Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1.

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

Activation of mitogen-activated protein (MAP) kinase pathway by pervanadate, a potent inhibitor of tyrosine phosphatases.

Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in signal transduction. The regulatory role of protein-tyrosine phosphatases (PTPs) in this process was explored by studying the effects of a powerful PTP inhibitor, pervanadate, on the activation of the mitogen-activated protein (MAP) kinase cascade. Treatment of HeLa cells with pervanadate resulted in a marked inhibition of PTP activity, accompanied by a drastic increase in tyrosine phosphorylation of cellular proteins. The increased tyrosine phosphorylation coincided with the activation of the MAP kinase cascade as indicated by enzymatic activity assays of MEK (MAP kinase/ERK-kinase) and MAP kinase and gel mobility shift analyses of Raf-1 and MAP kinase. The activation was sustained but reversible. Upon removal of pervanadate, both tyrosine phosphorylation and MAP kinase activation declined to basal levels. Therefore, inhibition of PTP activity is sufficient per se to initiate a complete MAP kinase activation program.

Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Identification and purification of a chicken brain neuroglia-associated protein.

The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.

A general peptide substrate for protein tyrosine phosphatases.

The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a C18 cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecular-weight PTPs exhibited Km values between 0.5 and 1.3 microM, whereas the receptor forms CD45 and RPTP alpha gave values of 14 and 35 microM, respectively. All PTPs displayed similar properties toward the peptide including a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to 10 ng in presence of protein substrates.

Suppression of v-fms-induced transformation by overexpression of a truncated T-cell protein tyrosine phosphatase.

Rat 2 cells stably transformed by murine v-fms (pB5 cells) were infected with retroviruses containing a human cDNA encoding either a full-length human T-cell protein tyrosine phosphatase (TC.PTP) or a truncated form (delta C11.PTP) in which an 11-kDa carboxy-terminal extension had been removed. This segment is responsible for enzyme localization and regulation. Clonal cell lines were isolated following G418 selection and their transforming properties analysed; pB5 cells containing the vector alone or TC.PTP remained transformed. These cells grew readily in soft agar, formed tumors in nude mice and were morphologically indistinguishable from the parental pB5 cells. In contrast, cells expressing delta C11.PTP showed dramatic changes in cell morphology, loss of anchorage-independent growth in soft agar and reduced or lack of tumor formation in nude mice. Both increases and decreases in tyrosine phosphorylation of specific proteins in the cells overexpressing the truncated enzyme were detected. These results indicate that coexpression of the deregulated, soluble tyrosine phosphatase with a constitutively active, oncogenic receptor tyrosine kinase leads to the suppression of the transformed phenotype.

Purification and characterization of a protein tyrosine phosphatase containing SH2 domains.

A protein tyrosine phosphatase (PTP) containing two SH2 domains (PTP1C) was purified to near homogeneity from an adenovirus expression system by a two-step chromatographic procedure with a yield of 67%. The purified enzyme behaves as a monomer of 68 kDa on gel filtration and is totally specific for phosphotyrosyl residues. Its optimal pH is around neutrality for protein substrates such as reduced, carboxyamidomethylated, maleylated (RCM)-lysozyme and myelin basic protein but below 5 for low molecular weight compounds such as para-nitrophenyl phosphate (p-NPP) and phosphotyrosine. Furthermore, with the protein substrates, it displays an activity less than 1% of that obtained with other known PTPs but comparable activities toward p-NPP and phosphotyrosine. Its responsiveness toward the usual PTP activators (e.g. spermine) or inhibitors (e.g. vanadate, molybdate, heparin, or Zn2+) varied considerably with the nature of the substrates involved. Limited digestion with trypsin caused the cleavage of a C-terminal segment of the enzyme, giving rise to a 63-kDa fragment; this cleavage resulted in an approximately 20- and 10-fold activation of the enzyme toward RCM-lysozyme and myelin basic protein, respectively.

Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase.

Epidermal growth factor stimulates the activity of several cytosolic serine/threonine protein kinases in quiescent Swiss 3T3 cells. Two of these, which use myelin basic protein (MBP) as substrate, act as kinase kinases in that they are able to activate a separate peptide kinase activity in vitro by a mechanism involving protein phosphorylation. In this study, we have identified two activities from extracts of epidermal growth factor-treated cells that stimulate an ATP-dependent activation of both of the MBP kinases, derived in their inactive precursor forms from extracts of untreated cells. The resulting MBP kinase activities are stable to further purification and can be inactivated with either tyrosine or serine/threonine protein phosphatases and then reactivated to their original levels of activity. Thus, we propose that the in vitro activation involves protein phosphorylation, stimulated by the action of novel MBP kinase activating factors that represent intermediate components in a growth factor-stimulated kinase cascade.

Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes.

A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.

CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity.

Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.

Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

Human placenta protein-tyrosine-phosphatase: amino acid sequence and relationship to a family of receptor-like proteins.

The amino acid sequence of the cytosolic human placenta protein-tyrosine-phosphatase 1B (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been determined. It consists of a single chain of 321 residues with an N-acetylated N-terminal methionine and an unusually proline-rich C-terminal region. The enzyme is structurally related to the two cytoplasmic domains of both the leukocyte common antigen CD45 and LAR, a CD45-like molecule with an external segment that resembles a neural cell adhesion molecule. A low molecular weight protein encoded by a cDNA clone from T cells also shows extensive sequence similarities. The present study defines homologous domains common to this diverse family of PTPases that includes both soluble and receptor-like transmembrane forms. The cysteinyl residues 121 and 215 of PTPase 1B are conserved among all members of the family and are candidates for involvement in catalysis since PTPase 1B is inactivated by thiol modifying reagents. Two segments rich in positively charged residues (residues 33-47 and 227-238) may provide sites of interaction with inhibitory anionic polymers such as heparin or poly(Glu/Tyr).

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.

Purification of the major protein-tyrosine-phosphatases of human placenta.

This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.

Characterization of the major protein-tyrosine-phosphatases of human placenta.

In the preceding article (Tonks, N. K., Diltz, C. D., and Fischer, E. H. (1988) J. Biol. Chem. 263, 6722-6730), the purification of the major protein-tyrosine-phosphatases from human placenta, some to apparent homogeneity, was described. This report compares the characteristics of these enzymes and clearly identifies at least two distinct protein-tyrosine-phosphatase catalytic subunits. All were absolutely specific for phosphotyrosyl residues and showed no activity on any of the phosphoseryl/phosphothreonyl-containing proteins tested; they exhibited a high affinity for substrate with Km values in the submicromolar range. All were absolutely dependent on sulfhydryl compounds and appeared to contain at least one highly reactive cysteinyl residue essential for activity. Subtypes 1A and 1B could be distinguished by their response to polyanionic and polycationic compounds. The 1B enzymes were activated by EDTA, spermine, spermidine, and myelin basic protein to a greater extent than the 1A subtypes. Furthermore, they were inhibited by approximately 2 orders of magnitude lower concentrations of heparin (IC50 approximately 20 nM) and 1:1 or 4:1 poly (glutamate/tyrosine) (IC50 approximately 50 nM) than the 1A subtypes. Surprisingly, inhibition by the glutamate/tyrosine copolymers was strictly noncompetitive. Peptide mapping following digestion with Achromobacter protease I or Staphylococcus aureus V8 protease supported the view that, whereas protein-tyrosine-phosphatase subtypes 1A and 1B are different, their soluble and particulate counterparts are closely related structurally and are distinct from serine/threonine phosphatases 1 and 2A.