Protein Tyrosine Phosphatase Epsilon Research Articles

Receptor Type Protein Tyrosine Phosphatase Epsilon (PTPRE) Plays an Oncogenic Role in Thyroid Carcinoma by Activating the AKT and ERK1/2 Signaling Pathway.

Thyroid carcinoma (TC) is a common malignant tumor in human and its incidence has been increasing in recent years. Studies have shown that receptor type protein tyrosine phosphatase epsilon (PTPRE) is a key regulator of tumorigenesis in cancer progression, but its role in TC has not been revealed. Here, in this work, we explored the essential role of PTPRE in TC progression. The expression of PTPRE in TC clinical samples and cell lines was detected by RT-qPCR and Western blot. Cell proliferation was measured by MTT and cell cycle analysis. Cell migration, invasion and epithelial-mesenchymal transition (EMT) were analyzed by wound healing, transwell, and immunofluorescent staining assays. AKT and ERK1/2 signaling pathway related protein level was analyzed by Western blot. PTPRE was highly expressed in TC clinical samples and cell lines, especially anaplastic thyroid carcinoma (ATC). High level of PTPRE was associated with tumor size and TNM stage. Upregulated PTPRE promoted cell proliferation, and enhanced the migration, invasion and EMT of TC cells, whereas the knockdown of PTPRE suppressed these behaviors. Importantly, we confirmed that the AKT and ERK1/2 signaling pathways were activated by PTPRE, reflected by the enhanced protein level of phosphorylated AKT and ERK1/2. Accordingly, we indicated that PTPRE plays an oncogenic role in TC progression via activating the AKT and ERK1/2 signaling pathway. These findings indicated that modulation of PTPRE expression may as a potential strategy to interfere with the progression of TC.

Blockage of the JAK/STAT3 signaling pathway in multiple myeloma by leelamine

BackgroundLeelamine (LEE) is a lipophilic diterpene amine phytochemical, which can be naturally extracted from pine's bark trees. It has been extensively studied recently for its promising chemopreventive and anti-cancer effects against various cancers such as that of prostate and breast. HypothesisWe examined the potential impact of LEE in affecting the activation of signal transducer and activator of transcription 3 (STAT3) and promoting apoptosis in human multiple myeloma (MM) cells. MethodsWe evaluated the effect of LEE on STAT3 signaling pathway in MM cells by using Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Thereafter, apoptosis was evaluated using cell cycle analysis and Annexin V assay. ResultsWe noted that LEE could attenuate the phosphorylation of STAT3 and other up-stream signaling molecules such as JAK1, JAK2, and Src activation in U266 and MM.1S cells. It also diminished STAT3 translocation into the nucleus and enhanced the expression of protein-tyrosine phosphatase epsilon (PTPε). Additionally, LEE caused cell cycle arrest and synergistically augmented the apoptotic actions of bortezomib against MM cells. ConclusionsOur data indicates that LEE could block STAT3 signaling cascade linked to tumorigenesis and can be used in combination with approved anti-cancer agents in attenuating MM growth and survival.

MiR-631 Inhibits Intrahepatic Metastasis of Hepatocellular Carcinoma by Targeting PTPRE.

MicroRNAs (miRNAs) have been reported to play critical roles in the pathological development of hepatocellular carcinoma (HCC), one of the most common cancers in the world. Our study aims to explore the expression, function and mechanism of miR-631 in HCC. Our findings are that expression of miR-631 is significantly down-regulated in HCC tissue compared with that in adjacent non-cancerous tissue, and low expression of miR-631 in HCC tissue is associated with cirrhosis, multiple tumors, incomplete tumor encapsulation, poor tumor differentiation, and high TNM stage. Our test results showed that miR-631 could inhibit migration, invasion, epithelial–mesenchymal transition (EMT) and intrahepatic metastasis of HCC. Receptor-type protein tyrosine phosphatase epsilon (PTPRE) as a downstream target of miR-631 could promote migration, invasion and EMT of HCC cells. Besides, the expression of PTPRE had a negative correlation with the expression of miR-631 both in vivo and in vitro, and increasing expression of PTPRE could reverse inhibitory effects of miR-631 in HCC cells. In sum, our study first demonstrated that miR-631 targeted PTPRE to inhibit intrahepatic metastasis in HCC. We gain insights from these findings into the mechanism of miRNAs regulation in HCC metastasis and further introduce a novel therapeutic target for HCC treatment.

Role of Protein Tyrosine Phosphatase Epsilon (PTPε) in Leukotriene D4-Induced CXCL8 Expression.

Phosphorylation on tyrosine residues is recognized as an important mechanism for connecting extracellular stimuli to cellular events and defines a variety of physiologic responses downstream of G protein-coupled receptor (GPCR) activation. To date, few protein tyrosine phosphatases (PTPs) have been shown to associate with GPCRs, and little is known about their role in GPCR signaling. To discover potential cysteinyl-leukotriene receptor (CysLT1R)-interacting proteins, we identified protein tyrosine phosphatase ε (PTPε) in a yeast two-hybrid assay. Since both proteins are closely linked to asthma, we further investigated their association. Using a human embryonic kidney cell line 293 (HEK-293) cell line stably transfected with the receptor (HEK-LT1), as well as human primary monocytes, we found that PTPε colocalized with CysLT1R in both resting and leukotriene D4 (LTD4)-stimulated cells. Cotransfection of HEK-LT1 with PTPε had no effect on CysLT1R expression or LTD4-induced internalization, but it inhibited LTD4-induced CXC chemokine 8 (CXCL8) promoter transactivation, protein expression, and secretion. Moreover, reduced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), but not of p38 or c-Jun-N-terminal kinase 1 or 2 mitogen-activated protein kinases (MAPKs), was observed upon LTD4 stimulation of HEK-LT1 coexpressing cytosolic (cyt-) PTPε, but not receptor (R) PTPε The increased interaction of cyt-PTPε and ERK1/2 after LTD4 stimulation was shown by coimmunoprecipitation. In addition, enhanced ERK1/2 phosphorylation and CXCL8 secretion were found in LTD4-stimulated human monocytes transfected with PTPε-specific siRNAs, adding support to a regulatory/inhibitory role of PTPε in CysLT1R signaling. Given that the prevalence of severe asthma is increasing, the identification of PTPε as a new potential therapeutic target may be of interest.

Tuning the Protein Phosphorylation by Receptor Type Protein Tyrosine Phosphatase Epsilon (PTPRE) in Normal and Cancer Cells.

Tyrosine phosphorylation is an important post-translation modification of proteins that is controlled by tyrosine kinases and phosphatases. Disruption of the balance between the activity of tyrosine kinases and phosphatases may result in diseases. Receptor type protein tyrosine phosphatase epsilon (PTPRE) is closely related with receptor type protein tyrosine phosphatase alpha (PTPRA). PTPRE has been studied in osteoclast cells, nerve cells, hematopoietic cells, cancer cells and others, and it has different functions among various tissues. In this review, we summarized the current knowledge about the regulation of PTPRE on cellular signal transduction and its function under normal and pathological conditions.

High-resolution crystal structures of the D1 and D2 domains of protein tyrosine phosphatase epsilon for structure-based drug design.

Here, new crystal structures are presented of the isolated membrane-proximal D1 and distal D2 domains of protein tyrosine phosphatase epsilon (PTPℇ), a protein tyrosine phosphatase that has been shown to play a positive role in the survival of human breast cancer cells. A triple mutant of the PTPℇ D2 domain (A455N/V457Y/E597D) was also constructed to reconstitute the residues of the PTPℇ D1 catalytic domain that are important for phosphatase activity, resulting in only a slight increase in the phosphatase activity compared with the native D2 protein. The structures reported here are of sufficient resolution for structure-based drug design, and a microarray-based assay for high-throughput screening to identify small-molecule inhibitors of the PTPℇ D1 domain is also described.

Identification of N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamide derivatives as novel protein tyrosine phosphatase epsilon inhibitors exhibiting anti-osteoclastic activity

Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro, two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering.

The Discovery of Novel Protein Tyrosine Phosphatase ε Inhibitors Using a High‐throughput Screening Approach

Protein tyrosine phosphatase epsilon (PTPε) is important for signal transduction in osteoclasts, and is considered to be an attractive drug target for the treatment of osteoporosis. We identified 11 potent PTPε inhibitors based on three chemical scaffolds through the high‐throughput screening of a chemical library. As these compounds are structurally diverse with high bioavailability, they warrant further investigation in the near future. The discovery of these inhibitors and the relationship between their structure and inhibitory activity toward PTPε is discussed in detail.

Abstract 3807: Capsazepine attenuates JAK/STAT3 signaling pathway, proliferation, survival of prostate carcinoma DU145 cells through induction of the protein tyrosine phosphatase epsilon (PTP-epsilon)

Abstract Constitutive activation of signal transducers and activators of transcription (STAT) 3 is a major distinguishing feature of various human cancer cells and its activation leads to survival, proliferation, and metastasis of tumor cells. Whether the anti-proliferative, pro-apoptotic, and anti-invasive effects of capsazepine (Capz), a synthetic analog of capsaicin, are linked to its capability to inhibit STAT3 activation was investigated. We found that Capz suppressed both constitutive and IL-6-inducible STAT3 activation in human prostate carcinoma cells. Capz also suppressed nuclear translocation and DNA binding activity of STAT3. The suppression was mediated through the inhibition of activation of the upstream kinases JAK1/2 and c-Src. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate treatment reversed the Capz-induced down-regulation of STAT3, thereby suggesting the involvement of a PTP. Capz induced the expression of PTPϵ protein and mRNA. Moreover, knockdown of PTPϵ by small interfering RNA suppressed the induction of PTPϵ, reversed the inhibition of STAT3 activation, suggesting the critical role of PTPϵ in its possible mechanism of action. Capz downregulated the expression of STAT3-regulated proliferative, antiapoptotic, angiogenetic, and metastatic gene products; and this correlated with suppression of cell proliferation and invasion, the accumulation of cells in sub-G1 phase of cell cycle, and induction of apoptosis. Overexpression of phosphorylated STAT3 led to the attenuation of Capz-mediated cleavage of PARP as compared to the control. Thus, overall, our results suggest that Capz is a novel inhibitor of STAT3 activation and thus may have a potential in negative regulation of growth, metastasis, and angiogenesis of tumor cells. Citation Format: Jong Hyun Lee, Chulwon Kim, Seok-Geun Lee, Junhee Lee, Jung-woo Lee, Kwang Seok Ahn. Capsazepine attenuates JAK/STAT3 signaling pathway, proliferation, survival of prostate carcinoma DU145 cells through induction of the protein tyrosine phosphatase epsilon (PTP-epsilon). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2015-3807

Abstract 5046: Protein tyrosine phosphatase epsilon (PTPE), a candidate tumor-suppressor in Wilms tumors of childhood, can regulate PI3K/AKT/mTOR pathway.

Abstract Wilms Tumor is the most common pediatric cancer of the genitourinary tract. It is a primitive multilineage malignant neoplasm of embryonic renal precursor cells and is often associated with persistent foci of embryonic renal tissue known as Nephrogenic Rests. However, early molecular events that orchestrate the progression of Nephrogenic Rests to Wilms Tumor are unknown. Protein tyrosine phosphorylation, a critical regulator of signaling pathways, is a reversible process controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The possible role of PTPs in Wilms Tumor has not been explored. Through immunohistochemical (IHC) analysis of tissue microarrays containing Wilms Tumors, Nephrogenic Rests, and normal kidney, we have identified downregulation of protein tyrosine phosphatase epsilon (PTPE) as a potential early event in Wilms tumorigenesis. IHC results showed strong staining for PTPE in 6 of 6 (100%) Nephrogenic Rests. In contrast, of the 51 Wilms Tumors studied, PTPE staining was absent in 2 (4%), weak in 26 (51%), moderate in 16 (31%), and strong in 7 (14%) compared to normal renal cortex. In addition, quantitative reverse-transcription PCR showed decreased PTPE expression in Wilms Tumor tissues compared to normal renal cortex. To identify potential targets of PTPE, we performed phosphokinase arrays and demonstrated that overexpression of PTPE in HEK293 cells results in dephosphorylation of Focal adhesion kinase (FAK), Lymphocyte-specific protein tyrosine kinase (LCK), and Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR). To validate the relevance of PTPE in Wilms Tumors, we established primary tumor cultures from nephrectomy specimens of children undergoing treatment for Wilms Tumor. Consistent with our IHC data, these primary cultures exhibit varying levels of PTPE expression. We have extended our results in these primary Wilms Tumor cells and found that overexpression of PTPE leads to dephosphorylation of FAK and LCK. We also probed downstream effector pathways and found that PTPE overexpression leads to dephosphorylation and inactivation of AKT (T308 and S375) and mTOR (S2448). Collectively, these PTPE targets have been established to control essential functions like cell proliferation, adhesion, and migration. In light of our results, we suggest that PTPE is a putative tumor suppressor in Wilms Tumor, acting to control signaling through FAK, LCK, and other SRC family proteins, as well as the PI3K/AKT/mTOR pathway. These results establish the utility of primary Wilms Tumor culture models, and provide the rationale for exploring SRC family kinase and AKT/mTOR pathway inhibitors as novel targeted agents for the treatment of Wilms Tumor. Citation Format: Abhay A. Shukla, Shama Khokhar, James F. Amatruda, Dinesh Rakheja. Protein tyrosine phosphatase epsilon (PTPE), a candidate tumor-suppressor in Wilms tumors of childhood, can regulate PI3K/AKT/mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5046. doi:10.1158/1538-7445.AM2013-5046

Protein Tyrosine Phosphatase Epsilon Affects Body Weight by Downregulating Leptin Signaling in a Phosphorylation-Dependent Manner

Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.

Protein Tyrosine Phosphatase Epsilon Regulates Integrin-mediated Podosome Stability in Osteoclasts by Activating Src

The nonreceptor isoform of tyrosine phosphatase epsilon (cyt-PTPe) supports osteoclast adhesion and activity in vivo, leading to increased bone mass in female mice lacking PTPe (EKO mice). The structure and organization of the podosomal adhesion structures of EKO osteoclasts are abnormal; the molecular mechanism behind this is unknown. We show here that EKO podosomes are disorganized, unusually stable, and reorganize poorly in response to physical contact. Phosphorylation and activities of Src, Pyk2, and Rac are decreased and Rho activity is increased in EKO osteoclasts, suggesting that integrin signaling is defective in these cells. Integrin activation regulates cyt-PTPe by inducing Src-dependent phosphorylation of cyt-PTPe at Y638. This phosphorylation event is crucial because wild-type-but not Y638F-cyt-PTPe binds and further activates Src and restores normal stability to podosomes in EKO osteoclasts. Increasing Src activity or inhibiting Rho or its downstream effector Rho kinase in EKO osteoclasts rescues their podosomal stability phenotype, indicating that cyt-PTPe affects podosome stability by functioning upstream of these molecules. We conclude that cyt-PTPe participates in a feedback loop that ensures proper Src activation downstream of integrins, thus linking integrin signaling with Src activation and accurate organization and stability of podosomes in osteoclasts.

Protein Tyrosine Phosphatase ε is a Negative Regulator of FcεRI‐mediated Mast Cell Responses

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase epsilon (PTPepsilon; Ptpre) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcepsilonRI). Bone marrow-derived mast cells (BMMC) from Ptpre(-/-) mice exhibited enhanced FcepsilonRI-induced Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca(2+) mobilization revealed that the absence of PTPepsilon leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPepsilon. In the event, Syk but not Lyn was hyperphosphorylated in PTPepsilon-deficient BMMC. Thus, PTPepsilon most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca(2+). Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre(-/-) mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPepsilon negatively regulates FcepsilonRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.

Protein-tyrosine Phosphatase ϵ Regulates Shc Signaling in a Kinase-specific Manner: INCREASING COHERENCE IN TYROSINE PHOSPHATASE SIGNALING

Individual protein tyrosine kinases and phosphatases target multiple substrates; this may generate conflicting signals, possibly within a single pathway. Protein-tyrosine phosphatase epsilon (PTPepsilon) performs two potentially opposing roles: in Neu-induced mammary tumors, PTPepsilon activates Src downstream of Neu, whereas in other systems PTPepsilon can indirectly down-regulate MAP kinase signaling. We now show that the latter effect is mediated at least in part via the adaptor protein Shc. PTPepsilon binds and dephosphorylates Shc in vivo, reducing the association of Shc with Grb2 and inhibiting downstream ERK activation. PTPepsilon binds Shc in a phosphotyrosine-independent manner mediated by the Shc PTB domain and aided by a sequence of 10 N-terminal residues in PTPepsilon. Surprisingly, PTPepsilon dephosphorylates Shc in a kinase-dependent manner; PTPepsilon targets Shc in the presence of Src but not in the presence of Neu. Using a series of point mutants of Shc and Neu, we show that Neu protects Shc from dephosphorylation by binding the PTB domain of Shc, most likely competing against PTPepsilon for binding the same domain. In agreement, PTPepsilon dephosphorylates Shc in mouse embryo fibroblasts but not in Neu-induced mammary tumor cells. We conclude that in the context of Neu-induced mammary tumor cells, Neu prevents PTPepsilon from targeting Shc and from reducing its promitogenic signal while phosphorylating PTPepsilon and directing it to activate Src in support of mitogenesis. In so doing, Neu contributes to the coherence of the promitogenic role of PTPepsilon in this system.

Protein tyrosine phosphatase epsilon and Neu-induced mammary tumorigenesis

Aberrant regulation of the phosphorylation of proteins on tyrosine residues is a well-established cause of cancer. Protein tyrosine phosphatases (PTPs) share in the crucial function of maintaining appropriate levels of phosphorylation of cellular proteins, making them potentially key players in regulating the transformation process. The receptor-type tyrosine phosphatase Epsilon (RPTPepsilon) participates in supporting the transformed phenotype of mammary tumor cells induced in vivo by the Neu tyrosine kinase. The phosphatase is overexpressed in mammary tumors induced in mice by a Neu transgene and expression of RPTPepsilon in mouse mammary glands leads to massive hyperplasia and associated tumorigenesis. Furthermore, cells isolated from mammary tumors induced by Neu in mice genetically lacking RPTPepsilon appear less transformed and proliferate less well than corresponding mammary tumor cells isolated from mice expressing the phosphatase. At the molecular level, RPTPepsilon dephosphorylates and activates Src and the related kinases Yes and Fyn, and the activities of these kinases are significantly reduced in tumor cells lacking RPTPepsilon. Restoring the activities of these kinases reveals that it is only the reduced activity of Src that causes the aberrant morphology and proliferation rate of tumor cells lacking RPTPepsilon. RPTPepsilon is primed to activate Src, and presumably related kinases, following its phosphorylation by Neu at Y695 within its C-terminus. This event is crucial in enabling RPTPepsilon to activate Src, but appears not to affect the activity of RPTPepsilon towards unrelated substrates. We conclude that a Neu-RPTPepsilon-Src pathway exists in mouse mammary tumor cells, in which Neu phosphorylates RPTPepsilon thereby driving the phosphatase to specifically activate Src family kinases and to assist in maintaining the transformed phenotype.

Neu-mediated phosphorylation of protein tyrosine phosphatase epsilon is critical for activation of Src in mammary tumor cells

The receptor-type protein tyrosine phosphatase epsilon (RPTPepsilon) activates c-Src in mammary tumor cells induced in vivo by Neu. Tumor cells lacking RPTPepsilon exhibit reduced c-Src activity, appear less transformed morphologically and proliferate slower in vitro and in vivo. Expression of Src rescues most of these phenotypes, indicating that c-Src activity is important for maintaining the transformed phenotype. However, the molecular mechanisms that control activation of c-Src by RPTPepsilon are unknown. We show that Neu induces phosphorylation of RPTPepsilon exclusively at its C-terminal Y695, and that this phosphorylation is required for activation of c-Src by RPTPepsilon. Phosphorylation of RPTPepsilon does not affect its activity toward another substrate, the voltage-gated potassium channel Kv2.1, suggesting that phosphorylation directs RPTPepsilon activity toward c-Src. Phosphorylation of RPTPepsilon reduces its dimerization at the cell membrane, although this does not affect its activity significantly. RPTPepsilon is subject to strong auto- and trans-dephosphorylation, suggesting that dephosphorylation limits the activation of c-Src downstream of Neu. We conclude that an Neu-RPTPepsilon-Src signaling pathway exists in mammary tumor cells, in which phosphorylation of RPTPepsilon by Neu directs RPTPepsilon to activate c-Src. Reversible phosphorylation of RPTPepsilon at Y695 may thus function as a 'molecular switch', which affects the substrate specificity of the phosphatase.

Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells

The receptor-type form of protein tyrosine phosphatase epsilon (RPTPε) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTPε appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTPε in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTPε is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTPε, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTPε. RPTPε also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTPε. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTPε-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTPε activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.

Dimerization in vivo and inhibition of the nonreceptor form of protein tyrosine phosphatase epsilon.

cyt-PTP epsilon is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTP epsilon enables analysis of phosphatase regulation in the absence of extracellular domains, which participate in dimerization and inactivation of the receptor-type phosphatases receptor-type protein tyrosine phosphatase alpha (RPTPalpha) and CD45. Using immunoprecipitation and gel filtration, we show that cyt-PTP epsilon forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTP epsilon readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTP epsilon dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTP epsilon, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTP epsilon is a major contributor to intermolecular binding and strongly interacts in a homotypic manner; the presence of D2 and the interactions that it mediates inhibit cyt-PTP epsilon activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTP epsilon lacking these regions constitutively dimerizes, and its activities in vitro towards para-nitrophenylphosphate and in vivo towards the Kv2.1 potassium channel are markedly reduced. We conclude that physiological signals can regulate dimerization and phosphorylation of cyt-PTP epsilon in the absence of direct interaction between the PTP and extracellular molecules. Furthermore, dimerization can be mediated by the D2 domain and does not strictly require the presence of PTP extracellular domains.

Reduced tumorigenicity of murine leukemia cells expressing protein-tyrosine phosphatase, PTPepsilon C.

Recently, we reported that a cytosolic isoform of protein-tyrosine phosphatase epsilon (PTP epsilon C), when overexpressed, inhibits terminal differentiation and apoptosis of murine M1 myeloblastic leukemia cells induced by interleukin-6. To determine whether these observed effects in vitro correspond to a tumorigenicity of PTP epsilon C-expresser (M1- epsilon C) cells in vivo, parent M1 and M1- epsilon C cells were intravenously inoculated into scid or nude mice, and survival of mice receiving these cell lines was monitored. Unexpectedly, both scid and nude mice inoculated with M1- epsilon C cells showed significantly prolonged survival time than those receiving parent M1 cells. While parent M1 cells inoculated by intravenous injection formed metastatic tumors in the spleen, expression of PTP epsilon C suppressed tumor development in the spleen. The results suggest a suppressive role of PTP epsilon C in tumorigenesis.

Characterization, expression and functional aspects of a novel protein tyrosine phosphatase epsilon isoform.

This report describes the identification and characterization of a novel cytoplasmic isoform of human protein tyrosine phosphatase epsilon (PTPepsilon). The novel isoform, denoted cyt-PTPepsilonPD1, displays only the N-terminal catalytic, active phosphatase domain 1 (PD1) which is common in all known PTPepsilon isoforms. In addition, it contains a unique 132-residue long C-terminal end with no known motifs or homology to other characterized proteins. RNAse protection assay on isolated leucocyte subpopulations and selected cell lines demonstrated highest expression of cyt-PTPepsilonPD1 in monocytes. The mRNA-encoding cyt-PTPepsilonPD1 is detected as distinct transcript(s) by Northern blot analysis and is a result of alternative splicing. cyt-PTPepsilonPD1 shows similar cellular localization in transfected cells, both in the cytoplasm and nucleus, as has been previously described for cytoplasmic PTPepsilon isoform. Our previous data suggest that the expression of cytoplasmic PTPepsilon inhibits the mitogen-activated protein kinase cascade through the extracellular signal-regulated kinase 1 and 2 pathway. A similar functional role is also presented here for cyt-PTPepsilonPD1, supporting our previous data suggesting that the catalytic first PD of PTPepsilon is responsible for this inhibition.