Publications

The cytokine RANKL (Receptor Activator of NFκB Ligand) is the key driver of differentiation of monocytes/macrophages to form multi-nucleated, bone-resorbing osteoclasts, a process that is accompanied by significant changes in gene expression. We show that exposure to RANKL rapidly down-regulates expression of Brain Acid Soluble Protein 1 (BASP1) in cultured primary mouse bone marrow macrophages (BMMs), and that this reduced expression is causally linked to the osteoclastogenic process in vitro. Knocking down BASP1 expression in BMMs or eliminating its expression in these cells or in RAW 264.7 cells enhanced RANKL-induced osteoclastogenesis, promoted cell-cell fusion, and generated cultures containing larger osteoclasts with increased mineral degrading abilities relative to controls. Expression of exogenous BASP1 in BMMs undergoing osteoclastogenic differentiation produced the opposite effects. Upon exposure to RANKL, primary mouse BMMs in which BASP1 had been knocked down exhibited increased expression of the key osteoclastogenic transcription factor Nfatc1and of its downstream target genes Dc-stamp, Ctsk, Itgb3, and Mmp9 relative to controls. The knock-down cells also exhibited increased sensitivity to the pro-osteoclastogenic effects of RANKL. We conclude that BASP1 is a negative regulator of RANKL-induced osteoclastogenesis, which down-regulates the pro-osteoclastogenic gene expression pattern induced by this cytokine. Decreased expression of BASP1 upon exposure of BMMs to RANKL removes a negative regulator of osteoclastogenesis and promotes this process.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo.

Formation of mature bone-resorbing cells through osteoclastogenesis is required for the continuous remodeling and repair of bone tissue. In aging and disease this process may become aberrant, resulting in excessive bone degradation and fragility fractures. Interaction of receptor-activator of nuclear factor-κB (RANK) with its ligand RANKL activates the main signaling pathway for osteoclastogenesis. However, compelling evidence indicates that this pathway may not be sufficient for the production of mature osteoclast cells and that co-stimulatory signals may be required for both the expression of osteoclast-specific genes and the activation of osteoclasts. Osteoclast-associated receptor (OSCAR), a regulator of osteoclast differentiation, provides one such co-stimulatory pathway. This review summarizes our present knowledge of osteoclastogenesis signaling and the role of OSCAR in the normal production of bone-resorbing cells and in bone disease. Understanding the signaling mechanism through this receptor and how it contributes to the production of mature osteoclasts may offer a more specific and targeted approach for pharmacological intervention against pathological bone resorption.

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.

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.

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.

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.

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.

Osteoclasts, specialized cells that degrade bone, are key components of the cellular system that regulates and maintains bone homeostasis. Aberrant function of osteoclasts can lead to pathological loss or gain of bone mass, such as in osteopetrosis, osteoporosis, and several types of cancer that metastasize to bone. Phosphorylation of osteoclast proteins on tyrosine residues is critical for formation of osteoclasts and for their proper function and responses to physiological signals. Here we describe preparation and growth of osteoclasts from bone marrow of mice, use of viral vectors to downregulate expression of endogenous proteins and to express exogenous proteins in osteoclasts, and analysis of signaling processes triggered by M-CSF, estrogen, and physical contact with matrix in these cells.

Female mice lacking protein tyrosine phosphatase ε (PTP ε) 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 ε with that of the closely related PTP α 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 α. The varying effects of either PTP on podosome organization in osteoclasts are caused by their distinct N-termini. Osteoclasts express the receptor-type PTP α (RPTPa), which is absent from podosomes, and the nonreceptor form of PTP ε (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 α enables them to function in osteoclasts. Serine 2 within this sequence regulates cyt-PTPe activity and its effects on podosomes. We conclude that PTPs α and ε 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.

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]

The obesity pandemic has focused attention in recent years to the physiological and molecular mechanisms that regulate body weight and glucose metabolism. Key signaling pathways that regulate both parameters depend heavily on reversible phosphorylation of proteins on tyrosine residues, a process regulated by the opposing activities of tyrosine kinases and tyrosine phosphatases. Here we review the roles of protein tyrosine phosphatase epsilon (PTPe) in regulating the leptin and insulin signaling pathways and through them—body weight and glucose metabolism. Mice lacking PTPe are leptin-hypersensitive and are protected from weight gain that follows a high-fat diet. PTPe helps downregulate leptin receptor signaling in the hypothalamus by dephosphorylating JAK2 following activation of the leptin receptor, thus inhibiting the receptor post-activation. PTPe is induced to perform this function after undergoing leptin receptor-induced phosphorylation at its C-terminal Y695. Mice lacking PTPe are also insulin-hypersensitive, indicating that PTPe downregulates signaling by this receptor as well. Studies in muscle cells confirm that PTPe inhibits insulin receptor signaling, possibly by targeting the receptor itself. These studies identify PTPe as a physiological inhibitor of both signaling pathways and as a factor in supporting the resistance to leptin and insulin that is established in obesity and in type-II diabetes, respectively.

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.

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.

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.

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.

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.

Individual protein tyrosine kinases and phosphatases target multiple substrates; this may generate conflicting signals, possibly within a single pathway. Protein-tyrosine phosphatase ε (PTPε) performs two potentially opposing roles: in Neu-induced mammary tumors, PTPε activates Src downstream of Neu, whereas in other systems PTPε 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. PTPε binds and dephosphorylates Shc in vivo, reducing the association of Shc with Grb2 and inhibiting downstream ERK activation. PTPε binds Shc in a phosphotyrosine-independent manner mediated by the Shc PTB domain and aided by a sequence of 10 N-terminal residues in PTPε. Surprisingly, PTPε dephosphorylates Shc in a kinase-dependent manner; PTPε 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 PTPε for binding the same domain. In agreement, PTPε 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 PTPε from targeting Shc and from reducing its promitogenic signal while phosphorylating PTPε and directing it to activate Src in support of mitogenesis. In so doing, Neu contributes to the coherence of the promitogenic role of PTPε in this system.

The receptor-type protein tyrosine phosphatase epsilon (RPTPε) activates c-Src in mammary tumor cells induced in vivo by Neu. Tumor cells lacking RPTPε 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 RPTPε are unknown. We show that Neu induces phosphorylation of RPTPε exclusively at its C-terminal Y695, and that this phosphorylation is required for activation of c-Src by RPTPε. Phosphorylation of RPTPε does not affect its activity toward another substrate, the voltage-gated potassium channel Kv2.1, suggesting that phosphorylation directs RPTPε activity toward c-Src. Phosphorylation of RPTPε reduces its dimerization at the cell membrane, although this does not affect its activity significantly. RPTPε 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-RPTPε-Src signaling pathway exists in mammary tumor cells, in which phosphorylation of RPTPε by Neu directs RPTPε to activate c-Src. Reversible phosphorylation of RPTPε at Y695 may thus function as a 'molecular switch', which affects the substrate specificity of the phosphatase.

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.

The platelet-derived growth factor (PDGF) β receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF β receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF β receptor, we compared PDGF β receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF β 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 Cγ1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cγ1 activity and migratory hyperresponsiveness to PDGF. PDGF β receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPε 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 β receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.

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.

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 ε (PTPε) 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 PTPε. PTPε inhibits events downstream of ERKs, such as transcriptional activation mediated by Elk1 or by the serum response element. PTPε 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 PTPε 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 PTPε. Nonetheless, PTPε is present in a molecular complex with ERK, providing PTPε with opportunity to act on ERK proteins also directly. We conclude that PTPε is a physiological inhibitor of ERK signaling. Slow induction of PTPε and its lack of nuclear translocation following mitogenic stimulation suggest that PTPε functions to prevent inappropriate activation and to terminate prolonged, rather than acute, activation of ERK in the cytosol.

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.

Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase ε (cyt-PTPε) can be found in cell nuclei, among other cellular locations, while p67 PTPε, a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTPε, is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTPε, 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-PTPε in cell nuclei. Of the four known protein forms of PTPε, cyt-PTPε 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-PTPε is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTPε which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTPε can be actively regulated by cells.

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 Atmdeficient 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.

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.

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.

The protein tyrosine phosphatase Epsilon (PTPε) gene gives rise to two physiologically-distinct protein products - a transmembranal, receptor-like form and cytoplasmic, non-receptor form. Previous studies have suggested a link between expression of transmembranal PTPε and transformation of mouse mammary epithelium specifically by ras or neu, although little is known about the underlying molecular mechanisms; cytoplasmic PTPε is believed to function mainly in hematopoietic tissues. As part of our efforts to understand PTPε function at the molecular level, we demonstrate here that both forms of PTPε 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 PTPε(Y638), and probably to additional phosphotyrosine residues in transmembranal PTPε. Through its SH2 domain, Grb2 can constitutively associate with transmembranal PTPε in mammary tumors initiated by ras or neu, and can be induced to associate with cytoplasmic PTPε in Jurkat T-cells following stimulation of T-cell receptor signaling by pervanadate. These findings indicate that tyrosine phosphorylation of PTPε and subsequent binding to Grb may link this phosphatase to downstream events which transduce signals from the cell membrane to its interior.

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.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, 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 p52 apoptotic pathways are not completely congruent. Finally- and in contrast to prior predictions-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.

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,

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.

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.

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 PC 12-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 of 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.

Down's 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 ATP 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.

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.