Cutting edge: the tyrosine phosphatase SHP-1 regulates thymocyte positive selection

The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development.     

Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells

The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1-deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.     

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.     

Substrate specificity of protein tyrosine phosphatase: differential behavior of SHP-1 and SHP-2 towards signal regulation protein SIRPalpha1

The substrate specificity of catalytic domains and the activation of full length protein tyrosine phosphatases, SHP-1 and SHP-2 have been investigated using synthetic phosphotyrosyl peptides derived from SIPRalpha1. We found that the catalytic domains of SHP-1 and SHP-2 exhibit different substrate specificity towards a longer trideca-peptide pY(469+3) ((-7)RPEDTLTpYADLDM(+5)) and not to the shorter decapeptide pY(469) ((-5)EDTLTpYADLD(+4)), the former being the substrate of SHP-2 only. Furthermore, the activation of full-length SHP-1 and not the SHP-2 by the deca/trideca-peptides suggested SIRPalpha 1 to be possibly acting as both an upstream activator and a substrate for SHP-1, and merely as the downstream substrate for SHP-2 in signaling events.     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

Role of SHP-2 tyrosine phosphatase in the DNA damage-induced cell death response

SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in the regulation of growth factor and cytokine signal transduction. Here we report a novel function of this phosphatase in DNA damage-induced cellular responses. Mutant embryonic fibroblast cells lacking functional SHP-2 showed significantly decreased apoptosis in response to DNA damage. Following cisplatin treatment, induction of p73 and its downstream effector p21(Cip1) was essentially blocked in SHP-2 mutant cells. Further investigation revealed that activation of the nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage induction of p73, was impaired in the mutant cells, suggesting a functional requirement of SHP-2 in c-Abl activation. Consistent with this observation, the effect of overexpression of c-Abl kinase in SHP-2 mutant cells on sensitizing the cells to DNA damage-induced death was abolished. Additionally, we found that in embryonic fibroblast cells 30-40% of SHP-2 was localized in the nuclei, and that a fraction of nuclear SHP-2 was constitutively associated with c-Abl via its SH3 domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was significantly enhanced in response to DNA damage. These results together suggest a novel nuclear function for SHP-2 phosphatase in the regulation of DNA damage-induced apoptotic responses.     

Determination of the molecular reach of the protein tyrosine phosphatase SHP-1

Immune receptors signal by recruiting (or tethering) enzymes to their cytoplasmic tails to catalyze reactions on substrates within reach. This is the case for the phosphatase SHP-1, which, upon tethering to inhibitory receptors, dephosphorylates diverse substrates to control T cell activation. Precisely how tethering regulates SHP-1 activity is incompletely understood. Here, we measure binding, catalysis, and molecular reach for tethered SHP-1 reactions. We determine the molecular reach of SHP-1 to be 13.0 nm, which is longer than the estimate from the allosterically active structure (5.3 nm), suggesting that SHP-1 can achieve a longer reach by exploring multiple active conformations. Using modeling, we show that when uniformly distributed, receptor-SHP-1 complexes can only reach 15% of substrates, but this increases to 90% when they are coclustered. When within reach, we show that membrane recruitment increases the activity of SHP-1 by a 1000-fold increase in local concentration. The work highlights how molecular reach regulates the activity of membrane-recruited SHP-1 with insights applicable to other membrane-tethered reactions.     

The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis

The protein tyrosine phosphatase SHP-1 is a well-known inhibitor of activation-promoting signaling cascades in hematopoietic cells but its potential role in insulin target tissues is unknown. Here we show that Ptpn6(me-v/me-v) (also known as viable motheaten) mice bearing a functionally deficient SHP-1 protein are markedly glucose tolerant and insulin sensitive as compared to wild-type littermates, as a result of enhanced insulin receptor signaling to IRS-PI3K-Akt in liver and muscle. Downregulation of SHP-1 activity in liver of normal mice by adenoviral expression of a catalytically inert mutant of SHP-1, or after small hairpin RNA-mediated SHP-1 silencing, further confirmed this phenotype. Tyrosine phosphorylation of CEACAM1, a modulator of hepatic insulin clearance, and clearance of serum [125I]-insulin were markedly increased in SHP-1-deficient mice or SHP-1-deficient hepatic cells in vitro. These findings show a novel role for SHP-1 in the regulation of glucose homeostasis through modulation of insulin signaling in liver and muscle as well as hepatic insulin clearance.     

Crystal structure of human protein tyrosine phosphatase SHP-1 in the open conformation

SHP‐1 belongs to the family of non‐receptor protein tyrosine phosphatases (PTPs) and generally acts as a negative regulator in a variety of cellular signaling pathways. Previously, the crystal structures of the tail‐truncated SHP‐1 and SHP‐2 revealed an autoinhibitory conformation. To understand the regulatory mechanism of SHP‐1, we have determined the crystal structure of the full‐length SHP‐1 at 3.1 Å. Although the tail was disordered in current structure, the huge conformational rearrangement of the N‐SH2 domain and the incorporation of sulfate ions into the ligand‐binding site of each domain indicate that the SHP‐1 is in the open conformation. The N‐SH2 domain in current structure is shifted away from the active site of the PTP domain to the other side of the C‐SH2 domain, resulting in exposure of the active site. Meanwhile, the C‐SH2 domain is twisted anticlockwise by about 110°. In addition, a set of new interactions between two SH2 domains and between the N‐SH2 and the catalytic domains is identified, which could be responsible for the stabilization of SHP‐1 in the open conformation. Based on the structural comparison, a model for the activation of SHP‐1 is proposed. J. Cell. Biochem. 112: 2062–2071, 2011. © 2011 Wiley‐Liss, Inc.     

A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation.

SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth.     

Association of tyrosine phosphatase SHP-2 with F-actin at low cell densities

SHP-2 is an intracellular SH2 domain-containing protein-tyrosine phosphatase with an essential role in cell signaling. Here we demonstrate that localization of SHP-2 is regulated by cell density in a cell adhesion-dependent manner. When cells were plated at low densities, SHP-2 was distributed in Triton X-100-insoluble fractions, whereas it was totally soluble when cells were plated at high densities or when low density cells approached confluency. In all cases, the total protein level of SHP-2 was not changed. Fluorescent cell staining revealed that SHP-2 was co-localized with actin stress fibers to the cell peripheral at low cell densities but was diffused in the entire cytoplasm at high cell densities. Transient transfection of cells with truncated forms of SHP-2 demonstrated that the catalytic domain of the enzyme was responsible for the density-regulated distribution of SHP-2, but the catalytic activity was not required. An in vitro co-sedimentation study demonstrated direct binding of full-length and SH2 domain-truncated forms of SHP-2 to F-actin. The data indicate that SHP-2 is regulated by cell density and that it may have a role in assembling and disassembling of the actin network.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.     

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.     

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.     

Regulating Vav1 phosphorylation by the SHP-1 tyrosine phosphatase is a fine-tuning mechanism for the negative regulation of DISC formation and Fas-mediated cell death signaling

The actin cytoskeleton association is required for caspase 8-independent Fas/CD95 receptor internalization, a critical step for an optimal death-inducing signaling complex formation along the endocytic pathway, leading to efficient activation of the caspase cascade and, ultimately, cell death. However, the way in which this initiation phase of Fas receptor signaling is regulated is still unknown. We report herein that, in B cells, upon Fas engagement, the tyrosine phosphatase SHP-1-regulated Vav dephosphorylation, by downmodulating the Fas-ezrin-actin linkage is a fine-tune switch-off mechanism that the cell uses as a way to terminate the receptor internalization, controlling therefore the time and extent of the DISC formation and cell death.