Protein Tyrosine Phosphatase Domain Research Articles

Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand.

SHP-1, a protein tyrosine phosphatase containing two tandem SH2 domains, is autoinhibited at rest by its N-terminal SH2 (N-SH2) domain. Relief from autoinhibition and a subsequent 10-60-fold increase in V(max) have been observed upon N-SH2 domain engagement by a specific phosphotyrosyl ligand or upon deletion of the SH2 domains to yield the catalytic PTPase domain. In this study, we observed that glycerol and propane-1,2-diols, at concentrations of 4-6 M, accelerated the k(cat) of the full length enzyme by 47-fold and of the PTPase domain by 8-fold. Glycerol also increases the rate of proteolytic cleavage between the SH2 and catalytic PTPase domains. In stopped-flow studies using p-nitrophenyl phosphate (pNPP) as a substrate, a burst of p-nitrophenolate in the full length enzyme was not observed; however, a 50-70% stoichiometric burst was observed with the PTPase domain. Rapid quench studies using [32P]pNPP as a substrate showed a very low level of covalent [32P]phosphocysteinyl enzyme intermediate accumulation: 0.06% in the full length enzyme and 1% in the PTP domain. Stimulation by glycerol reduced the accumulating levels of phosphocysteinyl enzyme in both cases of full length SHP-1 and the PTPase domain; however, glycerol is not acting as a cosubstrate since no glycerophosphate product was detectable. It is likely that, for full length SHP-1, with pNPP as a model substrate, enzyme-substrate complex (ES) accumulates in its basal autoinhibited state, whereas enzyme-product complex (EP(i)) accumulates in its pY ligand-bound activated state. Glycerol probably relaxes the compact structure of SHP-1 and the PTP domain, thereby accelerating the catalytic rates in both cases by increasing forward reaction rates of ES and EP(i).

Comparative Kinetic Analysis and Substrate Specificity of the Tandem Catalytic Domains of the Receptor-like Protein-tyrosine Phosphatase α

The catalytic activity and substrate specificity of protein-tyrosine phosphatase alpha (PTPalpha) is primarily controlled by the membrane proximal catalytic domain (D1). The membrane distal (D2) domain of PTPalpha by itself is a genuine PTPase, possessing catalytic activity comparable to that of D1 using aryl phosphates as substrates. Surprisingly, kcat and kcat/Km for the D2-catalyzed hydrolysis of phosphotyrosine-containing peptides are several orders of magnitude reduced in comparison with those of D1. Substitution of the putative general acid/base Glu-690 in D2 by an Asp, which is invariably found in the WPD motifs in all cytoplasmic PTPases and all the D1 domains of receptor-like PTPases, only increases the kcat for D2 by 4-fold. Thus the much reduced D2 activity toward peptide substrates may be due to structural differences in the active sites other than the general acid/base. Alternatively, the D2 domain may have a functional active site with a highly stringent substrate specificity. PTPalpha display modest peptide substrate selectivity and are sensitive to charges adjacent to phosphotyrosine. In the sequence context of DADEpYLIPQQG (where pY stands for phosphotyrosine), the minimal sizes recognized by PTPalpha are either ADEpYLI or DADEpY-NH2.

Both SH2 Domains Are Involved in Interaction of SHP-1 with the Epidermal Growth Factor Receptor but Cannot Confer Receptor-directed Activity to SHP-1/SHP-2 Chimera

The previously demonstrated functional and physical interaction of the SH2 domain protein-tyrosine phosphatase SHP-1 with the epidermal growth factor (EGF) receptor (Tomic, S., Greiser, U., Lammers, R., Kharitonenkov, A., Imyanitov, E., Ullrich, A., and Böhmer, F. D. (1995) J. Biol. Chem. 270, 21277-21284) was investigated with respect to the involved structural elements of SHP-1. Various mutants of SHP-1 were transiently expressed in 293 or COS-7 cells and analyzed for their capacity to associate with immobilized autophosphorylated EGF receptor in vitro and to dephosphorylate coexpressed EGF receptor in intact cells. Inactivating point mutation of the C-terminal SH2 domain reduced the association weakly, point mutation of the N-terminal SH2 domain reduced association strongly and the respective double mutation abolished association totally. The capacity of SHP-1 to dephosphorylate coexpressed EGF receptor was impaired by all point mutations. Truncation of the N-terminal or of both SH2 domains strongly reduced or abolished association, respectively, but the truncated SHP-1 derivatives still dephosphorylated coexpressed EGF receptor effectively. Various chimeric protein-tyrosine phosphatases constructed from SHP-1 and the closely homologous SHP-2 dephosphorylated the EGF receptor when they contained the catalytic domain of SHP-1. As native SHP-2, the chimera lacked activity toward the receptor when they contained the catalytic domain of SHP-2, despite their capacity to associate with the receptor and to dephosphorylate an artificial phosphopeptide. We conclude that the differential interaction of SHP-1 and SHP-2 with the EGF receptor is due to the specificity of the respective catalytic domains rather than to the specificity of the SH2 domains. Functional interaction of native SHP-1 with the EGF receptor requires association mediated by both SH2 domains.

Cloning and Characterization of Fetal Liver Phosphatase 1, a Nuclear Protein Tyrosine Phosphatase Isolated From Hematopoietic Stem Cells

We report the isolation of cDNAs encoding protein tyrosine phosphatases (PTPs) from highly purified hematopoietic stem cell populations. One such cDNA encodes a novel PTP, designated fetal liver phosphatase 1 (FLP1), which consists of one PTP domain followed by a carboxy terminal domain of 160 amino acids. Northern blot and in situ hybridization analysis showed that expression of FLP1 mRNA is restricted to thymus in 15.5-day-old and 17.5-day-old mouse embryos and to kidney and hematopoietic tissues in adult mice. Furthermore, polymerase chain reaction-based analysis shows that FLP1 is expressed in hematopoietic stem cells as well as in more mature hematopoietic cells. Peptide antisera against FLP1 immunoprecipitated a 48-kD protein that is localized in the nuclei of Ba/F3 lymphoid cells. We have analyzed the effects of overexpressing either wild-type FLP1 or a functionally inactive mutant of FLP1 in hematopoietic cells. In the progenitor K562 cell line, cells ectopically expressing functional FLP1 differentiated normally to megakaryocytes after induction with tetradecanoyl phorbol acetate (TPA). In contrast, when K562 transfectants expressing an inactive mutant FLP1 protein were treated with TPA, the characteristic cell spreading and substrate adhesion that accompany megakaryocytic differentiation did not occur. We show that, in these cells, the induction of the differentiation marker alphaIIb beta3 is not affected. However, both constitutive and TPA-induced expression of alpha2 integrin, a late megakaryocytic marker, are inhibited. These results suggest that the expression of an inactive form of FLP1 affects late signaling events of K562 megakaryocytic differentiation.

The N-terminal domains of tensin and auxilin are phosphatase homologues.

Tensin, an actin filament capping protein, and auxilin, a component of receptor-mediated endocytosis, are known to have 350 residue regions of significant sequence similarity near their N-termini (Schröder et al., 1995, Eur J Biochem 228:297-304). Here we demonstrate that these regions are homologous, not only to each other, but also to the catalytic domain of a putative protein tyrosine phosphatase (PTP) from Saccharomyces cerevisiae and to other PTPs. We propose that the PTP-like portion of the homology region of tensin and auxilin represents a distinct domain. A detailed sequence comparison indicates that the PTP-like domain in tensin is unlikely to exhibit phosphatase activity, whereas in auxilin it may possess a different phosphatase specificity from tyrosine phosphatases. It is probable that the PTP-like domains in tensin and auxilin mediate binding interactions with phosphorylated polypeptides; they may therefore represent members of a distinct class of phosphopeptide recognition domain.

Cloning and Characterization of Islet Cell Antigen-related Protein-tyrosine Phosphatase (PTP), a Novel Receptor-like PTP and Autoantigen in Insulin-dependent Diabetes

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium.

A number of bacterial pathogens have evolved sophisticated strategies to subvert host-cell signal-transduction pathways for their own benefit. These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells. In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence. Sequence analysis revealed that a carboxy-terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases. Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine. An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases. In addition, an amino-terminal region of SptP exhibited sequence similarity to the ribosyltransferase exoenzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp. The modular nature of this effector protein may allow multiple interactions with host-cell signalling functions.

Molecular Cloning of Phogrin, a Protein-tyrosine Phosphatase Homologue Localized to Insulin Secretory Granule Membranes

An insulin granule membrane protein-tyrosine phosphatase (PTP) homologue, phogrin, was cloned by expression screening of a rat insulinoma cDNA library. The 3723-base pair cDNA encoded a transmembrane glycoprotein of 1004 amino acids (Mr 111876) that underwent post-translational proteolysis to 60-64-kDa products after a 30-min delay. The kinetics of proteolytic conversion (t1/2 = 45 min) and turnover (t1/2 = 12 h) were consistent with sorting and conversion in a late compartment of the secretory pathway. Studies on the native beta-cell protein suggested that the COOH-terminal PTP domain was on the cytosolic face of the secretory granule. The lumenal segment was comprised of a protease-resistant globular domain of around 25 kDa. Its localization and topology is thus consistent with a transmembrane receptor function related to granule biogenesis, exocytosis, or subsequent membrane recovery, and it should prove to be a useful cell biological marker for the granule membrane. High expression of the mRNA (5.4 kilobases) and protein was evident in islets, pancreatic alpha- and beta-cell tumor lines, brain cells, and other cells of neuroendocrine lineage. It is closely related to the diabetic autoantigen ICA512 (IA-2) (42% identity overall; 80% in the 260-amino acid PTP domain) and thus a potential target of autoimmunity in diabetes mellitus.

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.

Multiple Roles of the Novel Protein Tyrosine Phosphatase PTP3 during Dictyostelium Growth and Development

PTP3, the third nonreceptor protein tyrosine phosphatase identified in Dictyostelium discoideum, has a single catalytic protein tyrosine phosphatase domain. Recombinant PTP3 exhibited phosphatase activity that was inhibited by vanadate. PTP3 is expressed at a moderate level during growth. The level of transcripts increased between growth and 8 h of development and declined thereafter. Expression of lacZ under the control of the PTP3 promoter indicated a spatial localization of PTP3 in the anterior-like and prestalk cell types. There are two copies of the PTP3 gene in this haploid organism. Disruption of one copy led to a slow-growth phenotype. We were unable to obtain a strain with disruptions in both PTP3 genes. Overexpression of wild-type PTP3 led to slower growth rates and the formation of large aggregation streams. These streams split into smaller aggregates, many of which then arrested in development. Overexpression of a catalytically inactive mutation (Cys to Ser) had no effect on growth rate; however, this strain also formed large aggregation streams that later split up into large and small mound structures and became fruiting bodies of various sizes. Antiphosphotyrosine Western blot (immunoblot) analysis of total cell proteins showed that the pattern of protein tyrosine phosphorylation was specifically altered in PTP3 mutants. Addition of growth medium to starving cells and a subsequent replacement with nonnutrient buffer led to reciprocal changes in the pattern of several phosphotyrosine proteins, including a protein of approximately 130 kDa. Analysis of strains overexpressing active or inactive PTP3 suggested that p130 is a potential substrate of PTP3. A transient posttranslational phosphorylation of PTP3 further supported the role of PTP3 in these processes. The data obtained strongly suggest new regulatory functions for PTP3 that are distinct from those described earlier for D. discoideum PTP1 and PTP2.

Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family.

Cleavage and subsequent release of the extracellular domains of receptor protein tyrosine phosphatases (RPTP) occur at high cell density and may have an important role in regulating their activity. Because cleavage of RPTP occurs at a target motif (RXK/RR) recognized by a family of subtilisin/kexin-like endoproteases, we postulated that members of the subtilisin family may have an important role in this cleavage. We show in this report that the membrane-associated RPTPmu--both in its full 200-kDa form and as a 100-kDa cleavage product--is upregulated 4- and 7-fold, respectively, as human umbilical vein endothelial cells (HUVEC) approach confluence. To determine whether RPTPmu cleavage depended on PC5 (a subtilisin/kexin like endoprotease present in endothelial cells), we transfected COS cells with expression plasmids coding for RPTPmu and PC5 or the closely related protease PACE4. PC5, but not PACE4, cleaved RPTPmu, and RPTPmu cleavage was absent in COS cells transfected with an expression plasmid encoding a mutant PC5 whose active-site serine had been mutated to alanine. We also performed RNA blot analysis to determine whether PC5 expression was affected by confluence in HUVEC. PC5 mRNA levels were upregulated by more than 30-fold when confluence in HUVEC increased from 25% to 100%. These results indicate that PC5 may have an important role in mediating the cleavage of RPTPmu in response to contact inhibition in HUVEC.

4'-O-[2-(2-fluoromalonyl)]-L-tyrosine: a phosphotyrosyl mimic for the preparation of signal transduction inhibitory peptides.

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.

Immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum: a light and electron microscopic study with a complementary DNA-generated polyclonal antibody.

The present study concerns the immunocytochemical localization of the striatal enriched protein tyrosine phosphatase in the rat striatum. A novel molecular biology technique allowed us to produce a complementary DNA-generated polyclonal antibody raised against the non-catalytic domain of the striatal enriched protein tyrosine phosphatase, which selectively recognized the striatal enriched protein tyrosine phosphatase protein with 46,000 mol. wt on western blots. Immunocytochemical analysis with the specific antibody revealed strong striatal enriched protein tyrosine phosphatase immunoreactivity in the striatum. Light microscopy showed striatal striatal enriched protein tyrosine phosphatase-immunopositive neurons to be of medium size (mean diameter of 14.4 microns), and to comprise approximately 80% of the total neuronal population in the striatum. These cells had round, triangular or polygonal cell bodies with relatively little cytoplasm. Nerve fibers stained positively for striatal enriched protein tyrosine phosphatase were also present in the globus pallidus and the substantia nigra, and the nigral labeling on the ipsilateral side almost disappeared subsequent to cerebral hemitransection, suggesting these immunolabeled structures to be striatal projections. Double-immunofluorescence analysis demonstrated separate populations of striatal enriched protein tyrosine phosphatase-positive cells and neurons stained for parvalbumin. Also, ultrastructural study showed that the striatal enriched protein tyrosine phosphatase-positive neurons (n = 50) possessed no nuclear indentations or intranuclear inclusions. Thus, most striatal striatal enriched protein tyrosine phosphatase-positive neurons were thought to be of the medium-sized spinous type. At the light microscopic level, stained striatal neurons exhibited striatal enriched protein tyrosine phosphatase immunolabeling in their somata, dendrites and axonal processes, but not in their nuclei. Electron microscopic observation showed strong striatal enriched protein tyrosine phosphatase-immunoreactivity on the inner surface of the plasmalemma, on the outer surfaces of mitochondria and on microtubules, particularly of dendrites. A heavy deposit of immunoreaction product was also present on postsynaptic densities in labeled dendrites, while a light deposit was seen on the synaptic vesicles of nerve terminals. The characteristic distribution profile of striatal enriched protein tyrosine phosphatase suggested that the enzyme may play a role in a variety of functional properties of striatal neurons, especially in postsynaptic signaling processes and in regulation of microtubular functions. On the basis of the present findings, we propose the following conclusions: (i) a protein tyrosine phosphorylation system regulated by striatal enriched protein tyrosine phosphatase is involved in certain specialized cellular processes (e.g. signal transduction cascades) of medium-sized spinous neurons distinct from those of other neuronal subsets in the striatum; (ii) a striatal medium spiny neuron is characterized by its expression of striatal enriched protein tyrosine phosphatase and, therefore, the enzyme is useful for detection of the distinct subset of striatal cells or for tracing their axonal projection fibers in the basal ganglia.

Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of neurocan and phosphacan.

We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

Complex-type Asparagine-linked Oligosaccharides on Phosphacan and Protein-tyrosine Phosphatase-ζ/β Mediate Their Binding to Neural Cell Adhesion Molecules and Tenascin

Phosphacan, a soluble nervous tissue-specific chondroitin sulfate proteoglycan, is an alternative splicing product representing the entire extracellular domain of a transmembrane receptor-type protein-tyrosine phosphatase (RPTP zeta/beta) that also occurs as a chondroitin sulfate proteoglycan in brain. We have previously demonstrated that phosphacan binds with high affinity to neural cell adhesion molecules (Ng-CAM/L1 and N-CAM) and to the extracellular matrix protein tenascin and that it is a potent inhibitor of cell adhesion and neurite outgrowth. Tryptic digests of 125I-labeled phosphacan contain two glycopeptides that bind to Ng-CAM/L1, N-CAM, and tenascin. The larger of these (17 kDa) begins at Gln-209 near the end of the carbonic anhydrase-like domain of phosphacan/RPTP zeta/beta, whereas a 13-kDa glycopeptide begins at His-361 located in the middle of the fibronectin type III-like domain. Treatment of phosphacan with peptide N-glycosidase under nondenaturing conditions reduced its binding the neural cell adhesion molecules and tenascin by 65-75%, whereas endo-beta-N-acetylglucosaminidase H had no effect, and peptide N-glycosidase treatment both decreased the molecular sizes of the tryptic peptides to congruent to 11 kDa and abolished their binding. Based on the amino acid sequence of phosphacan, it can be concluded that each of the tryptic peptides contains one potential N-glycosylation site (at Asn-232 and Asn-381), and analyses of the isolated glycopeptides demonstrated the presence of sialylated complex-type oligosaccharides. Our results therefore indicate that the interactions of phosphacan/RPTP zeta/beta with neural cell adhesion molecules and tenascin are mediated by asparagine-linked oligosaccharides present in their carbonic anhydrase- and fibronectin type III-like domains.

Distinct Isoforms of the CD45 Protein-tyrosine Phosphatase Differentially Regulate Interleukin 2 Secretion and Activation Signal Pathways Involving Vav in T Cells

The CD45 family of transmembrane protein-tyrosine phosphatases plays a crucial role in the regulation of lymphocyte activation by coupling activation signals from antigen receptors to the signal transduction apparatus. Multiple CD45 isoforms, generated through regulated alternative mRNA splicing, differ only in the length and glycosylation of their extracellular domains. Differential distribution of these isoforms defines subsets of T cells having distinct functions and activation requirements. While the requirement for the intracellular protein-tyrosine phosphatase domains has been documented, the physiological role of the extracellular domains remains elusive. Here we report the generation of CD45-antisense transfected Jurkat T cell clones that lack CD45 or have been reconstituted to uniquely express either the smallest, CD45(0), or the largest, CD45(ABC), isoform. These cells exhibited marked isoform-dependent differences in IL-2 production and tyrosine phosphorylation of cellular proteins, including Vav after anti-CD3 stimulation. These results demonstrate that the distinct CD45 extracellular domains differentially regulate T cell receptor-mediated signaling pathways. Furthermore, these findings suggest that alterations in CD45 isoform expression by individual T cells during thymic ontogeny and after antigen exposure in the periphery directly affects the signaling pathways utilized.

The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions.

Focal adhesions are sites of cell-extracellular matrix interactions that function in anchoring stress fibers to the plasma membrane and in adhesion-mediated signal transduction. Both focal adhesion structure and signaling ability involve protein tyrosine phosphorylation. LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion-like ectodomain and two intracellular protein tyrosine phosphatase domains. We have identified a novel cytoplasmic 160 kDa phosphoserine protein termed LAR-interacting protein 1 (LIP.1), which binds to the LAR membrane-distal D2 protein tyrosine phosphatase domain and appears to localize LAR to focal adhesions. Both LAR and LIP.1 decorate the ends of focal adhesions most proximal to the cell nucleus and are excluded from the distal ends of focal adhesions, thus localizing to regions of focal adhesions presumably undergoing disassembly. We propose that LAR and LIP.1 may regulate the disassembly of focal adhesions and thus help orchestrate cell-matrix interactions.

Nucleotide sequence and molecular variants of rat receptor-type protein tyrosine phosphatase-/β

We have previously described the cloning of phosphacan, a chondroitin sulfate proteoglycan of nervous tissue which interacts with neurons, glia, neural cell adhesion molecules, and tenascin, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase. We now report the complete cDNA and deduced amino acid sequences of the rat transmembrane phosphatase, and demonstrate that the phosphatase and the extracellular proteoglycan have different 3'-untranslated regions. Northern analysis showed three probable splice variants, comprising the extracellular proteoglycan (phosphacan) and long and short forms of the transmembrane phosphatase. PCR studies of rat genomic DNA indicated that there are no introns at the putative 5' and 3' splice sites or in the 2.6 kb segment which is deleted in the short transmembrane protein. Using variant-specific riboprobes corresponding to sequences in the 3'-untranslated region of phosphacan and in the first or second phosphatase domains of the transmembrane protein, in situ hybridization histochemistry of embryonic rat brain and spinal cord and early postnatal cerebellum demonstrated identical localizations of phosphacan and phosphatase mRNAs.

Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3.

Human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ; also denoted HPTP zeta or RPTP beta) has a large extracellular region with the N-terminal carbonic anhydrase-like domain and a cytoplasmic region with two tandemly located protein tyrosine phosphatase domains. One of the characteristics of PTPRZ is its preferential expression in the central nervous system. We localized the human PTPRZ gene to chromosome band 7q31.3 by somatic cell hybrid mapping and fluorescence in situ hybridization.

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.