Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondria protein with an N-terminal mitochondrial targeting sequence and induces apoptosis

Apoptosis is a genetically determined cell suicide program. Mitochondria play a central role in this process and various molecules have been shown to regulate apoptosis in this organelle. In the present study, we firstly identified that protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondrial protein, which may induce apoptosis in HEK293T and HeLa cell lines. PTPIP51 transfection resulted in the externalization of phosphatidylserine (PS), activation of caspase-3, cleavage of PARP, and condensation of nuclear DNA. Further investigation revealed that PTPIP51 over-expression caused a decrease in mitochondrial membrane potential and release of cytochrome c, suggesting that it may be involved in a mitochondria/cytochrome c mediated apoptosis pathway. We also found that a putative TM domain near the N terminus of PTPIP51 is required for its targeting to mitochondria, as evidenced by the finding that deletion of the PTPIP51 TM domain prevented the protein's mitochondiral localization. Furthermore, this deletion significantly influenced the ability of PTPIP51 to induce apoptosis. Taken together, the results of the present study suggest that PTPIP51 is a mitochondrial protein with apoptosis-inducing function and that the N-terminal TM domain is required for both the correct targeting of the protein to mitochondria and its apoptotic functions.     

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.     

Protein tyrosine phosphatase-1B and T-cell protein tyrosine phosphatase regulate IGF-2-induced MCF-7 cell migration

The protein tyrosine phosphatase-1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) have been implicated in down-regulation of tyrosine kinase receptors, conferring anti-oncogenic functions to these PTPases. However, recent work has shown that PTP1B is positively implicated in oncogenic properties of breast cancer cells by regulating the ERK pathway. Here, we studied the function of PTP1B and TC-PTP in IGF-2-induced growth, survival and migration of MCF-7 breast cancer cells. Using siRNA, we showed that reduction in the expression of these PTPases decreased cell growth and ERK phosphorylation. Reduction in the expression of these PTPases did not impair IGF-2 effects on cell survival to acute treatment with 4-OH Tamoxifen. In contrast, IGF-2-induced MCF-7 cell migration was markedly impaired by reduction of PTP1B or TC-PTP expression, independently of the ERK pathway. This novel finding reinforces the potential role of these PTPases as therapeutic targets for treatment of breast cancer.     

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.     

microRNA-135a protects against myocardial ischemia-reperfusion injury in rats by targeting protein tyrosine phosphatase 1B

microRNAs are an emerging class of molecules that regulate pathogenesis of cardiovascular diseases. Here we aim to elucidate the effects and mechanism of miR-135a, a previously reported regulator of ischemia-reperfusion (I/R) injury, in myocardial I/R injury. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of miR-135a was significantly decreased both in the rat I/R group and H9c2 cells subjected to hypoxia/reoxygenation. Overexpression of miR-135a in vivo markedly decreased the infarct size and inhibited the I/R-induced cardiomyocyte apoptosis. Overexpression of miR-135a in H9c2 also exerted antiapoptosis effects. Furthermore, bioinformatics analysis, luciferase activity, and the Western blot assay indicated that protein tyrosine phosphatase 1B (PTP1B) is a direct target of miR-135a. In addition, the expression of proapoptotic-related genes, such as p53, Bax, and cleaved caspase3, were decreased in association with the downregulation of PTP1B. In summary, this study demonstrates that miR-135a exerts protective effects against myocardial I/R injury by targeting PTP1B.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Synthesis and characterization of 5,7-dihydroxyflavanone derivatives as novel protein tyrosine phosphatase 1B inhibitors

A series of 5,7-dihydroxyflavanone derivatives were synthesized and identified as reversible and competitive protein tyrosine phosphatase (PTP) 1B inhibitors with IC₅₀ values in the micromolar range. Compound 4k had the most potent in vitro inhibition activity against PTP1B (IC₅₀ = 2.37?±?0.37 μM) and the greatest selectivity (3.7-fold) for PTP1B relative to T-cell protein tyrosine phosphatase. Cell-based studies revealed that 4k was membrane-permeable and enhanced insulin receptor tyrosine phosphorylation in CHO/hIR cells.     

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca²⁺/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,³²P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca²⁺/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.     

Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B)

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes.     

Evaluation of licorice flavonoids as protein tyrosine phosphatase 1B inhibitors

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator in insulin- and leptin-signaling cascades as well as a positive regulator in tumorigenesis, and much attention has been paid to PTP1B inhibitors as potential therapies for diabetes, obesity, and cancer. In the present study, the screening of a compound library of licorice flavonoids allowed for the discovery of several compounds, including licoagrone (3), licoagrodin (4), licoagroaurone (5), and isobavachalcone (6), as new PTP1B inhibitors. It was revealed that these compounds inhibit the activity of PTP1B in different modes and with different selectivities and that they exhibit different cellular activity in the insulin-signaling pathway. Glycybenzofuran (1), a competitive PTP1B inhibitor, showed both excellent inhibitory selectivity against PTP1B and cellular activity on the insulin-stimulated Akt phosphorylation level. The similarity of its action profiling in the insulin-signaling pathway suggested its potential as a new anti-insulin-resistant drug candidate.     

PTPIP51 in Protein Interactions: Regulation and In Situ Interacting Partners

This study investigated the regulation of 14-3-3β binding to PTPIP51 by the tyrosine phosphorylation status of PTPIP51. The tyrosine 176 residue is phosphorylated by c-Src. Up to now, nothing is known about the impact of such well-established phosphorylation events on the interaction profile of PTPIP51 with its partners of the mitogen-activated protein kinase (MAPK) pathway. In human keratinocytes the PTPIP51 phosphorylation was varied by inhibiting the phosphatase activity, thus enhancing the phosphorylation of PTPIP51. Differential blocking of Src kinase family members (despite c-Src) by PP2 increased the activity of c-Src and the tyrosine phosphorylation of PTPIP51 at position 176, which is the substrate of c-Src kinase. The amount of PTPIP51 interactions with 14-3-3β, Raf-1, PTP1B and c-Src was evaluated and the resulting data were compared to an untreated control group. The increased phosphorylation level resulted in a sharp drop of the 14-3-3β/PTPIP51 and 14-3-3β/Raf-1 interaction. Besides the 14-3-3 interaction of PTPIP51, the interaction with the two MAPK modulators, protein kinase A (PKA) and diacylglycerol kinase alpha (DAGKα), are also regulated by the tyrosine phosphorylation status of PTPIP51. Additional immunostaining experiments were done investigating the functional implication on these interactions of the phosphorylation in apoptotic processes. In the pervanadate- and PP2-treated HaCaT cells, higher amounts of apoptotic cells were not detected as compared to the control group. The presented data confirms a tyrosine phosphorylation-dependent interaction of PTPIP51 with 14-3-3β and Raf-1 in vivo and a tyrosine-dependent interaction profile with DAGKα and PKA. The non-interaction of PTPIP51 with 14-3-3 is not sufficient for triggering apoptosis.     

Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression

Leptin has been shown to improve insulin sensitivity and glucose metabolism in obese diabetic ob/ob mice, yet the mechanisms remain poorly defined. We found that 2 d of leptin treatment improved fasting but not postprandial glucose homeostasis, suggesting enhanced hepatic insulin sensitivity. Consistent with this hypothesis, leptin improved in vivo insulin receptor (IR) activation in liver, but not in skeletal muscle or fat. To explore the cellular mechanism by which leptin up-regulates hepatic IR activation, we examined the expression of the protein tyrosine phosphatase PTP1B, recently implicated as an important negative regulator of insulin signaling. Unexpectedly, liver PTP1B protein abundance was increased by leptin to levels similar to lean controls, whereas levels in muscle and fat remained unchanged. The ability of leptin to augment liver IR activation and PTP1B expression was also observed in vitro in human hepatoma cells (HepG2). However, overexpression of PTP1B in HepG2 cells led to diminished insulin-induced IR phosphorylation, supporting the role of PTP1B as a negative regulator of IR activation in hepatocytes. Collectively, our results suggest that leptin acutely improves hepatic insulin sensitivity in vivo with concomitant increases in PTP1B expression possibly serving to counterregulate insulin action and to maintain insulin signaling in proper balance.     

Protein tyrosine phosphatase interacting protein 51—a jack-of-all-trades protein

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts both in vitro and in vivo with PTP1B, a protein tyrosine phosphatase involved in cellular regulation. PTPIP51 is known to be expressed in many different types of tissues. It is involved in cellular processes such as proliferation, differentiation and apoptosis. Nevertheless, the exact cellular function of PTPIP51 is still unknown. The present review summarizes our current knowledge of the PTPIP51 gene and its mRNA and protein structure.     

Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols

Protein tyrosine phosphatases (PTPases) contain an active site cysteine which when oxidized leads to loss of phosphatase activity and accumulation of phosphoproteins. For example, oxidants produced following EGF stimulation inhibit PTP1B and enhance EGF receptor phosphorylation. Because NO-derived species also modify reactive thiols, we postulated that NO would reversibly inhibit PTP1B. In our studies we exposed A431 or Jurkat cells to NO donors and measured PTP1B activity or used 3-maleimidylpropionylbiocytin (MPB) to measure thiol redox status. Nitrosothiols led to a rapid inhibition of PTP1B through a mechanism that was greatly enhanced by addition of cysteine to the medium. Analysis of thiol oxidation status using immunoprecipitated PTP1B showed modification consistent with loss of activity. Both enzyme inhibition and modification were reversible in intact cells or after addition of DTT to cell lysates. While DTT reversed oxidation, ascorbate did not, suggesting that formation of a mixed disulfide (possibly glutathionylation) rather than S-nitrosylation accounts for PTP1B inhibition. Importantly, PTP1B inhibition by nitrosothiols led to EGF receptor phosphorylation even in the absence of exogenously added EGF. These findings suggest an important role for NO in modulating signaling pathways since inhibition of PTPases could potentially enhance or prolong activity of phosphoproteins.     

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.     

Computational revelation of binding mechanisms of inhibitors to endocellular protein tyrosine phosphatase 1B using molecular dynamics simulations

Endocellular protein tyrosine phosphatase 1B (PTP1B) is one of the most promising target for designing and developing drugs to cure type-II diabetes and obesity. Molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy methods were applied to study binding differences of three inhibitors (ID: 901, 941, and 968) to PTP1B, the calculated results show that the inhibitor 901 has the strongest binding ability to PTP1B among the current inhibitors. Principal component (PC) analysis was also carried out to investigate the conformational change of PTP1B, and the results indicate that the associations of inhibitors with PTP1B generate a significant effect on the motion of the WPD-loop. Free energy decomposition method was applied to study the contributions of individual residues to inhibitor bindings, it is found that three inhibitors can generate hydrogen bonding interactions and hydrophobic interactions with different residues of PTP1B, which provide important forces for associations of inhibitors with PTP1B. This research is expected to give a meaningfully theoretical guidance to design and develop of effective drugs curing type-II diabetes and obesity.     

The novel protein PTPIP51 exhibits tissue- and cell-specific expression

The expression patterns of both mRNA and protein of the novel protein tyrosine phosphatase interacting protein 51 (PTPIP51) were studied in various organs by in situ hybridization, immunoblotting, and immunocytochemistry. The protein was found in all mammalian species investigated: guinea pig, rat, mouse, pig, and human. The presence of the protein was, however, restricted to specific organs. High levels of PTPIP51 were found in epidermis and seminiferous epithelium. The expression appears to be associated with distinct stages of differentiation. While basal cells in the epidermis and spermatogonia showed no perceptible amount of PTPIP51, keratinocytes of suprabasal layers and differentiating first-order spermatocytes up to spermatids exhibited high expression. In skeletal muscle, the presence of PTPIP51 was restricted to fibers of the fast twitch type. In surface epithelia containing ciliated cells, the protein was associated with the microtubular structures responsible for ciliary movement. Furthermore, specific structures of the central nervous system, for example, neurons of the hippocampal region, ganglion cells of the autonomic nervous system, and axons of the peripheral nervous system showed a distinct staining pattern with the antibody to PTPIP51. Our data suggest that PTPIP51 might be involved in the regulation of cellular processes associated with differentiation, movement, or cytoskeletal organization.Tobias Kajosch died on August 9th 2004     

The protein tyrosine phosphatase alpha modifies insulin secretion in INS-1E cells

Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.     

Identification of novel imidazole flavonoids as potent and selective inhibitors of protein tyrosine phosphatase

A series of imidazole flavonoids as new type of protein tyrosine phosphatase inhibitors were synthesized and characterized. Most of them gave potent protein phosphatase 1B (PTP1B) inhibitory activities. Especially, compound 11a could effectively inhibit PTP1B with an IC₅₀ value of 0.63 μM accompanied with high selectivity ratio (9.5-fold) over T-cell protein tyrosine phosphatase (TCPTP). This compound is cell permeable with relatively low cytotoxicity. The high binding affinity and selectivity was disclosed by molecular modeling and dynamics studies. The structural features essential for activity were confirmed by quantum chemical studies.