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.     

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.     

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.     

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.     

Protein tyrosine phosphatases as novel targets in breast cancer therapy

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.     

Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling

Receptor tyrosine kinases (RTKs) control a wide variety of processes in multicellular organisms, including proliferation, differentiation, migration and survival. Their activity is tightly controlled through the coordinated action of both positive and negative regulators that function at multiple levels of the signal transduction cascade, and at different time points within the growth-factor-induced response. When this process goes awry, the outcome can be developmental defects and malignancy. Sprouty (Spry) proteins represent a major class of ligand-inducible inhibitors of RTK-dependent signaling pathways. New biochemical and genetic evidence indicates specific roles of the Spry genes in development and multiple modes of action of the Spry proteins in regulation of the RTK-induced response.     

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.     

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.     

Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo

We have investigated the regulation of protein tyrosine phosphatases (PTPs) by reactive oxygen species (ROS) in a cellular environment. We demonstrate that multiple PTPs were reversibly oxidized and inactivated following treatment of Rat-1 cells with H(2)O(2) and that inhibition of PTP function was important for ROS-induced mitogenesis. Furthermore, we show transient oxidation of the SH2 domain containing PTP, SHP-2, in response to PDGF that requires association with the PDGFR. Our results indicate that SHP-2 inhibits PDGFR signaling and suggest a mechanism by which autophosphorylation of the PDGFR occurs despite its association with SHP-2. The data suggest that several PTPs may be regulated by oxidation and that characterization of this process may define novel links between specific PTPs and particular signaling pathways in vivo.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2

B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2.     

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.     

An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases

Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.     

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.