Ari Elson

Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell–rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9–dependent manner; however, RANKL did not induce mobilization in young female PTPε-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Ligand‐induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c‐Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c‐Cbl, here we report that defective Tyr1045 of EGFR, an inducible c‐Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand‐induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization‐defective mutant receptor imply that c‐Cbl‐mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand‐induced ubiquitylation, especially in the presence of an overexpressed c‐Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c‐Cbl complex to Grb2‐specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well‐characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c‐Cbl‐dependent sorting of active tyrosine kinases to destruction.

Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase.

ABSTRACT 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.

Hypomyelination and increased activity of voltage‐gated K + channels in mice lacking protein tyrosine phosphatase ϵ

Protein tyrosine phosphatase epsilon (PTPϵ) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTPϵ 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 α‐subunits in sciatic nerve tissue and in primary Schwann cells. PTPϵ markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate‐trapping mutant of PTPϵ, and PTPϵ profoundly reduces Src‐ or Fyn‐stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTPϵ antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.

Protein tyrosine phosphatases in health and disease

Protein tyrosine phosphatases (PTPs) represent a super‐family of enzymes that play essential roles in normal development and physiology. In this review, we will discuss the PTPs that have a causative role in hereditary diseases in humans. In addition, recent progress in the development and analysis of animal models expressing mutant PTPs will be presented. The impact of PTP signaling on health and disease will be exemplified for the fields of bone development, synaptogenesis and central nervous system diseases. Collectively, research on PTPs since the late 1980s yielded the cogent view that development of PTP‐directed therapeutic tools is essential to further combat human disease.

Protein tyrosine phosphatases: functional inferences from mouse models and human diseases

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.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha 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 cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon 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 PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Generation of novel cytoplasmic forms of protein tyrosine phosphatase epsilon by proteolytic processing and translational control

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.

Elevated Cu/Zn‐SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm‐deficient mice

Patients with the genetic disorder ataxia‐telangiectasia (A‐T) display a pleiotropic phenotype that includes neurodegeneration, immunodeficiency, cancer predisposition and hypersensitivity to ionizing radiation. The gene responsible is ATM, and Atm‐knockout mice recapitulate most features of A‐T. In order to study the involvement of oxidative stress in the A‐T phenotype, we examined mice deficient for Atm and overexpressing human Cu/Zn superoxide dismutase (SOD1). We report that elevated levels of SOD1 exacerbate specific features of the murine Atm‐ deficient phenotype, including abnormalities in hematopoiesis and radiosensitivity. The data are consistent with the possibility that oxidative stress contributes to some of the clinical features associated with the A‐T phenotype.

Dimerization In Vivo and Inhibition of the Nonreceptor Form of Protein Tyrosine Phosphatase Epsilon

ABSTRACT 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 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-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.