Protein phosphatase 1α interacting proteins in the human brain

Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved.     

The receptor-like protein tyrosine phosphatase α: a role in cell proliferation and oncogenesis

The transmembrane nature of the receptor-like protein tyrosine phosphatases (PTPases) suggests that they transduce as yet unidentified extracellular signals to intracellular events via a phosphotyrosyl-protein dephosphorylation step, although little is known of their regulation and cellular activities. Structure/function studies of PTPα demonstrate that both catalytic domains are required for full enzymatic efficiency and that interdomain interactions may modulate PTPα activity and specificity. Overexpression of PTPα results in cell transformation and tumorigenesis, likely as a consequence of the ability of PTPα to dephosphorylate and activate the c-src tyrosine kinase. This suggests a role for PTPα in normal cell proliferation. PTPα is so far unique among the PTPases in terms of its oncogenic potential, and overexpression or deregulation of PTPα may be involved in the genesis, progression or maintenance of certain tumor states.     

Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A

Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.     

Protein tyrosine phosphatase 1B inhibitors as antidiabetic agents – A brief review

Diabetes mellitus and obesity are one of the most common health issues spread throughout world and raised the medical attention to find the new effective agents to treat these disease state. Occurrence of the drug resistance to the insulin and leptin receptor is also challenging major issues. The molecules that can overcome this resistance problem could be effective for the treatment of both type II diabetes and obesity. Protein Tyrosine Phosphatase (PTP) has emerged as new promising targets for therapeutic purpose in recent years. Protein Tyrosine Phosphatase 1B (PTP 1B) act as a negative regulator of insulin and leptin receptor signalling pathways. Several approaches have been successfully applied to find out potent and selective inhibitors. This article reviews PTP 1B inhibitors; natural, synthetic and semi-synthetic that showed inhibition towards enzyme as a major target for the management of type II diabetes. These studies could be contributing the future development of PTP 1B inhibitors as drugs.     

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.     

Structural basis of interaction between protein tyrosine phosphatase PCP-2 and β-catenin

PCP-2 is a member of receptor-like protein tyrosine phosphatase of the MAM domain family. To investigate which part of PCP-2 was involved in its interaction with β-catenin, we constructed various deletion mutants of PCP-2. These PCP-2 mutants and wild-type PCP-2 were co-transfected into BHK-21 cells with β-catenin individually. An in vivo binding assay revealed that the expression of wild-type PCP-2, PCP-2 DC1C2 (deleted PCP-2 without both PTP domains) and PCP-2 ΔC2 (deleted PCP-2 without the second PTP domain) could be immunoprecipitated by anti-catenin antibody in every co-transfection, but PCP-2 EXT (deleted PCP-2 without the juxtamembrane region and both PTP domains) was missing, which implied that PCP-2 and b-catenin could associate directly and the juxtamembrane region in PCP-2 was sufficient for the process.     

Purification and characterization of the human protein tyrosine phosphatase,PTPμ, from a baculovirus expression system

The receptor like PTPase, PTP, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment *80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP was membrane associated while the truncated form was recovered in the soluble fraction. PTP preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a K_m of 400 nM and a V_max of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn⁺⁺ and Mn⁺⁺ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.     

Protein tyrosine nitration--functional alteration or just a biomarker?

Protein 3-nitrotyrosine is a posttranslational modification found in many pathological conditions from acute to chronic diseases. Could 3-nitrotyrosine formation participate on the basis of these diseases or is it just a marker connected with the associated nitroxidative stress? In vitro and in vivo data, including proteomic research, show that protein tyrosine nitration is a selective process where only a small amount of proteins is found nitrated and one or a few tyrosine residues are modified in each. Accumulating data suggest a strong link between protein 3-nitrotyrosine and the mechanism involved in disease development. In this review, we analyze the factors determining protein 3-nitrotyrosine formation, the functional and biological outcome associated with protein tyrosine nitration, and the fate of the nitrated proteins.     

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [³²P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M_r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A_o of protein phosphatase 2A.     

A protein tyrosine phosphatase inhibitor,pervanadate, inhibits angiotensin II-Induced β-arrestin cleavage

β-Arrestins turn off G protein-mediated signals and initiate distinct G protein-independent signaling pathways. We previously demonstrated that angiotensin AT₁ receptor-bound β-arrestin 1 is cleaved after Phe³⁸⁸ upon angiotensin II stimulation. The mechanism and signaling pathway of angiotensin II-induced β-arrestin cleavage remain largely unknown. Here, we show that protein Tyr phosphatase activity is involved in the regulation of β-arrestin 1 cleavage. Tagging of green fluorescent protein (GFP) either to the N-terminus or C-terminus of β-arrestin 1 induced conformational changes and the cleavage of β-arrestin 1 without angiotensin AT₁ receptor activation. Orthovanadate and molybdate, inhibitors of protein Tyr phosphatase, attenuated the cleavage of C-terminal GFP-tagged β-arrestin 1 in vitro. The inhibitory effects of okadaic acid and pyrophosphate, which are inhibitors of protein Ser/Thr phosphatase, were less than those of protein Tyr phosphatase inhibitors. Cell-permeable pervanadate inhibited angiotensin II-induced cleavage of β-arrestin 1 in COS-1 cells. Our findings suggest that Tyr phosphorylation signaling is involved in the regulation of angiotensin II-induced β-arrestin cleavage.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations

Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.     

Expression of the transmembrane protein tyrosine phosphatase RPTPα in human oral squamous cell carcinoma

 Little is known about the role of protein-tyrosine phosphatases (PTPs), the cellular counterparts of protein-tyrosine kinases, both for normal growth regulation and for its dysregulation in cancer. The receptor-like PTPα (RPTPα) may play a positive role in growth regulation and has been shown to be overexpressed in colon carcinoma. An RNA/RNA in situ hybridisation protocol for RPTPα as well as RPTPα immunohistochemistry was developed to evaluate RPTPα expression in oral squamous cell carcinomas (OSCCs) of different histological grade and to reveal the synthetically active cells and their tissue distribution. In well-differentiated OSCC (G1), RPTPα mRNA could be detected by in situ hybridisation exclusively in stroma cells (fibro/myofibroblasts and inflammatory cells). A higher histological grade (G2/G3) was associated with an increased number of RPTPα-synthesising carcinoma cells haphazardly distributed within invading tumour areas. Consistent results were obtained by immunocytochemistry. Thus, both carcinoma dedifferentiation and stroma recruitment and activation seem to be associated with an upregulation of RPTPα expression in OSCC. The results speak in favour of the important role of activation of stroma fibro/myofibroblasts influencing the biological behaviour of epithelial tumours and also suggest that elevated RPTPα expression may be a more general marker for proliferating or dedifferentiated cells. Accepted: 2 February 1999     

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.     

The SHP—2 tyrosine phosphatase：Signaling mechanisms and biological functions

Cellular biological avtivities are tightly controlled by intracellular signaling processes initiated by extracellular signals.Protein tyrosine phosphatases,which remove phosphate groups from phosphorylated signaling molecules,play equally important tyrosine roles as protein tyrosine kinases in signal transduction.SHP-2 a cytoplasmic SH2 domain containing protein tyrosine phosphatase,is involved in the signaling pathways of a variety of growth factors and cytokines.Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus,and is a critical intracellular regulator in mediating cell proliferation and differentiation.