Molecular cloning and characterization of a novel human protein phosphatase,LMW-DSP3

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

Molecular cloning of rat cytolysin

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.     

Purification and identification of a novel subunit of protein serine/threonine phosphatase 4

The catalytic subunit of protein serine/threonine phosphatase 4 (PP4C) has greater than 65% amino acid identity to the catalytic subunit of protein phosphatase 2A (PP2AC). Despite this high homology, PP4 does not appear to associate with known PP2A regulatory subunits. As a first step toward characterization of PP4 holoenzymes and identification of putative PP4 regulatory subunits, PP4 was purified from bovine testis soluble extracts. PP4 existed in two complexes of approximately 270-300 and 400-450 kDa as determined by gel filtration chromatography. The smaller PP4 complex was purified by sequential phenyl-Sepharose, Source 15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final product contained two major proteins: the PP4 catalytic subunit plus a protein that migrated as a doublet of 120-125 kDa on SDS-polyacrylamide gel electrophoresis. The associated protein, termed PP4R1, and PP4C also bound to microcystin-Sepharose. Mass spectrometry analysis of the purified complex revealed two major peaks, at 35 (PP4C) and 105 kDa (PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A subunit of PP2A (PP2AA). The PP4R1 cDNA clone engineered with an N-terminal Myc tag was expressed in COS M6 cells and PP4C co-immunoprecipitated with Myc-tagged PP4R1. These data indicate that one form of PP4 is similar to the core complex of PP2A in that it consists of a catalytic subunit and a "PP2AA-like" structural subunit.     

The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1.

By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.     

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product.

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.     

Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis

Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.     

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).     

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.     

Molecular cloning of bovine cardiac muscle heat-shock protein 70 kDa and its phosphorylation by cAMP-dependent protein kinase in vitro

The 70-kDa heat-shock protein (Hsp70) has been cloned and sequenced from bovine cardiac muscle. On the basis of sequence features, the gene corresponds to the cytoplasmic form of Hsp70. This cardiac Hsp70 cDNA clone has an open reading frame of 1926 bp coding for 641 amino acids and a predicted molecular mass of 70.25 kDa. Comparison of the amino acid sequence revealed an extensive sequence identity with other species of Hsp70. Escherichia coli expressed cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. Notably, we observed that Hsp70 directly interacts with CaN using a pull-down assay. Furthermore, expressed cardiac-specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation resulted in the incorporation of 0.1 mol of phosphate per mol of Hsp70. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. This is the first demonstration that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70.     

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

A gene encoding, prespore-cell-inducing factor in Dictyostelium discoideum

Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum induce amoebae to differentiate into prespore cells when they are incubated at a very low cell density in submerged monolayer culture. Previously, we purified one of them, a glycoprotein factor with an apparent molecular mass of 106 kDa, and we named it psi factor (psi, prespore-inducing factor). Based on the partial amino acid sequence of the purified psi factor, we have isolated the corresponding cDNA clone, which is expressed maximally at the loose mound stage. The cDNA encodes a novel protein and the predicted molecular mass of the mature secreted protein is 60 kDa. Knockout mutant strains of the psi factor gene, psiA(-), were created by targeted integration. Although these mutant strains appear to develop normally, CM from these mutants showed reduced prespore-cell-inducing activity. Rescuing the mutant strains by expression of psi factor under control of a constitutive promoter causes overproduction of psi factor protein and CM from such cells showed a 20-fold higher level of prespore-cell-inducing activity than that from wild-type cells. Further, CM from parental cells induced prespore cell division, while that from psiA null strains showed no cell division inducing activity. Our results indicate that psi factor protein is a novel type of growth factor that does not belong to any of the families of growth factor so far identified in animals.     

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.     

Cloning and recombinant expression of the La RNA-binding protein from Trypanosoma brucei

We report the isolation, cloning and recombinant expression of a Trypanosoma brucei homolog of the La RNA-binding protein. Based on peptide sequence information we have isolated a cDNA clone which encodes a protein of 335 amino acids with a predicted molecular weight of 37.7 kDa. The amino acid sequence fits the domain structure of known La proteins and contains a putative ATP-binding site located in the COOH-terminal domain. The cDNA was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and the recombinant protein displayed RNA-binding activity in an electrophoretic mobility shift assay.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.