demonstrates that RPTPalpha and cluster of differentiation (CD)45 dimerize. Their catalytic activity is regulated by dimerization and structural evidence from RPTPalpha supports dimerization-induced inhibition of catalytic activity. The crystal structures of CD45 and leukocyte common antigen related (LAR) indicate that dimerization would result in a steric clash. Here, we investigate dimerization of four RPTPs. We demonstrate that LAR and RPTPmu dimerized constitutively, which is likely to be due to their ectodomains. To investigate the role of the cytoplasmic domain in dimerization we generated RPTPalpha ectodomain (EDalpha)/RPTP chimeras and found that -- similarly to native RPTPalpha -- oxidation stabilized their dimerization. Limited tryptic proteolysis demonstrated that oxidation induced conformational changes in the cytoplasmic domains of these RPTPs, indicating that the cytoplasmic domains are not rigid structures, but rather that there is flexibility. Moreover, oxidation induced changes in the rotational coupling of dimers of full length EDalpha/RPTP chimeras in living cells, which were largely dependent on the catalytic cysteine in the membrane-distal protein-tyrosine phosphatase domain of RPTPalpha and LAR. Our results provide new evidence for redox regulation of dimerized RPTPs.     

The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.     

Prediction of substrate sites for protein phosphatases 1B,SHP-1, and SHP-2 based on sequence features

Tyrosine phosphorylation plays crucial roles in numerous physiological processes. The level of phosphorylation state depends on the combined action of protein tyrosine kinases and protein tyrosine phosphatases. Detection of possible phosphorylation and dephosphorylation sites can provide useful information to the functional studies of relevant proteins. Several studies have focused on the identification of protein tyrosine kinase substrates. However, compared with protein tyrosine kinases, the prediction of protein tyrosine phosphatase substrates involved in the balance of protein phosphorylation level falls behind. This paper described a method that utilized the k-nearest neighbor algorithm to identity the substrate sites of three protein tyrosine phosphatases based on the sequence features of manually collected dephosphorylation sites. In the performance evaluation, both sensitivities and specificities could reach above 75 % for all three protein tyrosine phosphatases. Finally, the method was applied on a set of known tyrosine phosphorylation sites to search for candidate substrates.     

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

蛋白酪氨酸磷酸酶SHP-1/SHP-2催化活性域的表达和动力学分析

为了分离纯化SHP-1/SHP-2催化活性域蛋白(分别命名为D1C/D2C), 并估测其动力学常数, 将已经构建好的D1C/D2C重组质粒转化Escherichia coli BL21菌株, 经IPTG诱导表达、菌体裂解缓冲液悬浮和超声波破碎后, 通过HPLC分离纯化D1C/D2C蛋白, 所得产物进行SDS-PAGE电泳检测。然后, 以pY作为去磷酸化反应的底物, 利用孔雀绿显色法, 通过双倒数作图法对纯化的D1C/D2C蛋白进行动力学分析。结果表明, 本试验已成功地表达了D1C和D2C蛋白, 主要以可溶性蛋白的形式表达; 利用HPLC技术可有效地对D1C/D2C蛋白进行分离纯化; D1C的相对分子质量为34.6 kD, 米氏常数Km=2.04 mmol/L, 催化常数Kcat=44.98 s, 特异性常数Kcat/Km=22.05 L/(mmol·s); D2C的相对分子质量为35.3 kD, 米氏常数Km=2.47 mmol/L, 催化常数Kcat=27.45 s, 特异性常数Kcat/Km=11.11 L/(mmol·s); D1C的磷酸酶活性较强于D2C。     

Antagonism or synergism. Role of tyrosine phosphatases SHP-1 and SHP-2 in growth factor signaling

SHP-1 and SHP-2 are two Src homology 2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer and SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with small interfering RNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 because it was abrogated by knockdown of SHP-2, and SHP-2, but not SHP-1, binds directly to tyrosine-phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for previously unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases.     

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates p120 catenin

A prominent tyrosine-phosphorylated protein of approximately 100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120(ctn)). Different p120(ctn) isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120(ctn) tyrosine phosphorylation. Despite strong phosphorylation, p120(ctn) isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120(ctn) in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120(ctn) exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120(ctn), which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120(ctn) interaction.     

Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31.

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP-1 and SHP-2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP-1 and SHP-2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC-gamma1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP-1, SHP-2, SHIP and PLC-gamma1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate-stimulated THP-1 cells. These results suggest that overlapping immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based activation motif-like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP-1, SHP-2, SHIP and PLC-gamma1.     

A SHPing tale: perspectives on the regulation of SHP-1 and SHP-2 tyrosine phosphatases by the C-terminal tail

Protein tyrosine phosphorylation is a ubiquitous signalling mechanism and is regulated by a balance between the action of kinases and phosphatases. The SH2 domain-containing phosphatases SHP-1 and SHP-2 are the best studied of the classical non-receptor tyrosine phosphatases, but it is intriguing that despite their close sequence and structural homology these two phosphatases play quite different cellular roles. In particular, whereas SHP-1 plays a largely negative signalling role suppressing cellular activation, SHP-2 plays a largely positive signalling role. Major sequence differences between the two molecules are apparent in the approximately 100 amino acid residues at the extreme C-terminus of the proteins, beyond the phosphatase catalytic domain. Here we review how the differences in the tails of these proteins may regulate their activities and explain some of their functional differences.     

Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome

PRL-1, -2, and -3 represent a novel class of protein-tyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL-1 and -3 accumulated in a collapsed compact structure around the microtubule-organizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole-like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation.     

The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.     

LST1/A is a myeloid leukocyte-specific transmembrane adaptor protein recruiting protein tyrosine phosphatases SHP-1 and SHP-2 to the plasma membrane

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.     

Recruitment of the protein-tyrosine phosphatase SHP-2 to the C-terminal tyrosine of the prolactin receptor and to the adaptor protein Gab2

The protein-tyrosine phosphatase SHP-2 modulates signaling events through receptor tyrosine kinases and cytokine receptors including the receptor for prolactin (PRLR). Here we investigated mechanisms of SHP-2 recruitment within the PRLR signaling complex. Using SHP-2 and PRLR immunoprecipitation studies in 293 cells and in the mouse mammary epithelial cell line HC11, we found that SHP-2 co-immunoprecipitates with the PRLR and that the C-terminal tyrosine of the PRLR plays a regulatory role in both the tyrosine phosphorylation and the recruitment of SHP-2. Our results further indicate that SHP-2 association to the PRLR occurs via the C-terminal SH2 domain of the phosphatase. In addition, we determined that the newly identified adaptor protein Gab2, but not Gab1, is specifically tyrosine phosphorylated and is able to recruit SHP-2 and phosphatidyinositol 3-kinase in response to PRLR activation. Together, these studies suggest the presence of dual recruitment sites for SHP-2; the first is to the C-terminal tyrosine of the PRLR and the second is to the adaptor protein Gab2.     

Insufficient phosphorylation prevents fc gamma RIIB from recruiting the SH2 domain-containing protein-tyrosine phosphatase SHP-1

Fc gamma RIIB are IgG receptors that inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. Inhibition depends on an immunoreceptor tyrosine-based inhibition motif (ITIM) that is phosphorylated upon Fc gamma RIIB coaggregation with ITAM-bearing receptors and recruits SH2 domain-containing phosphatases. Agarose bead-coated phosphorylated ITIM peptides (pITIMs) bind in vitro the single-SH2 inositol 5-phosphatases (SHIP1 and SHIP2) and the two-SH2 protein tyrosine phosphatases (SHP-1 and SHP-2). Phosphorylated Fc gamma RIIB, however, recruit selectively SHIP1/2 in vivo. We aimed here at explaining this discordance. We found that beads coated with low amounts of pITIM bound in vitro SHIP1, but not SHP-1, i.e. behaved as phosphorylated Fc gamma RIIB in vivo. The reason is that SHP-1 requires its two SH2 domains to bind on adjacent pITIMs. Consequently, the binding of SHP-1, but not of SHIP1, increased with pITIM density on beads. When trying to increase Fc gamma RIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for Fc gamma RIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by Fc gamma RIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that Fc gamma RIIB phosphorylation may not be sufficient in vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate Fc gamma RIIB might enable SHP-1 recruitment.