Two Dictyostelium tyrosine kinase–like kinases function in parallel,stress-induced STAT activation pathways

When Dictyostelium cells are hyperosmotically stressed, STATc is activated by tyrosine phosphorylation. Unusually, activation is regulated by serine phosphorylation and consequent inhibition of a tyrosine phosphatase: PTP3. The identity of the cognate tyrosine kinase is unknown, and we show that two tyrosine kinase–like (TKL) enzymes, Pyk2 and Pyk3, share this function; thus, for stress-induced STATc activation, single null mutants are only marginally impaired, but the double mutant is nonactivatable. When cells are stressed, Pyk2 and Pyk3 undergo increased autocatalytic tyrosine phosphorylation. The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action. The signaling pathways that activate Pyk2 and Pyk3 are only partially overlapping, and there may be a structural basis for this difference because Pyk3 contains both a TKL domain and a pseudokinase domain. The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain. The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.     

The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.     

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Involvement of the membrane distal catalytic domain in pervanadate-induced tyrosine phosphorylation of receptor protein-tyrosine phosphatase alpha

Receptor protein-tyrosine phosphatase alpha, RPTPalpha, is a typical transmembrane protein-tyrosine phosphatase (PTP) with two cytoplasmic catalytic domains. RPTPalpha became strongly phosphorylated on tyrosine upon treatment of cells with the PTP inhibitor pervanadate. Surprisingly, mutation of the catalytic site Cys in the membrane distal PTP domain (D2), but not of the membrane proximal PTP domain (D1) that harbors the majority of the PTP activity, almost completely abolished pervanadate-induced tyrosine phosphorylation. Pervanadate-induced RPTPalpha tyrosine phosphorylation was not restricted to Tyr789, a known phosphorylation site. Cotransfection of wild-type RPTPalpha did not potentiate tyrosine phosphorylation of inactive RPTPalpha-C433SC723S, suggesting that RPTPalpha-mediated activation of kinase(s) does not underlie the observed effects. Mapping experiments indicated that pervanadate-induced tyrosine phosphorylation sites localized predominantly, but not exclusively, to the C-terminus. Our results demonstrate that RPTPalpha-D2 played a role in pervanadate-induced tyrosine phosphorylation of RPTPalpha, which may suggest that RPTPalpha-D2 is involved in protein-protein interactions.     

Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.     

Protein tyrosine phosphatase 1B deficiency potentiates PERK/eIF2α signaling in brown adipocytes

Background

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and body mass, and has been implicated in endoplasmic reticulum (ER) stress. Herein, we assess the role of PTP1B in ER stress in brown adipocytes, which are key regulators of thermogenesis and metabolic response.

Methodology/Principal Findings

To determine the role of PTP1B in ER stress, we utilized brown adipose tissue (BAT) from mice with adipose-specific PTP1B deletion, and brown adipocytes deficient in PTP1B and reconstituted with PTP1B wild type (WT) or the substrate-trapping PTP1B D181A (D/A) mutant. PTP1B deficiency led to upregulation of PERK-eIF2α phosphorylation and IRE1α-XBP1 sub-arms of the unfolded protein response. In addition, PTP1B deficiency sensitized differentiated brown adipocytes to chemical-induced ER stress. Moreover, PERK activation and tyrosine phosphorylation were increased in BAT and adipocytes lacking PTP1B. Increased PERK activity resulted in the induction of eIF2α phosphorylation at Ser51 and better translatability of ATF4 mRNA in response to ER stress. At the molecular level, we demonstrate direct interaction between PTP1B and PERK and identify PERK Tyr615 as a mediator of this association.

Conclusions

Collectively, the data demonstrate that PTP1B is a physiologically-relevant modulator of ER stress in brown adipocytes and that PTP1B deficiency modulates PERK-eIF2α phosphorylation and protein synthesis.     

Glycogen synthase kinase-3β regulates Tyr307 phosphorylation of protein phosphatase-2A via protein tyrosine phosphatase 1B but not Src

GSK-3β (glycogen synthase kinase-3β), a crucial tau kinase, negatively regulates PP2A (protein phosphatase 2A), the most active tau phosphatase that is suppressed in the brain in AD (Alzheimer's disease). However, the molecular mechanism is not understood. In the present study we found that activation of GSK-3β stimulates the inhibitory phosphorylation of PP2A at Tyr307 (pY307-PP2A), whereas inhibition of GSK-3β decreased the level of pY307-PP2A both in vitro and in vivo. GSK-3β is a serine/threonine kinase that can not phosphorylate tyrosine directly, therefore we measured PTP1B (protein tyrosine phosphatase 1B) and Src (a tyrosine kinase) activities. We found that GSK-3β can modulate both PTP1B and Src protein levels, but it only inhibits PTP1B activity, with no effect on Src. Furthermore, only knockdown of PTP1B but not Src by siRNA (small interfering RNA) eliminates the effects of GSK-3β on PP2A. GSK-3β phosphorylates PTP1B at serine residues, and activation of GSK-3β reduces the mRNA level of PTP1B. Additionally, we also observed that GSK-3 negatively regulates the protein and mRNA levels of PP2A, and knockdown of CREB (cAMP-response-element-binding protein) abolishes the increase in PP2A induced by GSK-3 inhibition. The results of the present study suggest that GSK-3β inhibits PP2A by increasing the inhibitory Tyr307 phosphorylation and decreasing the expression of PP2A, and the mechanism involves inhibition of PTP1B and CREB.     

Receptor protein tyrosine phosphatase alpha participates in the m1 muscarinic acetylcholine receptor-dependent regulation of Kv1.2 channel activity.

The phosphorylation state of a given tyrosine residue is determined by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. However, little is known about the functional interaction of these opposing activities at the level of an identified effector molecule. G protein-coupled receptors (GPCRs), including the m1 muscarinic acetylcholine receptor (mAChR), regulate a tyrosine kinase activity that phosphorylates and suppresses current generated by the Kv1.2 potassium channel. We examined the possibility that PTPs also participate in this signaling pathway since the tyrosine phosphatase inhibitor vanadate increases the extent of both Kv1.2 phosphorylation and suppression. We show that an endogenous transmembrane tyrosine phosphatase, receptor tyrosine phosphatase alpha (RPTPalpha), becomes tyrosine phosphorylated and co-immunoprecipitates with Kv1.2 in a manner dependent on m1 receptor activation. The N- and C-termini of Kv1.2 are shown to bind RPTPalpha in vitro. Overexpression of RPTPalpha in Xenopus oocytes increases resting Kv1.2 current. Biochemical and electrophysiological analysis reveals that recruiting RPTPalpha to Kv1.2 functionally reverses the tyrosine kinase-induced phosphorylation and suppression of Kv1.2 current in mammalian cells. Taken together, these results identify RPTPalpha as a new target of m1 mAChR signaling and reveal a novel regulatory mechanism whereby GPCR-mediated suppression of a potassium channel depends on the coordinate and parallel regulation of PTK and PTP activities.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with -tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of -tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that -tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.     

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA /GSK-3α in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 μM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA /GSK-3α was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA /GSK-3α immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA /GSK-3α can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA /GSK-3α. J. Cell. Physiol. 171:95–103, 1997. © 1997 Wiley-Liss, Inc.     

Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.     

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

BACKGROUND/ AIMS: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. METHODS: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. RESULTS: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). CONCLUSION: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.