Role and interrelationship of PTPases and H2O2 in light/dark-regulated stomatal movement in Vicia faba

Role and interrelationship of protein tyrosine phosphatases (PTPases) and H2O2 in light/dark-regulated stomatal movement in Vicia faba were investigated by epidermal strip bioassay, laser-scanning confocal microscopy and assays of PTPase activity. Our results indicate that phenylarsine oxide (PAO), a specific inhibitor of PTPases, ascorbic acid (ASA), an important reducing substrate for H2O2 removal, and catalase (CAT), one of the H2O2 scavenging enzymes, did not cause any change of stomatal aperture in light, but remarkably prevented dark-induced stomatal closure. Exogenous H2O2 had no obvious effect on stomatal aperture in the dark, but significantly induced stomatal closure in light. Both PTPase activity in epidermal strips and endogenous H2O2 level in guard cells in the dark were higher than those in light. The results showed that both PTPases and H2O2 mediate light/dark-regulated stomatal movement, that dark-induced stomatal closure requires the activation of PTPases and the enhancement of H2O2 levels in guard cells, and stomatal opening caused by light is associated with the inactivation of PTPases and the reduction of H2O2 levels in guard cells. Additionally, like ASA and CAT, PAO abolished dark-, exogenous H2O2-induced stomatal closure and dichlorofluorescein fluorescence in guard cells, indicating that activation of PTPases can enhance H2O2 levels probably via suppressing the decrease of H2O2 levels in guard cells. On the other hand, similar to PAO, ASA and CAT evidently prevented dark-, exogenous H2O2-induced stomatal closure and obviously inactivated PTPases in the dark. However, exogenous H2O2 significantly activated PTPases in light. The results show that H2O2 can induce activation of PTPases. Taken together, the present results provide evidence that both H2O2 and PTPases are involved in light/dark-regulated stomatal movement, and the interaction between H2O2 and PTPases plays a pivotal role in light/dark signal transduction process in guard cells.

A Noncatalytic, Ras-Independent Function of SHP-2 Is Essential in Hematopoietic Progenitors.

SHP-2 is a nonreceptor protein tyrosine phosphatase (PTPase) that is essential for embryonic hematopoiesis. SHP-2 acts as a signal relay molecule downstream of diverse growth factor receptors, and potentiates the activity of the Ras/Raf/MAPK pathway. Epistasis studies in model organisms indicate that SHP-2 can act upstream, downstream or parallel to Ras; however, most mammalian systems place SHP-2 upstream of Ras activation. Activating mutations in PTPN11, the gene encoding SHP-2, comprise the most common genetic lesion in juvenile myelomonocytic leukemia (JMML). Other etiologies of JMML include activating mutations in NRAS or KRAS2 and inactivation of the tumor suppressor NF1, which encodes a negative regulator of Ras, supporting a model in which SHP-2 activates Ras in hematopoietic progenitors. We tested the hypothesis that SHP-2 is essential in hematopoiesis because it is required for Ras activation. To do this, we bred mice with conditional hyperactive KrasG12D and inactive Ptpn11flox alleles, in conjunction with the inducible Mx1-Cre transgene. Myeloid progenitors in LSL-KrasG12D; Ptpn11flox/flox; Mx1-Cre mice never deleted both Ptpn11 alleles despite efficient Cre induction and expression of activated K-RasG12D. This indicates selective pressure to retain SHP-2 despite Ras activation and implies that Ptpn11 loss is epistatic to KrasG12D. To test this directly, we acutely disrupted Ptpn11 and expressed KrasG12D in fetal liver cells using Cre-expressing retroviruses, and tested them for myeloid colony-forming activity. This confirmed the need for SHP-2 in myeloid progenitors, and KrasG12D again failed to alleviate this requirement. Surprisingly, expression of either wild type or PTPase-deficient SHP-2 rescued colony growth and restored the aberrant growth of KrasG12D mutant cells. To test whether hematopoiesis in vivo is independent of SHP-2 PTPase function, we created chimeric Mx1-Cre, Ptpn11flox/flox mice in which a small fraction of bone marrow expressed exogenous SHP-2 and GFP. These mice were treated with a short course of pIpC to induce Cre and disrupt Ptpn11, and the contribution of SHP-2 expressing cells in the peripheral blood was monitored by flow cytometry. As expected, exogenous SHP-2 expression in Mx1-Cre, Ptpn11flox/flox bone marrow conferred a strong and durable (>16 wks) competitive advantage after pIpC treatment. Expression of PTPase-deficient SHP-2 also conferred a strong advantage lasting at least several weeks, and these mice are being aged to determine the duration of this response. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro, and suggest that SHP-2 is needed for maintenance of adult hematopoietic stem cells. In contrast to nearly all prior studies in mammalian cells, our data support a model in which SHP-2 has essential hematopoietic functions downstream or parallel to Ras activation. Furthermore, SHP-2 PTPase activity is not required in myeloid progenitors, a finding which also contradicts widely accepted models of SHP-2 function and may indicate that SHP-2 serves an adaptor role. Because PTPase activity is required by leukemogenic but not wild type SHP-2, pharmacologic PTPase inhibition may selectively target neoplastic hematopoietic progenitors.

Acetaldehyde dissociates the PTP1B–E-cadherin–β-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

Interactions between E-cadherin, β-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell–cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, β-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and β-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and β-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and β-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of β-catenin on tyrosine residues, and abolished the interaction of β-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of β-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and β-catenin was reduced by tyrosine phosphorylation of β-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)–PTP1B. The pairwise binding study showed that GST–E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of β-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, β-catenin and PTP1B by a phosphorylation-dependent mechanism.

Membrane-bound acid phosphatase (MAP) from Entamoeba histolytica has phosphotyrosine phosphatase activity and disrupts the actin cytoskeleton of host cells

Protein tyrosine phosphatases (PTPases) have been described as virulence factors in different pathogenic microorganisms. The pathogenic process by Entamoeba histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis. Lytic enzymes may participate during the second part of this process. In this work, we determined that purified membrane-bound acid phosphatase (MAP) from E. histolytica trophozoites has PTPase activity. The enzyme specifically dephosphorylated O-phospho-L-tyrosine at optimum pH of 5·0, with little activity towards O-phospho-L-serine, O-phospho-L-threonine, and ATP. It was inhibited by ammonium molybdate and sodium tungstate, and trifluoperazine did not show any effect. A monoclonal antibody against the catalytic domain of the human placental PTPase 1B, cross-reacted with a 55 kDa molecule present in the solubilized fraction. The interaction of the amoebic PTPase with HeLa cells resulted in the alteration of the cell actin cytoskeleton by disruption of the actin stress fibres.

Protein Tyrosine Phosphatase Activity in Insulin-Resistant RodentPsammomys Obesus

Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied inPsammomys obesus(sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) β-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat.Western blotting of tissue membrane fractions inPsammomysshowed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabeticPsammomysrose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance,Psammomysis characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes.

TSH-induced differentiated functions correlate with enhancement of phosphotyrosine phosphatase activity in thyroid cells. Effect of phorbol 12-myristate 13-acetate

TSH-treated pig thyroid cells reorganize into follicle-like structures and exhibit differentiated functions. TSH also induces a phosphotyrosine phosphatase (PTPase) activity evaluated by phosphorylated substrate hydrolysis. Incubation of thyrocytes with various concentrations of 8-bromo-cyclic AMP or forskolin induces an increase of PTPase activity in a dose-dependent manner. During the culture period, adenylyl cyclase sensitivity, protein binding iodine and PTPase activity progressively increase from the first to the fourth day of the culture. Chronic treatment with phorbol 12-myristate 13-acetate (PMA) significantly inhibits PTPase activity during the first 24 h following PMA addition. GF 109203X, a specific inhibitor of protein kinase C, abolishes the inhibitory effect of PMA. Electrophoresis of membrane extracts allowed us to demonstrate a phosphatase activity at 111 kDa (p111). Vanadate inhibits this activity, indicating that p111 is a PTPase. This p111 is significantly reduced in PMA-treated cells. These data suggest that PTPase activity evidenced at 111 kDa is correlated with a differentiated state of primary cultured pig thyroid cells induced by TSH.

Distinct Functions of the Two Protein Tyrosine Phosphatase Domains of LAR (Leukocyte Common Antigen-Related) on Tyrosine Dephosphorylation of Insulin Receptor

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.