(2019) Genes. 10, 8, 568. Abstract
Background: HER2 (human epidermal growth factor 2)-positive breast cancer is an aggressive type of breast cancer characterized by the overexpression of the receptor-type protein tyrosine kinase HER2 or amplification of the HER2 gene. It is commonly treated by the drug trastuzumab (Herceptin), but resistance to its action frequently develops and limits its therapeutic benefit. Dual-specificity phosphatases (DUSPs) were previously highlighted as central regulators of HER2 signaling; therefore, understanding their role is crucial to designing new strategies to improve the efficacy of Herceptin treatment. We investigated whether inhibiting certain DUSPs re-sensitized Herceptin-resistant breast cancer cells to the drug. We built a series of kinetic models incorporating the key players of HER2 signaling pathways and simulating a range of inhibition intensities. The simulation results were compared to live tumor cells in culture, and showed good agreement with the experimental analyses. In particular, we observed that Herceptin-resistant DUSP16-silenced breast cancer cells became more responsive to the drug when treated for 72 h with Herceptin, showing a decrease in resistance, in agreement with the model predictions. Overall, we showed that the kinetic modeling of signaling pathways is able to generate predictions that assist experimental research in the identification of potential targets for cancer treatment.
(2019) Computational Biology and Chemistry. 80, p. 138-146 Abstract
Background: Breast cancer remains the most lethal type of cancer for women. A significant proportion of breast cancer cases are characterised by overexpression of the human epidermal growth factor receptor 2 protein (HER2). These cancers are commonly treated by Herceptin (Trastuzumab), but resistance to drug treatment frequently develops in tumour cells. Dual-specificity phosphatases (DUSPs) are thought to play a role in the mechanism of resistance, since some of them were reported to be overexpressed in tumours resistant to Herceptin.Results: We used a systems biology approach to investigate how DUSP overexpression could favour cell proliferation and to predict how this mechanism could be reversed by targeted inhibition of selected DUSPs. We measured the expression of 20 DUSP genes in two breast cancer cell lines following long-term (6 months) exposure to Herceptin, after confirming that these cells had become resistant to the drug. We constructed several Boolean models including specific substrates of each DUSP, and showed that our models correctly account for resistance when overexpressed DUSPs were kept activated. We then simulated inhibition of both individual and combinations of DUSPs, and determined conditions under which the resistance could be reversed.Conclusions: These results show how a combination of experimental analysis and modelling help to understand cell survival mechanisms in breast cancer tumours, and crucially enable us to generate testable predictions potentially leading to new treatments of resistant tumours.
Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo(2019) FASEB Journal. 33, 4, p. 5101-5111 Abstract
Understanding how body weight is regulated at the molecular level is essential for treating obesity. We show that female mice genetically lacking protein tyrosine phosphatase (PTP) receptor type α (PTPRA) exhibit reduced weight and adiposity and increased energy expenditure, and are more resistant to diet-induced obesity than matched wild-type control mice. These mice also exhibit reduced levels of circulating leptin and are leptin hypersensitive, suggesting that PTPRA inhibits leptin signaling in the hypothalamus. Male and female PTPRA-deficient mice fed a high-fat diet were leaner and displayed increased metabolic rates and lower circulating leptin levels, indicating that the effects of loss of PTPRA persist in the obese state. Molecularly, PTPRA down-regulates leptin receptor signaling by dephosphorylating the receptor-associated kinase JAK2, with which the phosphatase associates constitutively. In contrast to the closely related tyrosine phosphatase ε, leptin induces only weak phosphorylation of PTPRA at its C-terminal regulatory site Y789, and this does not affect the activity of PTPRA toward JAK2. PTPRA is therefore an inhibitor of hypothalamic leptin signaling in vivo and may prevent premature activation of leptin signaling, as well as return signaling to baseline after exposure to leptin.-Cohen-Sharir, Y., Kuperman, Y., Apelblat, D., den Hertog, J., Spiegel, I., Knobler, H., Elson, A. Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo.
Phosphorylation of the phosphatase PTPROt at Tyr(399) is a molecular switch that controls osteoclast activity and bone mass in vivo(2019) Science Signaling. 12, 563, aau0240. Abstract
Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr(527) and its activating residue Tyr(416). Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr(399), in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr(399) caused PTPROt to dephosphorylate Src at the activating site pTyr(416). In contrast, phosphorylation of PTPROt at Tyr(399) enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr(527), thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr(399) is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.
(2019) Biochimica Et Biophysica Acta-Molecular Cell Research. 1866, 1, p. 114-123 Abstract
Maintaining the proper balance between osteoblast-mediated production of bone and its degradation by osteoclasts is essential for health. Osteoclasts are giant phagocytic cells that are formed by fusion of monocyte-macrophage precursor cells; mature osteoclasts adhere to bone tightly and secrete protons and proteases that degrade its matrix. Phosphorylation of tyrosine residues in proteins, which is regulated by the biochemically antagonistic activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is central in regulating the production of osteoclasts and their bone-resorbing activity. Here we review the roles of individual PTPs of the classical and dual-specificity sub-families that are known to support these processes (SHP2, cyt-PTPe, PTPRO, PTP-PEST, CD45) or to inhibit them (SHP1, PTEN, MKP1). Characterizing the functions of PTPs in osteoclasts is essential for complete molecular level understanding of bone resorption and for designing novel therapeutic approaches for treating bone disease.
Regulation of dual specificity phosphatases in breast cancer during initial treatment with Herceptin: a Boolean model analysis(2018) BMC Systems Biology. 12, Suppl 1, p. 13-24 11. Abstract
Background: 25% of breast cancer patients suffer from aggressive HER2-positive tumours that are characterised by overexpression of the HER2 protein or by its increased tyrosine kinase activity. Herceptin is a major drug used to treat HER2 positive breast cancer. Understanding the molecular events that occur when breast cancer cells are exposed to Herceptin is therefore of significant importance. Dual specificity phosphatases (DUSPs) are central regulators of cell signalling that function downstream of HER2, but their role in the cellular response to Herceptin is mostly unknown. This study aims to model the initial effects of Herceptin exposure on DUSPs in HER2-positive breast cancer cells using Boolean modelling.Results: We experimentally measured expression time courses of 21 different DUSPs between 0 and 24 h following Herceptin treatment of human MDA-MB-453 HER2-positive breast cancer cells. We clustered these time courses into patterns of similar dynamics over time. In parallel, we built a series of Boolean models representing the known regulatory mechanisms of DUSPs and then demonstrated that the dynamics predicted by the models is in agreement with the experimental data. Furthermore, we used the models to predict regulatory mechanisms of DUSPs, where these mechanisms were partially known.Conclusions: Boolean modelling is a powerful technique to investigate and understand signalling pathways. We obtained an understanding of different regulatory pathways in breast cancer and new insights on how these signalling pathways are activated. This method can be generalized to other drugs and longer time courses to better understand how resistance to drugs develops in cancer cells over time.
(2018) International Journal of Biochemistry and Cell Biology. 96, p. 135-147 Abstract
Protein tyrosine phosphorylation is critical for proper function of cells and organisms. Phosphorylation is regulated by the concerted but generically opposing activities of tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs), which ensure its proper regulation, reversibility, and ability to respond to changing physiological situations. Historically, PTKs have been associated mainly with oncogenic and pro-tumorigenic activities, leading to the generalization that protein dephosphorylation is anti-oncogenic and hence that PTPs are tumor-suppressors. In many cases PTPs do suppress tumorigenesis. However, a growing body of evidence indicates that PTPs act as dominant oncogenes and drive cell transformation in a number of contexts, while in others PTPs support transformation that is driven by other oncogenes. This review summarizes the known transforming and tumor-promoting activities of the classical, tyrosine specific PTPs and highlights their potential as drug targets for cancer therapy.
R51Q SNX10 induces osteopetrosis by promoting uncontrolled fusion of monocytes to form giant, non-functional osteoclasts(2018) BioRxiv. Abstract
The molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.
The PTPROt tyrosine phosphatase functions as an obligate haploinsufficient tumor suppressor in vivo in B-cell chronic lymphocytic leukemia(2017) Oncogene. 36, 26, p. 3686-3694 Abstract
The tyrosine phosphatase PTPROt is a suggested tumor suppressor (TS) in B-cell chronic lymphocytic leukemia (CLL), and its expression is reduced in this disease. In order to examine how reduced PTPROt expression affects CLL in vivo we induced CLL in PTPROt-targeted mice. Unexpectedly, loss of both Ptprot alleles delayed disease detection and progression and lengthened survival relative to mice carrying two intact alleles, indicating that PTPROt fulfills a novel tumor-promoting role in CLL. Tumor cells from mice lacking PTPROt exhibited reduced B-cell receptor (BCR)-induced signaling, as well as increased apoptosis and autophagy. Inhibition of BCR/Src signaling in CLL cells induced their apoptosis, indicating that these findings are linked causally. These results suggest a cell-autonomous mechanism for the weakened CLL phenotype of PTPROt-deficient mice and uncover non-redundant roles for PTPROt in support of BCR signaling and survival of CLL cells. In contrast, loss of only one Ptprot allele induced earlier detection and progression of CLL and reduced survival, consistent with a tumor-suppressing role for PTPROt. Tumor cells from mice lacking one or both Ptprot allele exhibited increased interleukin-10 (IL-10) expression and signaling, factors known to support CLL; cells lacking one Ptprot alleles exhibited normal BCR signaling and cell death rates. We conclude that loss of one Ptprot allele promotes CLL, most likely by activating IL-10 signaling. Loss of both Ptprot alleles also reduces BCR signaling and increases cell death rates, offsetting the IL-10 effects and reducing the severity of the disease. PTPROt thus functions as an obligate haploinsufficient TS in CLL, where its expression levels determine its role as a promoter or inhibitor of the tumorigenic process in mice. Partial loss of PTPROt generates the strongest disease phenotype, suggesting that its intermediate expression levels in CLL are selected for.
(2016) Biochemical Society Transactions. 44, 5, p. 1295-1303 Abstract
Protein tyrosine phosphatases (PTPs) perform specific functions in vivo, despite being vastly outnumbered by their substrates. Because of this and due to the central roles PTPs play in regulating cellular function, PTP activity is regulated by a large variety of molecular mechanisms. We review evidence that indicates that the divergent C-terminal tail sequences (C-terminal domains, CTDs) of receptor-type PTPs (RPTPs) help regulate RPTP function by controlling intermolecular associations in a way that is itself subject to physiological regulation. We propose that the CTD of each RPTP defines an 'interaction code' that helps determine molecules it will interact with under various physiological conditions, thus helping to regulate and diversify PTP function.
Receptor Protein Tyrosine Phosphatase alpha-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice(2016) Arthritis & Rheumatology. 68, 2, p. 359-369 Abstract
Objective. During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase alpha (RPTP alpha), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTP alpha mediates FLS aggressiveness and RA pathogenesis.Methods. Through RPTP alpha knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTP alpha-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation.Results. RPTP alpha was enriched in the RA synovial lining. RPTP alpha knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y-527 and decreased phosphorylation of FAK on stimulatory Y-397. Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTP alpha. RPTP alpha-KO mice were protected from arthritis development, which was due to radioresistant cells.Conclusion. By regulating the phosphorylation of Src and FAK, RPTP alpha mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.
Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase epsilon in Osteoclasts(2014) Journal of Biological Chemistry. 289, 52, p. 36048-36058 Abstract
The non-receptor isoform of protein-tyrosine phosphatase epsilon (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.[Alternate title: Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase ϵ in Osteoclasts]
(2014) Molecular Biology of the Cell. 25, 11, p. 1808-1818 Abstract
Female mice lacking protein tyrosine phosphatase epsilon (PTP epsilon) are mildly osteopetrotic. Osteoclasts from these mice resorb bone matrix poorly, and the structure, stability, and cellular organization of their podosomal adhesion structures are abnormal. Here we compare the role of PTP epsilon with that of the closely related PTP alpha in osteoclasts. We show that bone mass and bone production and resorption, as well as production, structure, function, and podosome organization of osteoclasts, are unchanged in mice lacking PTP alpha. The varying effects of either PTP on podosome organization in osteoclasts are caused by their distinct N-termini. Osteoclasts express the receptor-type PTP alpha (RPTPa), which is absent from podosomes, and the nonreceptor form of PTP epsilon (cyt-PTPe), which is present in these structures. The presence of the unique 12 N-terminal residues of cyt-PTPe is essential for podosome regulation; attaching this sequence to the catalytic domains of PTP alpha enables them to function in osteoclasts. Serine 2 within this sequence regulates cyt-PTPe activity and its effects on podosomes. We conclude that PTPs alpha and epsilon play distinct roles in osteoclasts and that the N-terminus of cyt-PTPe, in particular serine 2, is critical for its function in these cells.
(2014) Journal of Biomedical Research. 28, 3, p. 157-168 Abstract
Obesity and the metabolic syndrome and their associated morbidities are major public health issues, whose prevalence will continue to increase in the foreseeable future. Aberrant signaling by the receptors for leptin and insulin plays a pivotal role in development of the metabolic syndrome. More complete molecular-level understanding of how both of these key signaling pathways are regulated is essential for full characterization of obesity, the metabolic syndrome, and type II diabetes, and for developing novel treatments for these diseases. Phosphorylation of proteins on tyrosine residues plays a key role in mediating the effects of leptin and insulin on their target cells. Here, we discuss the molecular methods by which protein tyrosine phosphatases, which are key physiological regulators of protein phosphorylation in vivo, affect signaling by the leptin and insulin receptors in their major target tissues.
(2013) Biochimica et Biophysica Acta - Reviews on Cancer. 1836, 2, p. 211-226 Abstract
Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed. (C) 2013 Elsevier B.V. All rights reserved.
(2013) FEBS Journal. 280, 2, p. 708-730 Abstract
Protein tyrosine phosphatases (PTPs) represent a super-family of enzymes that play essential roles in normal development and physiology. In this review, we will discuss the PTPs that have a causative role in hereditary diseases in humans. In addition, recent progress in the development and analysis of animal models expressing mutant PTPs will be presented. The impact of PTP signaling on health and disease will be exemplified for the fields of bone development, synaptogenesis and central nervous system diseases. Collectively, research on PTPs since the late 1980's yielded the cogent view that development of PTP-directed therapeutic tools is essential to further combat human disease.
Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation(2012) Molecular and Cellular Biology. 32, 19, p. 3913-3924 Abstract
Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.
Multifaceted Modulation of K+ Channels by Protein-tyrosine Phosphatase epsilon Tunes Neuronal Excitability(2012) Journal of Biological Chemistry. 287, 33, p. 27614-27628 Abstract
Non-receptor-tyrosine kinases (protein-tyrosine kinases) and non-receptor tyrosine phosphatases (PTPs) have been implicated in the regulation of ion channels, neuronal excitability, and synaptic plasticity. We previously showed that protein-tyrosine kinases such as Src kinase and PTPs such as PTP alpha and PTP epsilon modulate the activity of delayed-rectifier K+ channels (IK). Here we show cultured cortical neurons from PTP epsilon knockout (EKO) mice to exhibit increased excitability when compared with wild type (WT) mice, with larger spike discharge frequency, enhanced fast after-hyperpolarization, increased after-depolarization, and reduced spike width. A decrease in IK and a rise in large-conductance Ca2+-activated K+ currents (mBK) were observed in EKO cortical neurons compared with WT. Parallel studies in transfected CHO cells indicate that Kv1.1, Kv1.2, Kv7.2/7.3, and mBK are plausible molecular correlates of this multifaceted modulation of K+ channels by PTP epsilon. In CHO cells, Kv1.1, Kv1.2, and Kv7.2/7.3 K+ currents were up-regulated by PTP epsilon, whereas mBK channel activity was reduced. The levels of tyrosine phosphorylation of Kv1.1, Kv1.2, Kv7.3, and mBK potassium channels were increased in the brain cortices of neonatal and adult EKO mice compared with WT, suggesting that PTP epsilon in the brain modulates these channel proteins. Our data indicate that in EKO mice, the lack of PTP epsilon-mediated dephosphorylation of Kv1.1, Kv1.2, and Kv7.3 leads to decreased IK density and enhanced after-depolarization. In addition, the deficient PTP epsilon-mediated dephosphorylation of mBK channels likely contributes to enhanced mBK and fast after-hyperpolarization, spike shortening, and consequent increase in neuronal excitability observed in cortical neurons from EKO mice.
Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase epsilon (PTP epsilon) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells(2012) Journal of Biological Chemistry. 287, 5, p. 3433-3444 Abstract
Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTP is an element of in human breast cancer cell lines. We found the up-regulation and activation of receptor PTP is an element of (RPTP is an element of) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTP is an element of in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTP is an element of displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTP is an element of is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.[Alternative title: Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ϵ (PTPϵ) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells]
Protein Tyrosine Phosphatase Epsilon Affects Body Weight by Downregulating Leptin Signaling in a Phosphorylation-Dependent Manner(2011) Cell Metabolism. 13, 5, p. 562-572 Abstract
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(2009) Molecular Biology of the Cell. 20, 20, p. 4324-4334 Abstract
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 epsilon is a Negative Regulator of Fc epsilon RI-mediated Mast Cell Responses(2009) Scandinavian Journal of Immunology. 69, 5, p. 401-411 Abstract
Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase epsilon (PTP epsilon; Ptpre) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (Fc epsilon RI). Bone marrow-derived mast cells (BMMC) from Ptpre(-/-) mice exhibited enhanced Fc epsilon RI-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 PTP epsilon 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 PTP epsilon. In the event, Syk but not Lyn was hyperphosphorylated in PTP epsilon-deficient BMMC. Thus, PTP epsilon 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 PTP epsilon negatively regulates Fc epsilon RI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.
PTP epsilon has a critical role in signaling transduction pathways and phosphoprotein network topology in red cells(2008) Proteomics. 8, 22, p. 4695-4708 Abstract
Protein tyrosine phosphatases (PTPs) are crucial components of cellular signal transduction pathways. Here, we report that red blood cells (RBCs) from mice lacking PTP epsilon (Ptpre(-/-)) exhibit (i) abnormal morphology; (ii) increased Ca2+-activated-K+ channel activity, which was partially blocked by the Src family kinases (SFKs) inhibitor PP1; and (iii) market perturbation of the RBC membrane tyrosine (Tyr-) phosphoproteome, indicating an alteration of RBC signal transduction pathways. Using the signaling network computational analysis of the Tyr-phosphoproteomic data, we identified seven topological clusters. We studied cluster 1 containing Fyn, SFK, and Syk another tyrosine kinase. In Ptpre(-/-) mouse RBCs, the activity of Fyn was increased while Syk kinase activity was decreased compared to wild-type RBCs, validating the network computational analysis, and indicating a novel signaling pathway, which involves Fyn and Syk in regulation of red cell morphology.
(2008) European Journal of Cell Biology. 87, 9-Aug, p. 479-490 Abstract
Osteoclasts are large cells derived from the monocyte-macrophage hematopoietic cell lineage. Their primary function is to degrade bone in Various physiological contexts. Osteoclasts adhere to bone via podosomes, specialized adhesion structures whose Structure and subcellular organization are affected by mechanical contact of the cell with bone matrix. Ample evidence indicates that reversible tyrosine phosphorylation of podosomal proteins plays a major role in determining the organization and dynamics of podosomes. Although roles of several tyrosine kinases are known in detail in this respect, little is known concerning the roles of protein tyrosine phosphatases (PTPs) in regulating osteoclast adhesion. Here we summarize available information concerning the known and hypothesized roles of the best-researched PTPs in osteoclasts - PTPRO, PTP epsilon, SHP-1, and PTP-PEST. Of these, PTPRO, PTP epsilon, and PTP-PEST appear to support osteoclast activity while SHP-1 inhibits it. Additional studies are required to provide full molecular details of the roles of these PTPs in regulating osteoclast adhesion, and to uncover additional PTPs that participate in this process. (C) 2008 Elsevier GmbH. All rights reserved.
(2008) Cancer and Metastasis Reviews. 27, 2, p. 193-203 Abstract
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 (RPTP epsilon) 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 RPTP epsilon in mouse mammary glands leads to massive hyperplasia and associated tumorigenesis. Furthermore, cells isolated from mammary tumors induced by Neu in mice genetically lacking RPTP epsilon appear less transformed and proliferate less well than corresponding mammary tumor cells isolated from mice expressing the phosphatase. At the molecular level, RPTP epsilon dephosphorylates and activates Src and the related kinases Yes and Fyn, and the activities of these kinases are significantly reduced in tumor cells lacking RPTP epsilon. 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 RPTP epsilon. RPTP epsilon 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 RPTP epsilon to activate Src, but appears not to affect the activity of RPTP epsilon towards unrelated substrates. We conclude that a Neu-RPTP epsilon-Src pathway exists in mouse mammary tumor cells, in which Neu phosphorylates RPTP epsilon thereby driving the phosphatase to specifically activate Src family kinases and to assist in maintaining the transformed phenotype.
(2008) FEBS Journal. 275, 5, p. 816-830 Abstract
Some 40-odd genes in mammals encode phosphotyrosine-specific, 'classical' protein tyrosine phosphatases. The generation of animal model systems and the study of various human disease states have begun to elucidate the important and diverse roles of protein tyrosine phosphatases in cellular signalling pathways, development and disease. Here, we provide an overview of those findings from mice and men, and indicate several novel approaches that are now being exploited to further our knowledge of this fascinating enzyme family.
Protein-tyrosine phosphatase epsilon regulates Shc signaling in a kinase-specific manner - Increasing coherence in tyrosine phosphatase signaling(2008) Journal of Biological Chemistry. 283, 8, p. 4612-4621 Abstract
Individual protein tyrosine kinases and phosphatases target multiple substrates; this may generate conflicting signals, possibly within a single pathway. Protein-tyrosine phosphatase epsilon (PTP epsilon) performs two potentially opposing roles: in Neu-induced mammary tumors, PTP epsilon activates Src downstream of Neu, whereas in other systems PTP epsilon 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. PTP epsilon binds and dephosphorylates Shc in vivo, reducing the association of Shc with Grb2 and inhibiting downstream ERK activation. PTP epsilon binds Shc in a phosphotyrosine-independent manner mediated by the Shc PTB domain and aided by a sequence of 10 N-terminal residues in PTP epsilon. Surprisingly, PTP epsilon dephosphorylates Shc in a kinase-dependent manner; PTP epsilon 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 PTP epsilon for binding the same domain. In agreement, PTP epsilon 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 PTP epsilon from targeting Shc and from reducing its promitogenic signal while phosphorylating PTP epsilon and directing it to activate Src in support of mitogenesis. In so doing, Neu contributes to the coherence of the promitogenic role of PTP epsilon in this system.
Cytosolic protein tyrosine phosphatase-epsilon is a negative regulator of insulin signaling in skeletal muscle(2008) Endocrinology. 149, 2, p. 605-614 Abstract
Whereas positive regulatory events triggered by insulin binding to insulin receptor (IR) have been well documented, the mechanism by which the activated IR is returned to the basal status is not completely understood. Recently studies focused on the involvement of protein tyrosine phosphatases (PTPs) and how they might influence IR signaling. In this study, we examined the possibility that cytosolic PTP epsilon (cytPTP epsilon) is involved in IR signaling. Studies were performed on L6 skeletal muscle cells. cytPTP epsilon was overexpressed by using pBABE retroviral expression vectors. In addition, we inhibited cytPTP epsilon by RNA silencing. We found that insulin induced rapid association of cytPTP epsilon with IR. Interestingly, this association appeared to occur in the plasma membrane and on stimulation with insulin the two proteins internalized together. Moreover, it appeared that almost all internalized IR was associated with cytPTP epsilon. We found that knockdown of cytPTP epsilon by RNA silencing increased insulin-induced tyrosine phosphorylation of IR and IR substrate (IRS)-1 as well as phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Moreover, overexpression of wild-type cytPTP epsilon reduced insulin-induced tyrosine phosphorylation of IR, IRS-1, and phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Finally, insulin-induced tyrosine phosphorylation of IR and IRS-1 was greater in skeletal muscle from mice lacking the cytPTP epsilon gene than that from wild-type control animals. We conclude that cytPTP epsilon serves as another major candidate negative regulator of IR signaling in skeletal muscle.
Association of tyrosine phosphatase epsilon with microtubules inhibits phosphatase activity and is regulated by the epidermal growth factor receptor(2007) Molecular and Cellular Biology. 27, 20, p. 7102-7112 Abstract
Protein tyrosine phosphatases (PTPs) are key mediators that link physiological cues with reversible changes in protein structure and function; nevertheless, significant details concerning their regulation in vivo remain unknown. We demonstrate that PTP epsilon associates with microtubules in vivo and is inhibited by them in a noncompetitive manner. Microtubule-associated proteins, which interact strongly with microtubules in vivo, significantly increase binding of PTPE to tubulin in vitro and further reduce phosphatase activity. Conversely, disruption of microtubule structures in cells reduces their association with PTP epsilon, alters the subcellular localization of the phosphatase, and increases its specific activity. Activation of the epidermal growth factor receptor (EGFR) increases the PTP epsilon-microtubule association in a manner dependent upon EGFR-induced phosphorylation of PTP epsilon at Y638 and upon microtubule integrity. These events are transient and occur with rapid kinetics similar to EGFR autophosphorylation, suggesting that activation of the EGFR transiently down-regulates PTP epsilon activity near the receptor by promoting the PTP epsilon-microtubule association. Tubulin also inhibits the tyrosine phosphatase PTP1B but not receptor-type PTP mu or the unrelated alkaline phosphatase. The data suggest that reversible association with microtubules is a novel, physiologically regulated mechanism for regulation of tyrosine phosphatase activity in cells.
Neu-mediated phosphorylation of protein tyrosine phosphatase epsilon is critical for activation of Src in mammary tumor cells(2007) Oncogene. 26, 49, p. 7028-7037 Abstract
The receptor-type protein tyrosine phosphatase epsilon (RPTPe) activates c-Src in mammary tumor cells induced in vivo by Neu. Tumor cells lacking RPTPe 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 RPTPe are unknown. We show that Neu induces phosphorylation of RPTPe exclusively at its C-terminal Y695, and that this phosphorylation is required for activation of c-Src by RPTPe. Phosphorylation of RPTPe does not affect its activity toward another substrate, the voltage-gated potassium channel Kv2.1, suggesting that phosphorylation directs RPTPe activity toward c-Src. Phosphorylation of RPTPe reduces its dimerization at the cell membrane, although this does not affect its activity significantly. RPTPe 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-RPTP epsilon-Src signaling pathway exists in mammary tumor cells, in which phosphorylation of RPTPe by Neu directs RPTPe to activate c-Src. Reversible phosphorylation of RPTPe at Y695 may thus function as a 'molecular switch', which affects the substrate specificity of the phosphatase.
Protein tyrosine phosphatases in osteoclasts(2007) Critical Reviews in Eukaryotic Gene Expression. 17, 1, p. 49-71 Abstract
Osteoclasts are large cells derived from the monocyte-macrophage hematopoietic cell lineage, whose primary function is to degrade bone in various physiological contexts. Reversible phosphorylation of tyrosine residues in proteins is known to play significant roles in regulating the function of osteoclasts, much as it does in other cell types. Protein tyrosine phosphatases (PTPs) are among the major regulators of this process, but significant gaps exist in our knowledge of which phosphatases function in osteoclasts and the nature of their precise cellular and molecular roles. We review here the roles of the four tyrosine phosphatases that are known currently to be expressed in osteoclasts-PTPRO, PTP epsilon (PTP epsilon), SHP-1, and PTP-PEST. Of these, PTPRO and PTP epsilon support osteoclast activity, whereas SHP-1 inhibits it. Much future research is required to uncover additional PTPs that function in osteoclasts and provide full molecular-level accounting of their respective roles in osteoclasts.
Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage-gated potassium channels in Schwann cells(2006) Molecular Biology of the Cell. 17, 10, p. 4330-4342 Abstract
Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTP epsilon and PTP alpha and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTP alpha inhibits Kv channels more strongly than PTP epsilon; this correlates with constitutive association of PTP alpha with Kv2.1, driven by membranal localization of PTP alpha. PTP alpha, but not PTP epsilon, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTP epsilon and PTP alpha differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo.
(2006) Nature Medicine. 12, 6, p. 657-664 Abstract
Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts ( OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTP epsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor ( SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.
Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase(2004) Molecular and Cellular Biology. 24, 5, p. 2190-2201 Abstract
The,platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.
(2004) Experimental Cell Research. 294, 1, p. 236-243 Abstract
The receptor-type form of protein tyrosine phosphatase epsilon (RPTPepsilon) 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 RPTPepsilon appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTPepsilon 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 RPTPepsilon is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTPepsilon, 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 PTPepsilon. RPTPepsilon also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTPepsilon. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTPepsilon-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTPepsilon activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells. (C) 2003 Elsevier Inc. All rights reserved..
Tyrosine phosphatase epsilon is a positive regulator of osteoclast function in vitro and in vivo(2004) Molecular Biology of the Cell. 15, 1, p. 234-244 Abstract
Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (
(2003) Molecular and Cellular Biology. 23, 15, p. 5460-5471 Abstract
cyt-PTPepsilon is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTPepsilon 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-PTPepsilon, forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTPepsilon readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTPepsilon dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTPepsilon, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTPepsilon 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-PTPepsilon activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTPepsilon 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-PTPepsilon 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.
Tyrosine phosphatase-epsilon activates Src and supports the transformed phenotype of Neu-induced mammary tumor cells(2003) Journal of Biological Chemistry. 278, 18, p. 15579-15586 Abstract
Few tyrosine phosphatases support, rather than inhibit, survival of tumor cells. We present genetic evidence that receptor-type protein-tyrosine phosphatase (RPTP)-epsilon performs such a function, as cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTPLepsilon appeared morphologically less transformed and exhibited reduced proliferation. We show that at the molecular level, RPTPepsilon activates Src, a known collaborator of Neu in mammary tumorigenesis. Lack of RPTPepsilon reduced Src activity and altered Src phosphorylation in tumor cells; RPTPepsilon dephosphorylated and activated Src; and Src bound a substrate-trapping mutant of RPTPepsilon. The altered morphology of tumor cells lacking RPTPepsilon was corrected by exogenous Src and exogenous RPTPepsilon or RPTPalpha; exogenous activated Src corrected also the growth rate phenotype. Together, these results suggest that the altered morphology of RPTPepsilon-deficient tumor cells is caused by reduced Src activity, caused, in turn, by lack of RPTPepsilon. Unexpectedly, the phenotype of RPTPepsilon-deficient tumor cells occurs despite expression of the related RPTPalpha, indicating that endogenous RPTPalpha does not compensate for the absence of RPTPepsilon in this case. We conclude that RPTPepsilon is a physiological activator of Src in Neuinduced mammary tumors and suggest that pharmacological inhibition of phosphatases that activate Src may be useful to augment direct pharmacological inhibition of Src.
Phosphorylation-dependent regulation of Kv2.1 channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon(2003) Journal of Biological Chemistry. 278, 19, p. 17509-17514 Abstract
Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and upregulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr124 is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr124 is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K+ currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr124 is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.
Protein tyrosine phosphatase epsilon inhibits signaling by mitogen-activated protein kinases(2003) Molecular Cancer Research. 1, 7, p. 541-550 Abstract
Mitogen-activated protein kinases (MAPKs) mediate signaling from the cell membrane to the nucleus following their phosphorylation at conserved threonine and tyrosine residues within their activation loops. We show that protein tyrosine phosphatase epsilon (PTPepsilon) inhibits ERK1 and ERK2 kinase activity and reduces their phosphorylation; in agreement, ERK phosphorylation is increased in fibroblasts and in mammary tumor cells from mice genetically lacking PTPepsilon. PTPepsilon inhibits events downstream of ERKs, such as transcriptional activation mediated by Elk1 or by the serum response element. PTPepsilon also inhibits transcriptional activation mediated by c-Jun and C/EBP binding protein (CHOP) but not that mediated by the unrelated NFkB, attesting that it is broadly active within the MAPK family but otherwise specific. The effect of PTPepsilon on ERKs is at least in part indirect because phosphorylation of the threonine residue in the ERK activation loop is reduced in the presence of PTPepsilon. Nonetheless, PTPE is present in a molecular complex with ERK, providing PTPepsilon with opportunity to act on ERK proteins also directly. We conclude that PTPepsilon is a physiological inhibitor of ERK signaling. Slow induction of PTPE and its lack of nuclear translocation following mitogenic stimulation suggest that PTPepsilon functions to prevent inappropriate activation and to terminate prolonged, rather than acute, activation of ERK in the cytosol.
(2003) p. 124-132 Abstract
The chapter presents the of whole mount analysis of mammary gland epithelial structures. Whole mount analysis provides good three-dimensional overviews of the dense structures (epithelial structures, lymph nodes, blood vessels, but not adipose tissue) within the mammary gland. Whole-mount analysis of mammary tissue helps in several studies of gene-targeted mice lacking specific PTPs. The chapter presents a brief overview of mouse mammary gland development. The increasing interest in determining the physiological roles of protein tyrosine phosphatases (PTPs) in vivo has resulted in the generation of numerous strains of mice in which specific PTP genes are inactivated. The chapter elaborates on the surgical techniques, which allows transplantation of epithelial structures or cells from one mouse to a gland in another mouse, from which epithelial structures are removed previously. Manipulation of this sort allows transplantation of epithelial structures from, for example, a gene-targeted mouse that does not survive for long, in a mouse from another strain that does survive. Such studies helps to determine whether a mammary phenotype is caused by defects that are epithelial cell-specific or endocrine in nature. The chapter describes whole mount procedure including the equipment and reagents needed; preparation of sample for analysis; and staining procedure.
Nuclear localization of non-receptor protein tyrosine phosphatase epsilon is regulated by its unique N-terminal domain(2002) Experimental Cell Research. 281, 2, p. 182-189 Abstract
Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase is an element of (cyt-PTPis an element of) can be found in cell nuclei, among other cellular locations, while p67 PTPis an element of, a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTPis an element of, is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTPis an element of, in particular residues R4, K5, and R9, are critical components for its nuclear localization. We also establish that increased oxidative stress enhances accumulation of cyt-PTPis an element of in cell nuclei. Of the four known protein forms of PTPis an element of, cyt-PTPis an element of is the only one which includes the extreme N-terminal sequence containing R4, K5, and R9. The role of the unique N terminus of cyt-PTPis an element of is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTPis an element of which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTPis an element of can be actively regulated by cells. (C) 2002 Elsevier Science (USA).
A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling(2002) EMBO Journal. 21, 3, p. 303-313 Abstract
Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.
Regulation of protein-tyrosine phosphatases alpha and epsilon by calpain-mediated proteolytic cleavage(2001) Journal of Biological Chemistry. 276, 34, p. 31772-31779 Abstract
The precise subcellular localization of non-receptor tyrosine phosphatases is a major factor in regulating their physiological functions. We have previously shown that cellular processing of protein-tyrosine phosphatase epsilon (PTP epsilon) generates a physiologically distinct, cytoplasmic form of this protein, p65 PTP epsilon. Here we describe a novel protein form of the related receptor-type tyrosine phosphatase alpha (RPTP alpha), p66 PTP alpha, which is detected in nearly all cell types where RPTPa is expressed. Both p66 PTP alpha and p65 PTP epsilon are produced by calpain-mediated proteolytic cleavage in vivo. Cleavage is inhibited in living cells by a variety of calpain inhibitors, can be induced in primary cortical neurons treated with calcium chloride, and is observed in lysates of brain or of cultured cells following addition of purified calpain. Cleavage occurs within the intracellular juxtamembrane domain of RPTPa, releasing the phosphatase catalytic domains from their membranal anchors and translocating them to the cytoplasm. Translocation reduces the ability of PTPa to act on membrane-associated substrates, as it loses its ability to dephosphorylate Src at its C-terminal regulatory site, and its ability to dephosphorylate the Kv2.1 voltage-gated potassium channel is severely impaired. In all, the data indicate that control of phosphatase function via post-translational processing occurs also among receptor-type phosphatases, and demonstrate the molecular complexity of regulating these parameters within the PTP alpha /PTP epsilon phosphatase subfamily.
Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice(2001) EMBO Journal. 20, 7, p. 1538-1546 Abstract
Patients with the genetic disorder ataxia-telangiectasia (A-T) display a pleiotropic phenotype that includes neurodegeneration, immunodeficiency, cancer predisposition and hypersensitivity to ionizing radiation. The gene responsible is ATM and Atm-knockout mice recapitulate most features of A-T. In order to study the involvement of oxidative stress in the A-T phenotype, we examined mice deficient for Atm and overexpressing human Cu/Zn superoxide dismutase (SOD1). We report that elevated levels of SOD1 exacerbate specific features of the murine Atm-deficient phenotype, including abnormalities in hematopoiesis and radiosensitivity. The data are consistent with the possibility that oxidative stress contributes to some of the clinical features associated with the A-T phenotype.
Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling(2001) Biochemical Journal. 354, p. 581-590 Abstract
To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTP epsilon and PTP alpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPc to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTP epsilon levels. Consistent with these results, expression levels and subcellular localization of PTP epsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTP epsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTP epsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTP epsilon map provide a molecular mechanism that determines PTP epsilon substrate selectivity and isoform-specific function.
Regulation of myelination by wild-type and novel forms of PTP epsilon(2001) p. 202-213 Abstract
Protein tyrosine phosphatase epsilon (PTPepsilon) is highly expressed in the nervous system; however, little is known about its physiological role. In order to study the physiological roles of the phosphatase in vivo we disrupted the single PTPe gene in mice. PTPepsilon-targeted mice exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed-rectifier, voltage-gated potassium (Kv) channels, and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells from Ptpre-deficient mice, In vitro studies demonstrate that catalytically active PTPepsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes of Kv2.1 in Xenopus oocytes, and profoundly reduces Src- or Fyn-stimulated tyrosine phosphorylation in transfected HEK 293 cells. The interaction of these potassium channels with PTPepsilon is direct, since a substrate-trapping mutant of the phosphatase associates with Kv2.1 in transfected cells. In addition, localization of PTPC at the cell membrane is important for its ability to act on Kv channels, as p65 and p67 - two naturally occurring truncated forms of PTPe which are exclusively cytoplasmic - are significantly less active towards Kv channels compared to the membrane associated forms of the enzyme. In all, PTPepsilon antagonizes activation of Kv channels by tyrosine kinases in vivo, most likely affecting Schwann cell function during a critical period of Schwann cell growth and myelination.
Generation of novel cytoplasmic forms of protein tyrosine phosphatase epsilon by proteolytic processing and translational control(2000) Oncogene. 19, 38, p. 4375-4384 Abstract
Two protein forms of tyrosine phosphatase epsilon (PTP epsilon) are known - receptor-like (tm-PTP epsilon) and non receptor-like (cyt-PTP epsilon), with each form possessing unique tissue-specific expression patterns, subcellular localization, and physiological functions. We describe two additional forms of PTP epsilon: protein - p67 and p65, p67 is produced by initiation of translation at an internal initiation codon of PTP epsilon mRNA molecules, while p65 is produced by specific proteolytic cleavage of larger PTP epsilon proteins. Cleavage is inhibited by MG132, but is proteasome-independent. In contrast with full-length tm-PTP epsilon and cyt-PTP epsilon, p67 and p65 are exclusively cytoplasmic, are not phosphorylated by Neu, and do not associate with Grb2 in unstimulated cells. p67 and p65 are catalytically active and can reduce Src-mediated phosphorylation of the Kv2.1 voltage-gated potassium channel, albeit with reduced efficiency which most likely results from their cytoplasmic localization. We also show that full-length cyt-PTP epsilon protein can be found at the cell membrane and in the nucleus and that it is the first 27 residues of cyt-PTP epsilon which determine this localization. p67 and p65 provide mechanisms for removing PTP epsilon activity from the cell membrane, possibly serving to down-regulate PTP epsilon activity there. PTP epsilon emerges as a family of four related proteins whose expression, subcellular localization and most likely physiological roles are subject to complex regulation at the transcriptional, translational and post-translational levels.
Hypomyelination and increased activity of voltage-gated K+ channels in mice lacking protein tyrosine phosphatase epsilon(2000) EMBO Journal. 19, 15, p. 4036-4045 Abstract
Protein tyrosine phosphatase epsilon (PTP epsilon) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTP epsilon exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed-rectifier, voltage-gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Ky channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells. PTP epsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate-trapping mutant of PTP epsilon, and PTP epsilon profoundly reduces Src- or Fyn-stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTP epsilon antagonizes activation of Ky channels by tyrosine kinases ii vive, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.
Protein tyrosine phosphatase epsilon increases the risk of mammary hyperplasia and mammary tumors in transgenic mice(1999) Oncogene. 18, 52, p. 7535-7542 Abstract
Accurate phosphorylation of tyrosine residues in proteins plays a central role in regulation of cellular function. Although connections between aberrant tyrosine kinase activity and malignancy are well-established, significantly less Is known about the roles of protein tyrosine phosphatases (PTPases) in tumorigenesis. We have previously shown that the transmembranal form of PTPase Epsilon (PTP epsilon) is upregulated in mouse mammary tumors initiated specifically by ras or neu, suggesting that PTP epsilon may play a role in transformation by these two oncogenes, In order to test this notion lit vivo, we created transgenic mice that express elevated le, els of PTP epsilon in their mammary epithelium by use of the MMTV promoter/enhancer, Following several cycles of pregnancy female MMTV-PTP epsilon mice uniformly de, eloped pronounced and persistent mammary hyperplasia which was accompanied by residual milk production. Solitary mammary tumors were often detected secondary to mammary hyperplasia. The sporadic nature of the tumors, the long latency period prior to their development, and low levels of transgene expression in the tumors indicate that PTP epsilon provides a necessary, but insufficient, signal for oncogenesis. The results provide genetic evidence that PTP epsilon plays an accessory role in production of mammary tumors in a manner consistent with its upregulation in mammary tumors induced by ras or neu.
The transmembranal and cytoplasmic forms of protein tyrosine phosphatase epsilon physically associate with the adaptor molecule Grb2(1999) Oncogene. 18, 36, p. 5024-5031 Abstract
The protein tyrosine phosphatase Epsilon (PTP epsilon) gene gives rise to two physiologically-distinct protein products-a transmembranal, receptor-like form and a cytoplasmic, non-receptor form. Previous studies have suggested a link between expression of transmembranal PTP epsilon and transformation of mouse mammary epithelium specifically by ras or neu, although little is known about the underlying molecular mechanisms; cytoplasmic PTP epsilon is believed to function mainly in hematopoietic tissues. As part of our efforts to understand PTP epsilon function at the molecular level, we demonstrate here that both forms of PTP epsilon associate with the adaptor molecule Grb2 in vivo. Binding is mediated by the SH2 domain of Grb2; this domain binds exclusively to the carboxy-terminal phosphotyrosine of cytoplasmic PTP epsilon(Y638), and probably to additional phosphotyrosine. residues in transmembranal PTP epsilon. Through its SH2 domain, Grb2 can constitutively associate with transmembranal PTP epsilon in mammary tumors initiated by ras or neu, and can be induced to associate with cytoplasmic PTP epsilon in Jurkat T-cells following stimulation of T-cell receptor signaling by pervanadate. These findings indicate that tyrosine phosphorylation of PTP epsilon and subsequent binding to Grb may link this phosphatase to downstream events which transduce signals from the cell membrane to its interior.
(1998) Proceedings of the National Academy of Sciences of the United States of America. 95, 21, p. 12653-12656 Abstract
Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction, Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease, To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum, A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor L-dopa, In addition, these mice were hypersensitive to the dopamine releasing drug D-amphetamine, These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.
Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice(1997) Proceedings of the National Academy of Sciences of the United States of America. 94, 26, p. 14590-14595 Abstract
Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype, p21(cip1/waf1) (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.
atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity(1997) Nature Genetics. 16, p. 397-401 Abstract
Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia(1) and Li-Fraumeni syndrome(2,3), respectively. The two genes are believed to interact in a number of pathways(4-6), including regulation of DNA damage-induced cell-cycle checkpoints(7), apoptosis and radiation sensitivity(8), and cellular proliferation(9). Atm-null mice(10-12), as well as those null for p53(13,14), develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions(4,6)-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.
Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints(1997) Cancer Research. 57, 9, p. 1664-1667 Abstract
Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G(1)-S tell cycle checkpoint control, and both afm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G(1)-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G(1)-S cell cycle control, and another more complex, involving aspects of growth regulation.
(1996) Proceedings of the National Academy of Sciences of the United States of America. 93, 23, p. 13084-13089 Abstract
We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm- deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm- deficient mice then exhibit many of the same symptoms found in ataxia- telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.
(1996) Genomics. 31, 3, p. 373-375 Abstract
We have mapped the mouse protein tyrosine phosphatase ∈ (PTP∈, gene symbol Ptpre) gene to the distal region of chromosome 7 by linkage analysis using two sets of multilocus genetic crosses. The human PTP∈ gene (gene symbol PTPRE) was mapped to chromosome 10q26 by fluorescence in situ hybridization. We have previously documented the existence of two isoforms of PTP∈ - a transmembranal, receptor-type isoform and a shorter, cytoplasmic one. Both isoforms have been suggested to arise from a single gene through the use of alternative promoters and 5′ exons. The identification of a single PTP∈ locus in both organisms is consistent with this suggestion,
Identification of a cytoplasmic, phorbol ester-inducible isoform of protein tyrosine phosphatase epsilon(1995) Proceedings of the National Academy of Sciences of the United States of America. 92, 26, p. 12235-12239 Abstract
The protein-tyrosine phosphatase ε (PTPε) is a transmembranal, receptor- type protein that possesses two phosphatase catalytic domains characteristic of transmembranal phosphatases. Here we demonstrate the existence of a nontransmembranal isoform of PTPε, PTPε-cytoplasmic. PTPε-cytoplasmic and the transmembranal isoform of PTPε have separate, nonoverlapping expression patterns. Further, the data clearly indicate that control of which of the two isoforms is to he expressed is initiated at the transcriptional level, suggesting that they have distinct physiological roles. PTPε-cytoplasmic mRNA is the product of a delayed early response gene in NIH 3T3 fibroblasts, and its transcription is regulated through a pathway that requires protein kinase C. The human homologue of PTPε-cytoplasmic has also been cloned and is strongly up-regulated in the early stages of phorbol 12-tetradecanoate 13- acetate-induced differentiation of HL-60 cells. Sequence analysis indicates and cellular fractionation experiments confirm that this isoform is a cytoplasmic molecule. PTPε-cytoplasmic is therefore the initial example to our knowledge of a nontransmembranal protein-tyrosine phosphatase that contains two tandem catalytic domains.
Protein-tyrosine Phosphatase Epsilon: An isoform specifically expressed in mouse mammary-tumors initiated by v-Ha-ras OR neu(1995) Journal of Biological Chemistry. 270, 44, p. 26116-26122 Abstract
Transgenic mice that overexpress v-Ha-ras, c-myc, c-neu or int-2 proto-oncogenes in the mammary epithelium develop breast tumors with morphologies that are characteristic of each initiating oncogene, Since these morphological differences reflect distinctive patterns of tumor-specific gene expression, the identification of the products of these genes might shed light on the mechanisms of transformation and/or the identity of target cells that are transformed by specific classes of oncogenes, By focusing on the tyrosine phosphorylation pathway, we have found that the transmembranal protein-tyrosine phosphatase epsilon (PTP epsilon) is highly expressed in murine mammary tumors initiated by c-neu and v-Ha-ras, but not in mammary tumors initiated by c-myc or int-2. This difference is striking and occurs both in primary tumors and in epithelial cells cultured from them, Moreover, PTP epsilon overexpression appears to be mammary tumor-specific in that it is not found in other ras-based tumors and cell lines, These observations suggest that PTP epsilon either plays a role in ras- and neu-mediated transformation of mammary epithelium or marks mammary epithelial cells particularly susceptible to transformation by these oncogenes, Because of its distinctive expression in these mammary tumors, we have further characterized murine PTP epsilon, cloning and determining the complete structures of its cDNAs and showing that it is a glycoprotein that is N-glycosylated in a tissue-specific manner.
Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice(1995) Genes and Development. 9, 21, p. 2712-2722 Abstract
We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812- amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.
The MMTV/c-myc transgene and p53 null alleles collaborate to induce T-cell lymphomas, but not mammary carcinomas in transgenic mice(1995) Oncogene. 11, 1, p. 181-190 Abstract
A number of properties of the cancer-related genes c-myc and p53 suggest that they might collaborate to induce tumorigenesis, To test this notion, we produced doubly heterozygotic mice bearing disrupted p53 alleles and a fusion transgene consisting of the mouse mammary tumor virus (MMTV) I;TR and the oncogene c-myc. Mice bearing both the MMTV/c-myc transgene and a single p53(-) allele develop very aggressive pre-T- and T-cell lymphomas with a significantly shorter latency than mice carrying either the p53- allele or the c-myc transgene alone, Moreover, every lymphoma occurring in these animals has lost or suffers an inactivation of its wild type p53 allele indicating that loss of p53 activity is necessary for this c-myc-accelerated lymphomagenesis. Nonetheless, p53 inactivation and expression of the MMTV/c-myc transgene are not sufficient for lymphoid transformation, Tumors that arise in homozygous p53(-) mice carrying the c-myc transgene are monoclonal, suggesting that at least one additional event is necessary for their transformation, Moreover, since mice bearing only the MMTV/c-myc transgene predominantly develop mammary carcinomas, it was surprising that the p53(-) allele failed to accelerate the incidence of mammary carcinomas, Further, in contrast to the lymphomas, only one in four mammary tumors that arose in the double heterozygotic mice had lost its wild type p53 allele, Apparently cell context influences the ability of c-myc and p53(-) to cooperate in inducing oncogenesis.
Overexpression of liver-type phosphofructokinase (PFKL) in transgenic-PFKL mice: implication for gene dosage in trisomy 21(1994) Biochemical Journal. 299, 2, p. 409-415 Abstract
The human liver-type subunit of the key glycolytic enzyme, phosphofructokinase (PFKL), is encoded by a gene residing on chromosome 21. This chromosome, when triplicated, causes the phenotypic expression of Down's syndrome (trisomy 21). Increased phosphofructokinase activity, a result of gene dosage, is commonly found in erythrocytes and fibroblasts from Down's syndrome patients. We describe the construction of transgenic mice overexpressing PFKL for use as a well-defined model system, in which the effects of PFKL overexpression in various tissues, and throughout development, can be studied. Mice transgenic for a murine PFKL 'gene cDNA' hybrid construct were found to overexpress PFKL in a tissue-specific manner resembling that of the endogenous enzyme. Although unchanged in adult brain, PFK specific activity was found to have been almost doubled in brains of embryonic transgenic-PFKL mice, suggesting that the extra copies of the PFKL gene are expressed during the developmental period. This pattern of overexpression of PFKL in brains of transgenic-PFKL mice suggests that gene-dosage effects may be temporally separated from some of their consequences, adding an additional layer of complexity to the analysis of gene dosage in trisomy 21.
Protein kinase C (PKC) level is increased in PC12 cells overexpressing transfected liver‐type phosphofructokinase(1994) Biology of the Cell. 81, 1, p. 23-29 Abstract
PC12 cells which overexpress transfected liver-type phosphofructokinase (PFKL) have previously been described as a model system for PFKL overexpression in Down's syndrome and have been shown to perform glycolysis at enhanced rates. Here we report that levels of protein kinase C (PKC) in PC12-PFKL cells were almost doubled, as estimated from in vitro activity and phorbol ester binding experiments and from an increase found in PKC-alpha mRNA levels. Most of the added PKC was found to be associated with the cellular membrane while the cytoplasmic levels of PKC were barely increased. The steady-state levels of 1,2-sn-diacylglycerol in PC12-PFKL cells were found to be unaltered, suggesting that enhanced glycolysis in these cells did not influence PKC by altering the amounts of this compound. PFKL is one of several genes known to be overexpressed in Down's syndrome. Upregulation df PKC due to PFKL overexpression could result in widespread disturbances of gene expression and play a part in causing some of the many symptoms of the disease.
Gene dosage and down's syndrome: Metabolic and enzymatic changes in PC12 cells overexpressing transfected human liver-type phosphofructokinase(1992) Somatic Cell and Molecular Genetics. 18, 2, p. 143-161 Abstract
Down syndrome (DS) is a human genetic disease caused by triplication of the distal third of chromosome 21 and overexpression of an unknown number of genes residing in it. The gene for the liver-type subunit of phosphofructokinase (PFKL), a key glycolytic enzyme, maps to this region and the product is overproduced in DS erythrocytes and fibroblasts. These facts, together with abnormalities which occur in DS glycolysis, make PFKL overexpression a candidate for causing some aspects of the DS phenotype. A cellular model for examining the consequences of PFKL overexpression in DS was constructed by transfecting rat PC12 cells with the human PFKL cDNA. Phosphofructokinase (PFK) isolated from PFKL-overexpressing clones was more inhibited by A TP and citrate and less activated by fructose-6-phosphate than control PFK; similar results were obtained when PFK preparations from DS and control fibroblasts were compared. In vivo NMR measurements determined that cells overexpressing PFKL performed glycolysis 40% faster than controls. These results show that overexpression of PFKL is the cause for altered biochemical regulatory characteristics of PFK in DS fibroblasts and can result in enhancement of glycolysis rates. It is also shown that increased gene dosage can exert its influence not merely by enhancing the amounts of gene products but also by altering their biochemical nature.
(1990) Genomics. 7, 1, p. 47-56 Abstract
We have isolated the gene for the human liver-type phosphofructokinase, from upstream to the 5′ mRNA terminus to beyond the polyadenylation site. The gene is at least 28 kb long and is divided into 22 exons; it contains conventional splice-junction sequences and one polyadenylation signal. Exons and introns are quite rich in G and C residues; some 60% of all nucleotides are either G or C. Five possible sites of polymorphism have been found. The gene structure reveals no signs of internal similarities despite protein sequence evidence which suggests that the PFK molecule is divided into two similar halves. The structure and organization of the human liver-type PFK gene are shown to be extremely similar to those of the rabbit muscle-type PFK.
The Primary Structure of Human Liver Type Phosphofructokinase and Its Comparison with Other Types of PFK(1989) DNA. 8, 10, p. 733-743 Abstract
The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5′ and 515 of 3′ noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (∼90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while the relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.