Purification and characterization of the cytoplasmic domain of human receptor-like protein tyrosine phosphatase RPTP mu

RPTP mu is a recently described receptor-like protein tyrosine phosphatase (PTP), the ectodomain of which mediates homophilic cell-cell adhesion. The cytoplasmic part contains two homologous PTP-like domains and a juxtamembrane region that is about twice as large as in other receptor-like PTPs. The entire 80-kDa cytoplasmic part of human RPTP mu was expressed in insect Sf9 cells and its enzymatic activity was characterized after purification to electrophoretic homogeneity. In addition, the effects of deletion and point mutations were analyzed following expression in Escherichia coli cells. The purified cytoplasmic part of RPTP mu displays high activity toward tyrosine-phosphorylated, modified lysozyme (Vmax 4500 nmol min-1 mg-1) and myelin basic protein (Vmax 8500 nmol min-1 mg-1) but negligible activity toward tyrosine-phosphorylated angiotensin or the nonapeptide, EDNDpYINASL, that serves as a good substrate for protein tyrosine phosphatase PTP1B. This suggests that RPTP mu and PTP1B have distinct substrate specificities. Catalytic activity is independent of Ca2+ (up to 1 mM) but is strongly inhibited by Zn2+, Mn2+, vanadate, phenylarsenic oxide, and heparin. The first of the two catalytic domains is 5-10 times less active than the expressed catalytic region containing both domains. Mutation of Cys 1095 to Ser in the first catalytic domain abolishes enzymatic activity when analyzed following expression in either E. coli or mammalian COS cells. Deletion of the first 53 amino acids from the juxtamembrane region reduces catalytic activity about 2-fold.     

PTP-NP, a new member of the receptor protein tyrosine phosphatase family, implicated in development of nervous system and pancreatic endocrine cells

The regulation of protein tyrosine phosphorylation is an important mechanism for developmental control. We describe here a new member of the protein tyrosine phosphatase (PTP) family, called PTP-NP (for neural and pancreatic). The cDNA sequence indicates a receptor-type transmembrane molecule. At early organogenesis, in situ hybridization with a probe for the PTP-NP extracellular region detects expression confined to the region of the developing pancreas, an organ of medical importance, but poorly understood with regard to molecular mechanisms of developmental control. This localized expression appears early, even before morphological differentiation of the pancreas, and is found in presumptive precursors of the endocrine cells by the earliest times that they can be distinguished. In neural development, an alternate RNA with a different or missing extracellular region is expressed transiently at early stages of neurogenesis and the full-length PTP-NP RNA appears later. To search for a ligand of PTP-NP, a fusion protein probe was made with the extracellular domain fused to an alkaline phosphatase tag. This probe bound strongly to pancreatic islets, providing evidence for a ligand-receptor interaction that could be involved in endocrine cell regulation. The results show PTP-NP is an especially early marker for pancreatic development and suggest it may be a receptor that could control the development of pancreatic endocrine cells.     

Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.     

CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes

CD148 is a new cluster of differentiation defined in the VI International Workshop on Leucocyte Differentiation Antigens. It has been identified as the hematopoietic form of a formerly described membrane protein tyrosine phosphatase called HPTP eta/ DEP-1. Previous data have demonstrated that this molecule is able to give rise to [Ca2+]i increase. In the present work we show its capability to induce protein tyrosine phosphorylation in human lymphocytes in spite of its intrinsic protein tyrosine phosphatase activity. The induction of kinase activity suggests the involvement of some protein tyrosine kinase based signaling pathway. The activation of this postulated kinase could be carried out through a direct association or via an adapter molecule.     

PPX, a novel protein serine/threonine phosphatase localized to centrosomes

The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa. PPX was expressed in the baculovirus system. Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A. However, PPX did not bind the 65 kDa regulatory subunit of PP2A. The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide. These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes. The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase. In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes. The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation.     

Cloning and expression of the chicken protein tyrosine phosphatase SH-PTP2

SH-PTP2 is a protein tyrosine phosphatase which contains two src homology 2 (SH2) domains. A partial cDNA clone encoding chicken SH-PTP2 was generated by RT-PCR and used as a probe to screen several chicken cDNA libraries. Two overlapping cDNA clones were identified and the nucleotide sequence of chicken SH-PTP2 containing the entire protein-coding region was determined. The deduced amino acid sequence shares 98% and 94% identity, respectively with the corresponding human and Xenopus proteins. Northern and Western blot analyses show that chicken SH-PTP2 is expressed ubiquitously like those of mammals and Xenopus. This suggests that chicken SH-PTP2 may have analogous biological roles to those of mammals.     

Molecular cloning, expression, and characterization of a novel human serine/threonine protein phosphatase, PP7, that is homologous to Drosophila retinal degeneration C gene product (rdgC)

A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of approximately 75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares < 35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 approximately equal to 250 microM). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin.     

Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development

We tried to identify a correlation between bacterial vaginosis and PROM with the aim of proposing a future screening for pregnant women at risk for premature rupture of membranes. We made two vaginal bacteriological tests on women pregnant at 24th and 34th week, and we repeated the same test when it proved positive for infection after therapy. We did 375 tests and our study group was composed of 249 pregnant women, aged between 24 and 35. We also evaluated some variable factors as ultrasonographic gestational age redetermination, number pregnancies of oral contraceptives or IUD use, presence of risk factors presence (recurrent abortion, previous PROM, glucose metabolism alteration, immunosuppression, condylomatosis). 171 tests proved positive (total positivity percentage: 47.2%). The most common micro-organism was Ureaplasma urealyticum (49.1%) that was related to PROM in 66.7%. This result contracts with affirmations of some England authors who think that Ureaplasma hasn't an important role in PROM. This fact, if related to the other data we obtained with our study, actually current, shows a strict correlation between PROM and bacterial vaginosis, but also a different distribution of vaginal infections in our country in comparison with English countries.     

Molecular cloning of a novel putative G protein-coupled receptor (GPCR21) which is expressed predominantly in mouse central nervous system

Agarose gel affinity electrophoresis has been used to demonstrate interactions between autologous IgG and specific erythrocyte membrane proteins. These binding phenomena are here further examined by combining affinity electrophoresis with affinity chromatography, absorption experiments, and immunoblotting. It is demonstrated that the interactions are highly dependent on polyreactive IgG binding favored by the low ionic strength conditions of the electrophoretic assay. Thus, about 25% of normal IgG under low ionic strength conditions bound to the purified cytoskeletal protein, spectrin, immobilized on Sepharose. This IgG reacted in affinity electrophoresis in a polyspecific fashion with the same array of membrane proteins as before the low ionic strength-affinity chromatography. Further, the binding seen in affinity electrophoresis, including the interaction with spectrin, was completely abolished by preabsorption of the IgG with spectrin-devoid membranes. The charge characteristics of an IgG subclass might be responsible for the observed binding. However, the observed precipitate formation suggested an interaction involving at least two binding sites on each molecule and the binding appears to require structurally intact IgG because reductive treatment with dithiothreitol diminished the reactivity considerably. Conclusively, under the conditions of affinity electrophoresis with ligand present in the gel, electrostatic interactions are amplified. The degree of binding of IgG to erythrocyte membrane proteins that take place under these conditions does not reflect binding which would occur to the same extent under physiological ionic strength conditions.     

Characterization of a novel CC chemokine, HCC-4, whose expression is increased by interleukin-10

We have identified and characterized a human beta (CC) chemokine, designated HCC-4, that is most closely related to HCC-1 and which demonstrates chemotactic activity for monocytes. Northern analysis of multiple tissue blots and of activated monocytes mRNA shows expression of a 500-bp mRNA. A 1,500-bp mRNA was highly expressed in monocytes activated 12 hours in the presence of interleukin-10 (IL-10) but was absent in monocytes activated for only 1 hour regardless of the presence or absence of IL-10. The upregulation of expression in the presence of IL-10 is in contrast to the downregulatory effects of IL-10 on expression of most other chemokines. Recombinant HCC-4 demonstrated chemotactic activity for human monocytes and THP-1 monocyte cells but not for resting lymphocytes or neutrophils. HCC-4 also induced a Ca2+ flux in THP-1 cells that was desensitized by prior exposure to RANTES. Taken together, these data indicate that HCC-4 is a novel chemokine whose expression is uniquely upregulated by IL-10.     

Developmental regulation of tyrosine phosphorylation of the NR2D NMDA glutamate receptor subunit in rat central nervous system

A subunit-specific antibody against the N-methyl-D-aspartate (NMDA) receptor NR2D protein along with an antiphosphotyrosine antibody were employed to examine the developmental profile of the tyrosine phosphorylation of NR2D and its regulation by a protein phosphatase inhibitor in rat brain. NMDA receptor proteins from the thalamus at postnatal days 1, 7, 21, and 49 were solubilized under denaturing conditions and used in immunoprecipitations with these antibodies followed by quantitative immunoblot analysis of NR2D protein in the resulting immunopellets. The results indicate that the NR2D subunit is tyrosine phosphorylated in the brain. The quantified data examining the developmental profile of tyrosine phosphorylation of NR2D in the thalamus show that the level of tyrosine phosphorylation of NR2D protein increases five- to sixfold during development. In addition, the protein phosphatase inhibitor pervanadate (vanadyl hydroperoxide) was found to increase tyrosine phosphorylation of NR2D subunit threefold in brain slices, implying an active cycle of phosphorylation and dephosphorylation in situ. These studies demonstrate developmentally regulated tyrosine phosphorylation of NR2D protein in vivo, suggesting that tyrosine phosphorylation may be important for regulating the functions of this NMDA receptor subunit in the mammalian central nervous system.     

Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.     

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.     

Identification of a novel polyproline recognition site in the cytoskeletal associated protein, proline serine threonine phosphatase interacting protein

Protein-protein interactions are often mediated by the recognition of proline-rich domains by SH3 or WW modules. Previously, we demonstrated that the PEST-type protein-tyrosine phosphatase, PTP HSCF (hematopoietic stem cell fraction), bound to a novel cytoskeletal associated protein, proline serine threonine phosphatase interacting protein (PST PIP), via an interaction between the proline-rich COOH terminus of the PTP and a site within the putative coiled-coil domain of PST PIP. Here we describe a more detailed analysis of this interaction. Earlier data suggested that the NH2 terminus of PST PIP was important for binding to the phosphatase, and deletion of the NH2-terminal 50 amino acids of the PST PIP resulted in an apparently misfolded protein that was incapable of binding PTP HSCF. To examine the region involved with binding to PTP HSCF, alanine-scanning mutants were produced at intervals throughout PST PIP. This analysis demonstrated that a tryptophan at position 232 was essential for binding in vitro. Transfection experiments demonstrated that the Trp232 mutant protein was capable of association with the cortical cytoskeleton but was not bound to PTP HSCF in vivo. Alanine scanning of a peptide derived from the COOH-terminal proline-rich domain of PTP HSCF revealed that a subset of prolines, as well as other residues, was required for efficient binding to PST PIP, and introduction of alanines at some of these positions in the protein resulted in decreased binding to PST PIP in vitro and in vivo. Analysis of in vivo tyrosine phosphorylation of the Trp232 mutant of PST PIP in the presence of v-Src revealed that this protein was phosphorylated more efficiently than the wild-type molecule. Thus, the interaction between PTP HSCF and PST PIP is mediated by a novel site in the cytoskeletal associated protein which interacts with residues within the proline-rich COOH terminus of the phosphatase.