Publications

The skeleton is a living, biological tissue responding to the biomechanical demands placed upon it throughout life. The individual bones creating this physiological system are each shaped by a multinucleated cell type - the osteoclast - that sculpts each bone in collaboration with local cellular partners, which offer chemical and even tactile feedback of many sorts. Unfortunately, the perturbation of osteoclast formation and function underpins a broad range of human skeletal pathologies, including osteopetrosis - a systemic pathology characterized by impaired osteoclast resorption leading to skeletal thickening, brittle bones, frailty, and lethality. Here, we describe a molecular dysfunction observed in murine and human models of two forms of osteoclast-rich, autosomal recessive osteopetrosis, and our approach for exploiting this molecular dysfunction to correct pathologic osteoclast hyperfusion and resorptive impairment. We find that La - a manager of osteoclast fusion and subsequent resorptive activity - is greatly elevated at the surface of osteoclasts upon loss of SNX10 or OSTM1. Using inhibitory antibodies, we suppress excessive La surface function in these mutant osteoclasts, impede osteopetrotic hyperfusion and restore osteoclast resorptive function. We share these observations as proofs-of-principle that osteoclast fusion represents a viable therapeutic target for addressing osteoclast dysfunction in diseases underpinned by excessive osteoclast multinucleation and perturbed resorptive function.Competing Interest StatementThe authors have declared no competing interest.United States-Israel Binational Science Foundation, 2021168Intramural Research Program of the National Institutes of HealthEunice Kennedy Shriver National Institute of Child Health and Human Development, R00-HD110609

Osteoclasts are specialized cells that degrade bone and are essential for bone formation and maintaining bone homeostasis. Excess or deficient activity of these cells can significantly alter bone mass, structure, and physical strength, leading to significant morbidity, as in osteoporosis or osteopetrosis, among many other diseases. Protein phosphorylation in osteoclasts plays critical roles in the signaling pathways that govern the production of osteoclasts and regulate their bone-resorbing activity. In this chapter, we describe the isolation of mouse splenocytes and their differentiation into mature osteoclasts on resorptive (e.g., bone) and non-resorptive (e.g., plastic or glass) surfaces, examining matrix resorption by osteoclasts, immunofluorescence staining of these cells, and knocking out genes by CRISPR in the mouse osteoclastogenic cell line RAW264.7.

Osteoclasts (OCLs) are hematopoietic cells whose physiological function is to degrade bone. OCLs are key players in the processes that determine and maintain the mass, shape, and physical properties of bone. OCLs adhere to bone tightly and degrade its matrix by secreting protons and proteases onto the underlying surface. The combination of low pH and proteases degrades the mineral and protein components of the matrix and forms a resorption pit; the degraded material is internalized by the cell and then secreted into the circulation. Insufficient or excessive activity of OCLs can lead to significant changes in bone and either cause or exacerbate symptoms of diseases, as in osteoporosis, osteopetrosis, and cancer-induced bone lysis. OCLs are derived from monocyte-macrophage precursor cells whose origins are in two distinct embryonic cell lineages - erythromyeloid progenitor cells of the yolk sac, and hematopoietic stem cells. OCLs are formed in a multi-stage process that is induced by the cytokines M-CSF and RANKL, during which the cells differentiate, fuse to form multi-nucleated cells, and then differentiate further to become mature, bone-resorbing OCLs. Recent studies indicate that OCLs can undergo fission in vivo to generate smaller cells, called \u201costeomorphs\u201d, that can be \u201cre-cycled\u201d by fusing with other cells to form new OCLs. In this review we describe OCLs and discuss their cellular origins and the cellular and molecular events that drive osteoclastogenesis.

Bone-resorbing osteoclasts (OCLs) are multinucleated phagocytes, whose central roles in regulating bone formation and homeostasis are critical for normal health and development. OCLs are produced from precursor monocytes in a multistage process that includes initial differentiation, cellcell fusion, and subsequent functional and morphological maturation; the molecular regulation of osteoclastogenesis is not fully understood. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as an essential regulator specifically of OCL maturation. Monocytes from PTPRJ-deficient (JKO) mice differentiate and fuse normally, but their maturation into functional OCLs and their ability to degrade bone are severely inhibited. In agreement, mice lacking PTPRJ throughout their bodies or only in OCLs exhibit increased bone mass due to reduced OCL-mediated bone resorption. We further show that PTPRJ promotes OCL maturation by dephosphorylating the M-CSF receptor (M-CSFR) and Cbl, thus reducing the ubiquitination and degradation of the key osteoclastogenic transcription factor NFATc1. Loss of PTPRJ increases ubiquitination of NFATc1 and reduces its amounts at later stages of osteoclastogenesis, thereby inhibiting OCL maturation. PTPRJ thus fulfills an essential and cell-autonomous role in promoting OCL maturation by balancing between the pro- and anti-osteoclastogenic activities of the M-CSFR and maintaining NFATc1 expression during late osteoclastogenesis.

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.

The R51Q mutation in sorting nexin 10 (SNX10) was shown to cause a lethal genetic disease in humans, namely autosomal recessive osteopetrosis (ARO). We describe here the first R51Q SNX10 knock-in mouse model and show that mice homozygous for this mutation exhibit massive, early-onset, and widespread osteopetrosis. The mutant mice exhibit multiple additional characteristics of the corresponding human disease, including stunted growth, failure to thrive, missing or impacted teeth, occasional osteomyelitis, and a significantly-reduced lifespan. Osteopetrosis in this model is the result of osteoclast inactivity that, in turn, is caused by absence of ruffled borders in the mutant osteoclasts and by their inability to secrete protons. These results confirm that the R51Q mutation in SNX10 is a causative factor in ARO and provide a model system for studying this rare disease.

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.

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.

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.

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 thembody 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.

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]

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.

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 PTPε 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 PTPε to tubulin in vitro and further reduce phosphatase activity. Conversely, disruption of microtubule structures in cells reduces their association with PTPε, alters the subcellular localization of the phosphatase, and increases its specific activity. Activation of the epidermal growth factor receptor (EGFR) increases the PTPε-microtubule association in a manner dependent upon EGFR-induced phosphorylation of PTPε 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 PTPε activity near the receptor by promoting the PTPε-microtubule association. Tubulin also inhibits the tyrosine phosphatase PTP1B but not receptor-type PTPμ 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.

Tyrosine phosphatases (PTPs) ε and α are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPε and PTPα 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. PTPα inhibits Kv channels more strongly than PTPε; this correlates with constitutive association of PTPα with Kv2.1, driven by membranal localization of PTPα. PTPα, but not PTPε, 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 PTPε and PTPα 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 receptor-type form of protein tyrosine phosphatase epsilon (RPTPε) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTPε appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTPε in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTPε is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTPε, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTPε. RPTPε also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTPε. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTPε-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTPε activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.

cyt-PTPε is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTPε 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 (RPTPα) and CD45. Using immunoprecipitation and gel filtration, we show that cyt-PTPε forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTPε readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTPε dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTPε, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTPε 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-PTPε activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTPε lacking these regions constitutively dimerizes, and its activities in vitro towards paranitrophenyl phosphate and in vivo towards the Kv2.1 potassium channel are markedly reduced. We conclude that physiological signals can regulate dimerization and phosphorylation of cyt-PTPε 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.

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cb1, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cb1, here we report that defective Tyr1045 of EGFR, an inducible c-Cb1 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-Cb1-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-Cb1. The underlying mechanism seems to involve recruitment of a Grb2 c-Cb1 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-Cb1-dependent sorting of active tyrosine kinases to destruction.

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 ε (PTPε) generates a physiologically distinct, cytoplasmic form of this protein, p65 PTPε. Here we describe a novel protein form of the related receptor-type tyrosine phosphatase α (RPTPα), p66 PTPα, which is detected in nearly all cell types where RPTPα is expressed. Both p66 PTPα and p65 PTPε 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 RPTPα, releasing the phosphatase catalytic domains from their membranal anchors and translocating them to the cytoplasm. Translocation reduces the ability of PTPα 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 PTPα/PTPε phosphatase subfamily.

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) 6 on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHKIR). The efficiency by which differently localized PTPε and PTPα 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 cytPTPε to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPε levels. Consistent with these results, expression levels and subcellular localization of PTPε were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPε stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPε 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 PTPε may provide a molecular mechanism that determines PTPε substrate selectivity and isoform-specific function.

Two protein forms of tyrosine phosphatase epsilon (PTPε) are known - receptor-like (tm-PTPε) and non receptor-like (cyt-PTPε), with each form possessing unique tissue-specific expression patterns, subcellular localization, and physiological functions. We describe two additional forms of PTPε protein - p67 and p65. p67 is produced by initiation of translation at an internal initiation codon of PTPε mRNA molecules, while p65 is produced by specific proteolytic cleavage of larger PTPε proteins. Cleavage is inhibited by MG132, but is proteasome-independent. In contrast with full-length tm-PTPε and cyt-PTPε, 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-PTPε protein can be found at the cell membrane and in the nucleus and that it is the first 27 residues of cyt-PTPε which determine this localization. p67 and p65 provide mechanisms for removing PTPε activity from the cell membrane, possibly serving to down-regulate PTPε activity there. PTPε 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.

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 phosphatase (PTPases) in tumorigenesis. We have previously shown that the transmembranal form of PTPase Epsilon (PTPε) is upregulated in mouse mammary tumors initiated specifically by ras or neu, suggesting that PTPε may play a role in transformation by these two oncogenes. In order to test this notion in vivo, we created transgenic mice that express elevated levels of PTPε in their mammary epithelium by use of the MMTV promoter/enhancer. Following several cycles of pregnancy female MMTV-PTPε mice uniformly developed 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 PTPε provides a necessary, but insufficient, signal for oncogenesis. The results provide genetic evidence that PTPε plays an accessory role in production of mammary tumors in a manner consistent with its upregulation in mammary tumors induced by ras or neu.

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.

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 G₁- S cell cycle checkpoint control, and both atm 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(cip1/waf1), showing that p53 is required for p21(cip1/waf1) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation- induced G₁-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between arm and p53, one linear, involving G₁-S cell cycle control, and another more complex, involving aspects of growth regulation.

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.

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.

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.

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.

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.

Free NcarbobenzyloxyphenylalanyltRNA is rapidly cleaved by the enzyme peptidyltRNA hydrolase. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed as rapidly as when free. The addition of 50 S ribosomal subunits to form the 70 S ribosomal binding complex protects the bound substrate from the enzyme. The degree of protection depends on the state of activity of the 50 S subunit. Under conditions favoring the formation of 70 S ribosomes, active 50 S subunits (capable of catalyzing the peptidyl transferase reaction) afford complete protection at one mole per mole of 30 S subunits. Inactive 50 S subunits also protect the substrate, but less effectively. The protection of bound substrate can be used to measure the amount of 70 S ribosomal binding complex formed and the rate of its formation. Using highly active hydrolase preparations, we have developed a rapid assay procedure and present several examples of its use. Under conditions favoring the association of ribosomal subunits, the formation of the 70 S complex may reach completion within a few seconds.

As previously reported, the 30 s ribosomal subunit can be reversibly converted from an active to an inactive form by relatively mild manipulations of the medium. The active form can bind Phe-tRNA non-enzymically in the cold in the presence of poly U; the inactive form cannot, even in a medium favourable for binding. The two forms are thought to differ in conformation. We have now found that only the active form of the 30 s subunit can bind the antibiotic dihydrostreptomycin in the cold. This binding is very rapid. If inactive ribosomes are incubated with dihydrostreptomycin at an elevated temperature in an activating medium, the antibiotic is bound, but much more slowly. Here, the rate limiting step is the conversion of the ribosome to the active form and not the binding of the antibiotic. Poly U markedly enhances the rate of ribosome activation, but has no effect on the rate of dihydrostreptomycin binding. A similar correlation between biological activity and antibiotic binding is seen with the 50 s subunit, which binds chloramphenicol, erythromycin and sparsomycin only when in an active form, i.e. when capable of catalysing peptide bond synthesis and the termination reaction of protein synthesis.