Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer

Previous study in our laboratory demonstrated suppression of the gene for protein tyrosine phosphatase receptor-type O (PTPRO) in primary and established rat hepatomas. The present study showed methylation-mediated silencing of this gene in primary human lung tumors and in several human lung cancer cell lines, one of the characteristics of many tumor-suppressor genes. The reduced expression of PTPRO in the primary lung tumors correlated with the methylation status of its CpG island. Demethylation of the gene by deoxy-5-azacytidine treatment led to its reactivation in a lung cancer line (A549). Overexpression of PTPRO in A549 cells inhibited anchorage-independent growth, delayed reentry of the cells into the cell cycle after release from cell-cycle arrest, and increased susceptibility of the cells to apoptosis. These data have demonstrated the growth-suppressor characteristics of PTPRO that are unique to a classical tumor suppressor.     

Protein tyrosine phosphatase 1B negatively regulates macrophage development through CSF-1 signaling

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolic phosphatase with the ability to dephosphorylate JAK2 and TYK2, and thereby down-regulate cytokine receptor signaling. Furthermore, PTP-1B levels are up-regulated in certain chronic myelogenous leukemia patients, which points to a potential role for PTP-1B in myeloid development. The results presented here show that the absence of PTP-1B affects murine myelopoiesis by modifying the ratio of monocytes to granulocytes in vivo. This bias toward monocytic development is at least in part due to a decreased threshold of response to CSF-1, because the PTP-1B -/- bone marrow presents no abnormalities at the granulocyte-monocyte progenitor level but produces significantly more monocytic colonies in the presence of CSF-1. This phenomenon is not due to an increase in receptor levels but rather to enhanced phosphorylation of the activation loop tyrosine. PTP-1B -/- cells display increased inflammatory activity in vitro and in vivo through the constitutive up-regulation of activation markers as well as increased sensitivity to endotoxin. Collectively, our data indicate that PTP-1B is an important modulator of myeloid differentiation and macrophage activation in vivo and provide a demonstration of a physiological role for PTP-1B in immune regulation.     

Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line

Protein tyrosine phosphatase 1B (PTP1B) has recently been implicated in the regulation of body weight. A surprising phenotype of PTP1B-deficient mice is their resistance to diet-induced obesity. Since leptin is one of the primary hormones involved in the regulation of body weight and energy homeostasis, we investigated whether PTP1B affects leptin receptor (lepR) signaling directly. A mouse hypothalamic cell line, GT1-7, was established as a suitable cell model for the study of leptin signaling. Stimulation of GT1-7 cells by leptin caused tyrosine phosphorylation of endogenous STAT3 and activation of a STAT-dependent luciferase reporter gene. Over-expression of PTP1B in GT1-7 cells resulted in a dose-dependent decrease in endogenous JAK2 and STAT3 tyrosine phosphorylation compared with cells transfected with lepR alone. Consistent with inhibition of JAK-STAT signaling, PTP1B over-expression caused a dose-dependent decrease in leptin-induced, STAT-dependent luciferase reporter gene activation in GT1-7 cells. Furthermore, over-expression of PTP1B led to a decrease in mRNA accumulation of suppressor-of-cytokine-signalling-3 (SOCS3) and c-fos, genes that are acutely induced by leptin. Using gene microarray analysis, we confirmed that PTP1B reduces the level of gene expression of SOCS3 and showed that the expression level of other leptin-regulated genes was affected. Genes up-regulated by leptin were decreased in cells over-expressing PTP1B. Conversely, the expression of genes down-regulated by leptin was enhanced by PTP1B over-expression in GT1-7 cells. Our findings indicate that PTP1B is a negative regulator of leptin signaling and suggest that PTP1B inhibitors might be efficacious in the treatment of obesity by increasing leptin sensitivity.     

Protein tyrosine phosphatase RQ is a phosphatidylinositol phosphatase that can regulate cell survival and proliferation

Protein tyrosine phosphatase RQ (PTPRQ) was initially identified as a protein tyrosine phosphatase (PTPase)-like protein that is upregulated in a model of renal injury. Here we present evidence that, like PTEN, the biologically important enzymatic activity of PTPRQ is as a phosphatidylinositol phosphatase (PIPase). The PIPase specificity of PTPRQ is broader than that of PTEN and depends on different amino acid residues in the catalytic domain. In vitro, the recombinant catalytic domain of PTPRQ has low PTPase activity against tyrosine-phosphorylated peptide and protein substrates but can dephosphorylate a broad range of phosphatidylinositol phosphates, including phosphatidylinositol 3,4,5-trisphosphate and most phosphatidylinositol monophosphates and diphosphates. Phosphate can be hydrolyzed from the D3 and D5 positions in the inositol ring. PTPRQ does not have either of the basic amino acids in the catalytic domain that are important for the PIPase activity of PTEN or the sequence motifs that are characteristic of type II phosphatidylinositol 5-phosphatases. Instead, the PIPase activity depends on the WPE sequence present in the catalytic cleft of PTPRQ, and in the "inactive" D2 domains of many dual-domain PTPases, in place of the WPD motif present in standard active PTPases. Overexpression of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. An E2171D mutation that retains or increases PTPase activity but eliminates PIPase activity, eliminates the inhibitory effects on proliferation and apoptosis. These results indicate that PTPRQ represents a subtype of the PTPases whose biological activities result from its PIPase activity rather than its PTPase activity.     

An ACTH-activated protein tyrosine phosphatase (PTP) is modulated by PKA-mediated phosphorylation

In adrenal cortex, ACTH regulation of steroidogenesis depends on PKA-dependent serine/threonine phosphorylation and also on the activity of protein tyrosine phosphatases (PTPs). In addition, ACTH increases total PTPs involving at least three soluble PTPs (50, 82 and 115 kDa). Serine/threonine phosphorylation of these enzymes themselves could be a regulatory mechanism of their activity since the increase of total PTP activity is dependent on PKA-activation. In this report we analyzed the effect of in vitro phospho-dephosphorylation processes on the activity displayed by the ACTH-activated PTP of 115 kDa. Using an in-gel PTP assay we demonstrate that dephosphorylation catalyzed by potato acid phosphatase (PAP) reduces the activity of the 115 kDa PTP present in ZF from ACTH-treated animals and PKA-mediated phosphorylation reverses this effect.     

CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptors in vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca²⁺ responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22 in vivo is likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.     

Altered lipid raft-associated proximal signaling and translocation of CD45 tyrosine phosphatase in B lymphocytes from patients with systemic lupus erythematosus

OBJECTIVE: B lymphocytes from patients with systemic lupus erythematosus (SLE) exhibit defective intracellular signaling, hyperactivity, and autoantibody production. Recent evidence indicates a reduced expression of Lyn kinase, a negative regulator of B cell signaling, and reduced translocation of Lyn into membrane signaling domains in SLE. The present study was undertaken to investigate the causes of this altered regulation of Lyn by assessing the expression levels of regulatory molecules and their translocation into the signaling domains of SLE B lymphocytes. METHODS: Blood was obtained from 48 patients with SLE and 28 healthy controls for the assessment of B lymphocytes. Levels and intracellular distribution of Lyn, CD45, COOH-terminal Src kinase (Csk), and c-Cbl were studied by Western blotting, confocal microscopy, and flow cytometry. The kinetics of signaling molecule translocation to the B cell receptor (BCR)-antigen synapse were investigated by confocal microscopy. RESULTS: A profound alteration in the expression and translocation of regulatory signaling molecules in membrane domains of B cells from patients with SLE was observed. B lymphocytes from SLE patients, but not those from healthy controls, expressed a low molecular weight isoform of CD45 in lipid raft signaling microdomains. Kinetic studies revealed that translocation of Lyn, CD45, Csk, and c-Cbl led to increased recruitment and retention of Lyn and CD45 in the BCR-antigen synapse in SLE B cells. CONCLUSION: The results provide evidence of altered expression and translocation/interaction of kinases and phosphatases in membrane signaling microdomains of B cells from patients with SLE. Altered translocation of CD45 correlated with reduced expression of Lyn, indicating that Lyn is a key molecule in the regulation of BCR-mediated signaling.     

Genetic reduction of striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder. Early in the pathophysiology of AD, synaptic function is disrupted by soluble Aβ oligomers, possibly through Aβ-mediated internalization of NMDA receptors. Striatal-enriched phosphatase (STEP) is a tyrosine phosphatase that regulates the internalization of NMDA receptors. Recent work shows that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Here, we use genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model and show that a decrease in STEP levels reverses cognitive and cellular deficits observed in these mice. Our results suggest that STEP inhibitors may prove therapeutic for this devastating disorder.     

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.     

Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.     

Lymphoid tyrosine phosphatase (LYP/PTPN22) Arg620Trp variant regulates insulin autoimmunity and progression to type 1 diabetes

Aims/hypothesis We analysed the contribution of the lymphoid protein tyrosine phosphatase (LYP) Arg620Trp variant (which corresponds to the PTPN22 C1858T polymorphism) to the emergence of beta-cell-specific humoral autoimmunity and progression to type 1 diabetes in man. We also explored the heterogeneity in the disease-predisposing effect of this polymorphism in relation to known disease loci, sex and age at disease onset.Subjects and methods A population-derived Finnish birth cohort with increased disease susceptibility conferred by HLA-DQB1 was monitored for the appearance of islet cell autoantibodies, and individuals found to be positive were tested for autoantibodies against insulin (IAA), glutamic acid decarboxylase and islet antigen-2 (n=574; mean follow-up time 4.9 years). Gene interaction effects on disease susceptibility were analysed in case–control and family series (546 patients, 538 controls, 245 nuclear families). All subjects were typed for HLA DR-DQ, insulin gene (INS), CTLA4 and PTPN22 C1858T polymorphisms.Results The PTPN22 1858TT genotype was associated with the appearance of IAA (adjusted hazard ratio=4.6, 95% CI 2.4–9.0; p=0.000013). PTPN22, INS and HLA-DRB1 had an additive effect on the emergence of IAA. The 1858TT and CT genotypes conferred an increased risk of developing additional autoantibodies or clinical disease (hazard ratio=4.1, 95% CI 1.5–11.6; and 1.6, 95% CI 1.1–2.4, respectively; p=0.003). The strong effect of PTPN22 on disease susceptibility (p=2.1×10⁻⁸) was more pronounced in males (p=0.021) and in subjects with non-DR4-DQ8/low-risk HLA genotypes (p=0.0004).Conclusions/interpretation In the pathogenesis of type 1 diabetes the underlying mechanism of the PTPN22 C1858T polymorphism appears to involve regulation of insulin-specific autoimmunity. Importantly, it strongly affects progression from prediabetes to clinical disease.     

Hematopoietic cell phosphatase associates with erythropoietin (Epo) receptor after Epo-induced receptor tyrosine phosphorylation: identification of potential binding sites

Erythropoietin (Epo) binding to its receptor (EpoR) induces tyrosine phosphorylation in responsive cells and this ability is required for a mitogenic response. One of the substrates of tyrosine phosphorylation is the Epo receptor (EpoR). The carboxyl region of EpoR cytoplasmic domain is required for EpoR phosphorylation and has been shown to negatively affect the response to Epo both in vivo and in cell lines. Hematopoietic cell phosphatase (HCP) has also been hypothesized to negatively regulate erythropoiesis, based on the hypersensitivity to Epo of erythroid lineage cells in moth-eaten mice that genetically lack HCP. In the studies presented here, we show that HCP binds the tyrosine phosphorylated Epo receptor through the amino-terminal src-homology 2 (SH2) domain of HCP. Using a series of phosphotyrosine-containing peptides, potential HCP binding sites in the cytoplasmic domain of the EpoR are identified. The results support the concept that, after Epo stimulation, phosphorylation of EpoR provides a docking site for HCP in the receptor complex. Recruitment of HCP to the complex and its subsequent dephosphorylation of substrates and/or associated kinases may be important to mitigate the ligand-induced mitogenic response.     

Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.     

Altered lipid raft–associated proximal signaling and translocation of CD45 tyrosine phosphatase in B lymphocytes from patients with systemic lupus erythematosus

Objective

B lymphocytes from patients with systemic lupus erythematosus (SLE) exhibit defective intracellular signaling, hyperactivity, and autoantibody production. Recent evidence indicates a reduced expression of Lyn kinase, a negative regulator of B cell signaling, and reduced translocation of Lyn into membrane signaling domains in SLE. The present study was undertaken to investigate the causes of this altered regulation of Lyn by assessing the expression levels of regulatory molecules and their translocation into the signaling domains of SLE B lymphocytes.

Methods

Blood was obtained from 48 patients with SLE and 28 healthy controls for the assessment of B lymphocytes. Levels and intracellular distribution of Lyn, CD45, COOH‐terminal Src kinase (Csk), and c‐Cbl were studied by Western blotting, confocal microscopy, and flow cytometry. The kinetics of signaling molecule translocation to the B cell receptor (BCR)–antigen synapse were investigated by confocal microscopy.

Results

A profound alteration in the expression and translocation of regulatory signaling molecules in membrane domains of B cells from patients with SLE was observed. B lymphocytes from SLE patients, but not those from healthy controls, expressed a low molecular weight isoform of CD45 in lipid raft signaling microdomains. Kinetic studies revealed that translocation of Lyn, CD45, Csk, and c‐Cbl led to increased recruitment and retention of Lyn and CD45 in the BCR–antigen synapse in SLE B cells.

Conclusion

The results provide evidence of altered expression and translocation/interaction of kinases and phosphatases in membrane signaling microdomains of B cells from patients with SLE. Altered translocation of CD45 correlated with reduced expression of Lyn, indicating that Lyn is a key molecule in the regulation of BCR‐mediated signaling.

     

Systemic Activin signaling independently regulates sugar homeostasis,cellular metabolism,and pH balance in Drosophila melanogaster

The ability to maintain cellular and physiological metabolic homeostasis is key for the survival of multicellular organisms in changing environmental conditions. However, our understanding of extracellular signaling pathways that modulate metabolic processes remains limited. In this study we show that the Activin-like ligand Dawdle (Daw) is a major regulator of systemic metabolic homeostasis and cellular metabolism in Drosophila. We find that loss of canonical Smad signaling downstream of Daw leads to defects in sugar and systemic pH homeostasis. Although Daw regulates sugar homeostasis by positively influencing insulin release, we find that the effect of Daw on pH balance is independent of its role in insulin signaling and is caused by accumulation of organic acids that are primarily tricarboxylic acid (TCA) cycle intermediates. RNA sequencing reveals that a number of TCA cycle enzymes and nuclear-encoded mitochondrial genes including genes involved in oxidative phosphorylation and β-oxidation are up-regulated in the daw mutants, indicating either a direct or indirect role of Daw in regulating these genes. These findings establish Activin signaling as a major metabolic regulator and uncover a functional link between TGF-β signaling, insulin signaling, and metabolism in Drosophila.Regulation of cellular metabolism and metabolic homeostasis is crucial for maintaining cellular and organismal physiology. Consequently, robust regulatory networks have evolved in most organisms to adapt and maintain a desired metabolic state depending on the environment and/or developmental stage (1, 2). Deciphering how components of these networks function to achieve homeostasis is essential for understanding normal physiology and the underlying mechanisms behind multifaceted disorders like metabolic syndrome and diabetes. Our understanding of these regulatory networks is largely confined to known metabolic pathways like the insulin signaling (IS) pathway, cellular target of rapamycin (TOR) pathway, and neuroendocrine signals. Nevertheless, additional extracellular signaling mechanisms that can regulate cellular and physiological metabolism must exist to allow integration of environmental, physiological, and developmental cues. In accordance with this possibility, involvement of classical developmental pathways in regulating physiological homeostasis has recently gained much attention (3, 4).One such developmental pathway that has been shown to affect several aspects of metabolism, including nutrient and energy homeostasis, is TGF-β signaling (5, 6). TGF-β signaling has recently been proposed to regulate mitochondrial biogenesis in mammals based on the observation that aberrant Activin signaling in mice can lead to changes in energy metabolism and mitochondrial gene expression (7). However, the study used a gene-replacement strategy where the mature domain of Activin-A was replaced with that of Activin-B (Inhiba^BK), thereby changing the relative levels of these two ligands. TGF-β ligands signal through distinct combinations of type I and type II receptors that are specific to different classes of ligands (8). Although both Activin-A and -B can signal redundantly through the type I receptor ALK4, Activin-B can also signal through ALK7 initiating unique biological responses (9). Hence, studies involving the Inhiba^BK mouse fail to distinguish individual contributions of Activin-A and Activin-B in the manifestation of the energy metabolism defects. Additionally, both Activin-A and -B have been implicated in regulation of IS (10). Because IS is known to impinge on both the TOR and AMPK signaling pathways, aberrant Activin signaling can potentially affect mitochondrial metabolism by affecting IS. Parsing out these mechanistic details is crucial for understanding the role of TGF-β/Activin signaling in metabolism and devising strategies to manipulate this pathway for therapeutic gain.In this study, we use Drosophila melanogaster to investigate the role of TGF-β/Activin signaling in regulation of metabolic homeostasis. Drosophila contains a highly conserved TGF-β signaling pathway. However, the number of signaling components in Drosophila is much smaller than vertebrates, allowing easy genetic manipulation of the pathway and reduces complications arising from functional redundancies between signaling molecules (8). Canonical TGF-β/Activin signaling in the fly is mediated by just three ligands, Activinβ (Actβ), Dawdle (Daw) [also called Alp (Activin-like peptide)], and Myoglianin (Myo), that signal through a single type I receptor, Babo, and a single intracellular R-Smad homolog, Smox (11). In addition, considerable parallels exist in the regulation of metabolic homeostasis between Drosophila and mammals providing a powerful genetic system to investigate the role of TGF-β signaling in metabolism and homeostasis (1, 2, 5).Here, we demonstrate that canonical TGF-β/Activin signaling, mediated by the Drosophila Activin homolog Daw, is a central metabolic regulator that impacts both mitochondrial metabolism and IS. Interestingly, we find that although Daw regulates IS by positively regulating release of Drosophila insulin-like peptides (Dilps), the effect of Daw on mitochondrial metabolism is independent of insulin/insulin-like growth factor signaling (IIS) and may be mediated by changes in expression of nuclear-encoded mitochondrial genes.     

Redox-sensitive signaling by angiotensin II involves oxidative inactivation and blunted phosphorylation of protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells from SHR

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II-induced SHP-2 effects were inhibited by valsartan (AT(1)R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II-mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT(1)R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II-stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II-induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2-dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II-AKT signaling.     

Interleukin 7 receptor ligation stimulates tyrosine phosphorylation, inositol phospholipid turnover, and clonal proliferation of human B-cell precursors.

Functional interleukin 7 (IL-7) receptors are expressed on the surface of multiphenotypic, biphenotypic, and immature B-lineage human lymphoid precursor cells with germ-line immunoglobulin heavy-chain genes but not on more mature B-lineage lymphoid cells with rearranged and/or expressed immunoglobulin heavy-chain genes. Thus, IL-7 may have an important regulatory role during the earliest stages of human B-cell ontogeny. The engagement of the surface IL-7 receptors on immature B-cell precursor cells with recombinant human IL-7 (rhIL-7) results in enhanced tyrosine phosphorylation of multiple phosphoproteins, stimulates inositol phospholipid turnover and DNA synthesis, and promotes their clonal proliferation. These effects are (i) specific for rhIL-7, since rhIL-3, rhIL-4, rhIL-5, rhIL-6, and recombinant human granulocyte colony-stimulating factor do not elicit similar activities on IL-7 receptor-positive human pro-B cells; and (ii) mediated by IL-7 receptors, since they are not observed in IL-7 receptor-negative B-lineage lymphoid cell populations. rhIL-7-induced tyrosine phosphorylation on the 35-, 53-, 55-, 62-, 69-, 76-, 94-, 150-, 170-, and 190-kDa substrates as well as rhIL-7-induced stimulation of inositol phospholipid turnover are abrogated by the tyrosine kinase inhibitor genistein. These results demonstrate that the IL-7 receptor on immature human B-cell precursor populations is intimately linked to a functional tyrosine kinase pathway and tyrosine phosphorylation is an important and perhaps mandatory step in the generation of the IL-7 receptor-linked transmembrane signal.     

Initial signaling of the fibronectin receptor (alpha5beta1 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation

BACKGROUND/AIMS: Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (alpha(5)beta(1)) receptor expression and interactions between alpha(5)beta(1) and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the alpha(5)beta(1) receptor initiates these changes. We aimed to determine the signaling cascade after alpha(5)beta(1) stimulation in activated HSC. METHODS: HSC were isolated from male Sprague-Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for alpha(5)beta(1)) or D-polylysine (inert control). HSC were stained with FTC-labeled antibodies against classes of signaling molecules. Tyrosine phosphorylation was blocked using genistein or herbimycin A. The fraction of beads with localized immunostaining (indicating accumulation of signaling protein) was determined. RESULTS: The majority of cytoskeletal proteins, Src substrates, Src kinases and members of the ERK and JNK signaling molecule families require actin cytoskeletal organization and tyrosine-kinase-mediated phosphorylation to accumulate. Several proteins (e.g. tensin, FAK) accumulated in the absence of tyrosine phosphorylation. CONCLUSIONS: The alpha(5)beta(1) integrin-ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by alpha(5)beta(1) are associated with cytoskeletal proteins and are independent of tyrosine phosphorylation. We suggest that there may be cytoskeletal changes that may be targeted to diminish HSC activation.