Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPepsilon) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPepsilon-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPepsilon adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPepsilon-deficient osteoclasts, indicating that loss of PTPepsilon does not cause widespread disruption of these signaling pathways. These results establish PTPepsilon as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.

The nonreceptor isoform of tyrosine phosphatase epsilon (cyt-PTPe) supports osteoclast adhesion and activity in vivo, leading to increased bone mass in female mice lacking PTPe (EKO mice). The structure and organization of the podosomal adhesion structures of EKO osteoclasts are abnormal; the molecular mechanism behind this is unknown. We show here that EKO podosomes are disorganized, unusually stable, and reorganize poorly in response to physical contact. Phosphorylation and activities of Src, Pyk2, and Rac are decreased and Rho activity is increased in EKO osteoclasts, suggesting that integrin signaling is defective in these cells. Integrin activation regulates cyt-PTPe by inducing Src-dependent phosphorylation of cyt-PTPe at Y638. This phosphorylation event is crucial because wild-type-but not Y638F-cyt-PTPe binds and further activates Src and restores normal stability to podosomes in EKO osteoclasts. Increasing Src activity or inhibiting Rho or its downstream effector Rho kinase in EKO osteoclasts rescues their podosomal stability phenotype, indicating that cyt-PTPe affects podosome stability by functioning upstream of these molecules. We conclude that cyt-PTPe participates in a feedback loop that ensures proper Src activation downstream of integrins, thus linking integrin signaling with Src activation and accurate organization and stability of podosomes in osteoclasts. INTRODUCTIONOsteoclasts are large multinucleated cells of hematopoietic origin that degrade bone matrix. To perform this function osteoclasts must adhere firmly to bone using specialized adhesion structures called podosomes (Geiger et al., 2001;Gimona and Buccione, 2006;Linder, 2007;Teitelbaum, 2007). Podosomes are punctate structures that contain an actin-rich core surrounded by a ring of associated proteins, which convey the signal generated by contact with matrix to the actin core and the actin cytoskeleton. Proteins present in the podosomal ring include integrins and associated molecules such as vinculin, paxillin, Cbl, Cas, Src, Pyk2, and the small GTPases Rho, Rac, and CDC42 (Linder and Aepfelbacher, 2003;Gimona, 2008).In addition to osteoclasts podosomes are found in, among other cell types, macrophages, dendritic cells, and in endothelial and epithelial cells (Linder, 2007;Gimona et al., 2008). In osteoclasts, however, the organization of podosomes is linked to the ability of the cell to fulfill its physiological role.In cultured osteoclasts that are not actively resorbing bone, podosomes are scattered at random. Podosomes can assemble into clusters that grow and transform into dynamic rings, which further expand to form a large superstructure at the cell periphery that is characteristic of mature, boneresorbing cells. In osteoclasts grown on degradable matrix, this peripheral, belt-like superstructure, referred to as the sealing zone, contains densely packed podosomes that are usually not individually discernible. In osteoclasts grown on nondegradable surface, podosomes are arranged in a lesscrowded sealing zone-like structure (SZL)...

cyt-PTP is a naturally occurring nonreceptor form of the receptor-type protein tyrosine phosphatase (PTP) epsilon. As such, cyt-PTP 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 (RPTP␣) and CD45. Using immunoprecipitation and gel filtration, we show that cyt-PTP forms dimers and higher-order associations in vivo, the first such demonstration among nonreceptor phosphatases. Although cyt-PTP readily dimerizes in the absence of exogenous stabilization, dimerization is increased by oxidative stress. Epidermal growth factor receptor stimulation can affect cyt-PTP dimerization and tyrosine phosphorylation in either direction, suggesting that cell surface receptors can relay extracellular signals to cyt-PTP, which lacks extracellular domains of its own. The inactive, membrane-distal (D2) phosphatase domain of cyt-PTP is a major contributor to intermolecular binding and strongly interacts in a homotypic manner; the presence of D2 and the interactions that it mediates inhibit cyt-PTP activity. Intermolecular binding is inhibited by the extreme C and N termini of D2. cyt-PTP lacking these regions constitutively dimerizes, and its activities in vitro towards para-nitrophenylphosphate and in vivo towards the Kv2.1 potassium channel are markedly reduced. We conclude that physiological signals can regulate dimerization and phosphorylation of cyt-PTP 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.Phosphorylation of tyrosine residues in proteins is a central mechanism for protein regulation and is well established as a master regulator of physiological processes. Tyrosine phosphorylation is reversible and is controlled by the generically opposing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs) (25). Receptor-type PTPs (RPTPs), which are a major structural subfamily of the PTP superfamily, are integral membrane proteins which possess extracellular domains of various lengths and typically two cytosolic phosphatase domains (2,12,49). Although their importance in regulating biological processes is well established, relatively little is known about how activities of RPTPs are regulated.Extracellular molecules have been suggested to bind RPTPs and to influence PTP activity or function, much as binding by pleiotrophin inhibits activity of PTP␤/ (36). However, little is known about how such binding affects signal transduction across cell membranes or RPTP activity (5, 41, 42, 53). Structural studies have suggested that ligand-induced dimerization inhibits activity of RPTPs by stabilizing homodimeric structures, in which the helix-turn-helix "wedge" domain of one molecule prevents access of substrates to the catalytic domain of its dimerization partner (6,34,51). Dimerizati...

Protein tyrosine phosphatases (PTPs) are key mediators that link physiological cues with reversible changes in protein structure and function; nevertheless, significant details concerning their regulation in vivo remain unknown. We demonstrate that PTP 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 PTP to tubulin in vitro and further reduce phosphatase activity. Conversely, disruption of microtubule structures in cells reduces their association with PTP, alters the subcellular localization of the phosphatase, and increases its specific activity. Activation of the epidermal growth factor receptor (EGFR) increases the PTP-microtubule association in a manner dependent upon EGFR-induced phosphorylation of PTP at Y638 and upon microtubule integrity. These events are transient and occur with rapid kinetics similar to EGFR autophosphorylation, suggesting that activation of the EGFR transiently down-regulates PTP activity near the receptor by promoting the PTP-microtubule association. Tubulin also inhibits the tyrosine phosphatase PTP1B but not receptor-type PTP 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.Reversible phosphorylation of tyrosine residues in proteins is a major regulator of protein structure and function. Tyrosine phosphorylation of proteins is regulated in part by the activity of members of the protein tyrosine phosphatase (PTP) superfamily, which currently includes over 100 genes in higher organisms. Of these, 38 genes encode "classical" tyrosine phosphatases (PTPs), which are strictly specific for phosphotyrosine. Products of this gene family all contain one or two copies of the PTP domain and are either receptor-type integral membrane proteins or non-receptor-type proteins (1, 3).The numbers of known tyrosine kinases and tyrosine phosphatases are similar and small compared to the numbers and complexities of their potential substrates (1). The apparent contradiction between this and the high degree of specificity that exists in signaling processes in vivo has made understanding the mechanisms by which PTP activity is regulated of paramount importance. PTP genes often produce several distinct protein products by alternative splicing or use of alternative promoters (3). At the protein level, PTP activity can be inhibited by dimerization (e.g., see references 22, 23, and 43) or by reversible oxidation of the key cysteine residue in the PTP catalytic domain (9, 44). Proteolysis and phosphorylation, which can affect subcellular localization, conformation, or the ability to bind other proteins can also activate or inhibit PTPs (e.g., see references 6, 13, 17, 48, and 49). Regulation of PTP activity by binding of extracellular ligands to receptor-type PTPs (RPTPs), which inhibits PTP activity...

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