Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.     

Regulation of NMDA receptors by the tyrosine kinase Fyn

The phosphorylation and trafficking of N-methyl-d-aspartate (NMDA) receptors are tightly regulated by the Src family tyrosine kinase Fyn, through dynamic interactions with various scaffolding proteins in the NMDA receptor complex. Fyn acts as a point of convergence for many signaling pathways that upregulate GluN2B-containing NMDA receptors. In the following review, we focus on Fyn signaling downstream of different G-protein-coupled receptors: the dopamine D1 receptor, and receptors cognate to the pituitary adenylate cyclase-activating polypeptide. The net result of activation of each of these signaling pathways is upregulation of GluN2B-containing NMDA receptors. The NMDA receptor is a major target of ethanol in the brain, and accumulating evidence suggests that Fyn mediates the effects of ethanol by regulating the phosphorylation of GluN2B NMDA receptor subunits. Furthermore, Fyn has been shown to regulate alcohol withdrawal and acute tolerance to ethanol through a GluN2B-dependent mechanism. In addition to its effects on NMDA receptor function, Fyn also modifies the threshold for synaptic plasticity at CA1 synapses, an effect that probably contributes to the effects of Fyn on spatial and contextual fear learning.     

Discovery of novel inhibitor of human leukocyte common antigen-related phosphatase

Human leukocyte common antigen-related phosphatase (LAR) may play a role in type 2 diabetes and cancer, and in the development of the nervous system, and it may be an attractive target for the treatment of diabetes and cancer. We identified eight hits from the random screening of LAR D1 with a high-throughput screening assay. Further validation of the eight hits showed that the meD insertion was associated with inhibition of LAR D1D2 and LAR D1Q. These data suggest that the inserted meD peptide influences the interaction of the enzyme and inhibitor, which is consistent with the kinetic catalysis constants of the substrate pNPP. Our data showed that Hit 1, the first published novel inhibitor of LAR, is a competitive inhibitor with a K(i) of 330 nM that displays obvious selectivity for LAR and mouse PTPsigma, but not for other protein tyrosine phosphatases.     

Purification and characterization of the catalytic domains of the human receptor-linked protein tyrosine phosphatases HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR).

Human HPTP beta, leukocyte common antigen (LCA), and leukocyte common antigen-related molecule (LAR) are transmembrane receptor-like proteins whose cytoplasmic regions contain either one (HPTP beta) or two (LCA and LAR) domains that are homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain 1 has enzymatic activity, the membrane-distal domain 2 of both LCA and LAR has no detectable catalytic activity. The cytoplasmic regions of HPTP beta, LCA, and LAR were expressed in Escherichia coli and purified to greater than 90% purity. Modulatory effects of various low molecular weight compounds and homo- and copolymers of amino acids were examined. Several polypeptides that contain a high proportion of tyrosine were strongly inhibitory to these PTPases. To determine a possible role for the LAR domain 2, the properties of recombinant LAR PTPases containing both domains 1 and 2 (LAR-D1D2) or only domain 1 (LAR-D1) were compared. In nearly all aspects examined, LAR-D1 and LAR-D1D2 were indistinguishable. However, polycationic polypeptides strongly stimulated the PTPase activity of LAR-D1D2, but not LAR-D1, using the peptide substrate Raytide. Thus, basic polypeptides seem to indirectly alter the catalytic activity of domain 1 by interacting with domain 2. This result suggests that domain 2 has a regulatory function.     

Specific dephosphorylation of the Lck tyrosine protein kinase at Tyr-394 by the SHP-1 protein-tyrosine phosphatase

The protein-tyrosine phosphatase SHP-1 has been shown to be a negative regulator of multiple signaling pathways in hematopoietic cells. In this study, we demonstrate that SHP-1 dephosphorylates the lymphoid-specific Src family kinase Lck at Tyr-394 when both are transiently co-expressed in nonlymphoid cells. We also demonstrate that a GST-SHP-1 fusion protein specifically dephosphorylates Lck at Tyr-394 in vitro. Because phosphorylation of Tyr-394 activates Lck, the fact that SHP-1 specifically dephosphorylates this site suggests that SHP-1 is a negative regulator of Lck. The failure of SHP-1 to inactivate Lck may contribute to some of the lymphoid abnormalities observed in motheaten mice.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6- phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti- phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.     

Regulation of expression and function of Lck tyrosine kinase by high cell density

For many types of cells, an increase in cell density leads to characteristic changes in intracellular signalling and cell function. It is unknown, however, whether cell density affects the function of T lymphocytes. It is presented here that aggregation of Jurkat T cells, murine thymocytes or human peripheral blood T cells, results in gradual modification of the Lck tyrosine kinase. Within one hour of aggregation, Lck in the detergent-insoluble lipid raft fraction is dephosphorylated mainly at the carboxy-terminal tyrosine. Further aggregation leads to gradual loss of Lck protein from both lipid raft and non-raft fractions which is accompanied by increased protein ubiquitination, a process that is more evident in the detergent-soluble fraction. In contrast, the expression of LAT, which like Lck distributes to raft and non-raft membrane, or Csk, a kinase with a structure similar to Lck, is not affected by cell aggregation. Dephosphorylation of lipid raft-associated Lck, albeit with reduced kinetics, is observed in aggregated Jurkat CD45-deficient cells as well, suggesting involvement of additional tyrosine phosphatases. Changes in Lck structure and expression correlate with reduced ability of aggregated cells to fully activate protein tyrosine phosphorylation after stimulation of the TCR, and with changes in the activation of down-stream signalling cascades.     

Regulation of integrin-mediated T cell adhesion by the transmembrane protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin alpha5beta1 (VLA-5), but not alpha4beta1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.     

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.     

Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase

It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.     

Direct suppression of TCR-mediated activation of extracellular signal-regulated kinase by leukocyte protein tyrosine phosphatase, a tyrosine-specific phosphatase.

Leukocyte protein tyrosine phosphatase (LC-PTP)/hemopoietic PTP is a human cytoplasmic PTP that is predominantly expressed in the hemopoietic cells. Recently, it was reported that hemopoietic PTP inhibited TCR-mediated signal transduction. However, the precise mechanism of the inhibition was not identified. Here we report that extracellular signal-regulated kinase (ERK) is the direct target of LC-PTP. LC-PTP dephosphorylated ERK2 in vitro. Expression of wild-type LC-PTP in 293T cells suppressed the phosphorylation of ERK2 by a mutant MEK1, which was constitutively active regardless of upstream activation signals. No suppression of the phosphorylation was observed by LC-PTPCS, a catalytically inactive mutant. In Jurkat cells, LC-PTP suppressed the ERK and p38 mitogen-activated protein kinase cascades. LC-PTP and LC-PTPCS made complexes with ERK1, ERK2, and p38alpha, but not with the gain-of-function sevenmaker ERK2 mutant (D321N). A small deletion (aa 1-46) in the N-terminal portion of LC-PTP or Arg to Ala substitutions at aa 41 and 42 resulted in the loss of ERK binding activity. These LC-PTP mutants revealed little inhibition of the ERK cascade activated by TCR cross-linking. On the other hand, the wild-type LC-PTP did not suppress the phosphorylation of sevenmaker ERK2 mutant. Thus, the complex formation of LC-PTP with ERK is the essential mechanism for the suppression. Taken collectively, these results indicate that LC-PTP suppresses mitogen-activated protein kinase directly in vivo.     

Leukocyte common antigen-related (LAR) tyrosine phosphatase positively regulates osteoblast differentiation by modulating extracellular signal-regulated kinase (ERK) activation

Protein tyrosine phosphatases (PTPs) are pivotal regulators of key cellular functions, including cell growth, differentiation, and adhesion. Previously, we reported that leukocyte common antigen-related (LAR) tyrosine phosphatase promotes osteoblast differentiation in MC3T3-E1 preosteoblast cells. In the present study, the mechanism of the regulatory action of LAR on osteoblast differentiation was investigated. The mineralization of extracellular matrix and calcium accumulation in MC3T3-E1 cells were markedly enhanced by LAR overexpression, and these effects were further increased by treatment with a MEK inhibitor. In addition, LAR overexpression dramatically reduced extracellular signal-regulated kinase (Erk) activation during osteoblast differentiation. In contrast, a marginal effect of the inactive LAR mutant on Erk activation was detected. Expression of osteoblast-related genes such as ALP, BSP, DLX5, OCN, and RUNX2, was increased by LAR overexpression during osteoblast differentiation. On the basis of these results, we propose that LAR functions as a positive regulator of osteoblast differentiation by modulating ERK activation. Therefore, LAR phosphatase could be used as a novel regulatory target protein in many bone-associated diseases, including osteoporosis.     

Regulation of protein tyrosine phosphatase 1B by sumoylation

Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.     

Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases

Signaling through receptor tyrosine kinases (RTKs) is a major mechanism for intercellular communication during development and in the adult organism, as well as in disease-associated processes. The phosphorylation status and signaling activity of RTKs is determined not only by the kinase activity of the RTK but also by the activities of protein tyrosine phosphatases (PTPs). This review discusses recently identified PTPs that negatively regulate various RTKs and the role of PTP inhibition in ligand-induced RTK activation. The contributions of PTPs to ligand-independent RTK activation and to RTK inactivation by other classes of receptors are also surveyed. Continued investigation into the involvement of PTPs in RTK regulation is likely to unravel previously unrecognized layers of RTK control and to suggest novel strategies for interference with disease-associated RTK signaling.     

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.     

Small molecule approach to studying protein tyrosine phosphatase

Understanding the function of protein tyrosine phosphatases (PTPs) is crucial to deciphering cellular signaling in higher organisms. Of the 100 putative PTPs in human genome, only a little is known about their precise biological functions. Thus establishing novel ways to study PTP function remains a top priority among researchers. Classical genetics and more recently the use of RNA interference (RNAi) for gene silencing remains a popular choice to study function. However, the one gene-one function hypothesis is now recognized as an oversimplified scenario, especially among the signaling proteins such as PTPs. Therefore, there is a need to understand gene function in an appropriate cellular context. Since proteins are the work horses of the cell, alteration of protein function by various means is a particularly attractive strategy. In this context, the chemical approach, where a small molecule is used to affect the function of the desired protein is increasingly being recognized as a method of choice. In this review, we describe how small molecules can be used to study the function of a prototypical PTP, PTP1B, which is a negative regulator in insulin signaling. This includes our initial strategies for finding the most potent and specific PTP1B inhibitor to date, synthesizing cell permeable analogues suitable for cellular studies, and using them to dissect the role of PTP1B in the insulin signaling pathway. This approach is potentially general and thus could be utilized to study the function of other PTPs.     

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

Inhibition of phosphorylase kinase, and tyrosine protein kinase activities by quercetin

Quercetin, a naturally occurring bioflavonoid inhibited the activities of phosphorylase kinase and a partially purified tyrosine protein kinase from rat lung. The inhibition was rapid and concentration dependent. Quercetin at 100 microM inhibited the activities of phosphorylase kinase and tyrosine protein kinase by about 95 and 80-90 percent respectively. ATP reversed the quercetin mediated inhibition of tyrosine protein kinase but not of phosphorylase kinase. These data suggest that quercetin has differential effect on different protein kinase activities and it may be used as a tool to probe the role of various protein kinases in cell function.