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.

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTP⑀ (cyt-PTP⑀) down-regulates channel activity and counters its phosphorylation and upregulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTP⑀.

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...

Tyrosine phosphatases (PTPs) and ␣ 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 and PTP␣ 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␣ inhibits Kv channels more strongly than PTP; this correlates with constitutive association of PTP␣ with Kv2.1, driven by membranal localization of PTP␣. PTP␣, but not PTP, 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 and PTP␣ 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. INTRODUCTIONReversible phosphorylation of tyrosine residues in proteins is a major mechanism for regulation of protein structure and function. Phosphorylation is regulated by the opposing activities of two superfamilies of enzymes-the protein tyrosine kinases (PTKs) and the protein tyrosine phosphatases (PTPs). More than 100 PTP genes are known in higher organisms, of which 38 belong to the classical, tyrosinespecific PTP family (Alonso et al., 2004;Andersen et al., 2004). The number of tyrosine phosphorylation sites in target proteins is significantly larger than the numbers of known PTPs or PTKs (Manning et al., 2002;Alonso et al., 2004). Each enzyme therefore interacts with several substrates, and a given substrate may be targeted by more than one kinase or phosphatase. Consequently, it is not always clear whether these enzymes fulfill molecular and physiological roles that are entirely independent or that overlap at least in part.Molecular and physiological studies of specific PTPs in recent years indicate that the issue of functional independence versus redundancy is complex. In many cases, specific substrates are currently known to be targeted by an individual PTP; in other cases, however, substrate sharing clearly occurs. A prime example of this is Src, which can be activated by dephosphorylation of its C-terminal inhibitory tyrosine by several PTPs, including PTP1B, SHP1, PTP␣, and PTP (Somani et al., 1997;Ponniah et al., 1999;Su et al., 1999;Bjorge et al., 2000;Gil-Henn and Elson, 2003;Pallen, 2003). Yet, targeting of a specific substrate by several PTPs may not necessarily imply that these PTPs are fully redundant in vi...

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helixhelix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNF␣, IL-1␤, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNF␣, IL-6, and IFN␥ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.cancer ͉ contact map prediction ͉ cytokines ͉ inflammation ͉ helix C onformational changes in proteins have an essential role in regulating activity. Natural and synthetic molecules that modulate such changes are of considerable biological importance. Such molecules include allosteric effectors that alter the rate of enzymecatalyzed reactions (1), molecules that shift the oligomerization equilibrium of proteins (2), and molecules that interfere with transmembrane helix-helix associations (3).Conformational changes that have been extensively studied are those that take place during viral-induced membrane fusion. This process is required for the propagation of enveloped viruses, and is facilitated by viral encoded type 1 integral membrane proteins (4, 5). Viral entry of enveloped viruses depends on a conformational change involving the formation of helix-helix interactions, i.e., 2 alpha helices that do not interact in the native (nonfusogenic) state, but do interact with each other when the protein folds into its fusion-active (fusogenic) form. Remarkably, synthetic peptides corresponding to some of these helical segments have an antiviral activity (6 -8), of which one has been developed for the treatment of HIV-1 infection (9, 10).We hypothesized that such a mode of inhibition could be also applied on nonviral proteins. The aim of the present study was to develop a computational tool for the detection of intramolecular helix-helix interactions and to use this tool for detecting such interacting helices in proteins of interest. This study focuses on secreted chaperones due to their biological and therapeutic relevance (11-14), and because conformational changes are known to take p...