Isolation of a novel protein kinase-encoding gene from yeast by oligodeoxyribonucleotide probing

We have identified a novel protein kinase-encoding gene, KIN3, in the genome of the budding yeast Saccharomyces cerevisiae. The gene was isolated from a library of cloned genomic fragments by probing with an oligodeoxyribonucleotide mixture corresponding to part of a highly-conserved region in the catalytic domain of protein serine-threonine kinases. KIN3 is unique in the yeast genome, maps to chromosome VI and is actively expressed in mitotically dividing cells to produce a 1400 nucleotide (nt) message. The nt sequence of KIN3 predicts a protein product of 43.4 kDa which contains all of the conserved elements found in known protein serine-threonine kinases, although the organisation of these elements in the KIN3 gene product differs significantly from the consensus. The function of the KIN3-encoded protein kinase is unclear although it appears not to be essential for growth, conjugation or sporulation.     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

A 63 kDa protein is secreted from BALB/c-3T3 cells entering the G1 phase from the G0 state

A 63 kDa protein is detectable in the culture fluid of mouse BALB/c-3T3 cells traversing from the G0 state to the G1 phase, whereas it is undetectable in the culture fluid of quiescent or growing BALB/c-3T3 cells. Secretion of the protein is maximal at 10 h after serum addition. G0-specific ts mutant cells (rat tsJT60) also secrete the 63 kDa protein only when the quiescent cells are stimulated by serum addition at permissive temperature. These facts indicate that the 63 kDa protein is secreted only from cells traversing from the G0 state to the G1 phase.     

Morphine enhances the phosphorylation of a 58 kDa protein in mouse brain membranes.

Morphine and [D-Ala2,D-Leu5]enkephalinamide enhance the phosphorylation of a 58 kDa protein in mouse brain synaptosomal membranes. The enhancement of phosphorylation was inhibited by naloxone, an antagonist of morphine. The phosphorylated 58 kDa protein was retained on wheat-germ-agglutinin-agarose and morphinone-Affi-Gel 401 columns and biospecifically eluted out from the columns with N-acetyl-D-glucosamine and naloxone respectively. These results suggest a strong possibility that the opiate-binding protein undergoes phosphorylation by endogenous protein kinase. Since the molecular mass of a mu-type opioid receptor in mouse brain is suggested to be 58 kDa, coincident with those of rat brain and neuroblastoma x glioma hybrid cells, it is conceivable that the phosphorylated 58 kDa protein is a mu-type receptor.     

Identification and immunological characterization of a novel 40-kDa protein linked to CD98 antigen.

Monoclonal antibodies (mAbs) were obtained from hybridoma clones established by cell fusion between mouse myeloma cells and spleen cells from a mouse immunized against an affinity-purified 40-kDa component of rat 125-kDa glycoprotein (GP125). Two mAbs designated as 3F2 and 6B4 detected a 40-kDa and a 125-kDa band under reducing and nonreducing conditions, respectively, in extracts prepared from rat, mouse and human tumor cells. Association of the 40-kDa protein with CD98 was revealed by sandwich-type enzyme-linked immunosorbent assay. The two mAbs were strongly reactive with various tumor cells and activated lymphocytes, but were only weakly reactive with resting lymphocytes. Confocal microscopy indicated colocalization of CD98 and the 40-kDa protein defined with 3F2 and 6B4 at the cell surface and perinuclear regions. On immunohistochemical analysis of frozen sections of rat tongue, the anti-rat CD98 mAb B3 selectively stained the basal layer and 3F2 stained the upper epithelial part in addition to the basal layer, indicating the existence of CD98-unlinked 40-kDa protein.     

Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein

By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions.     

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization.

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.     

Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes

Hepatocytes prepared from 18-day-old mouse embryos were grown in serum-free medium and reached confluence after two days in culture. The total amount of the 26 kDa gap junction protein decreased in these cells during the first 24 h in culture and increased again between day 1 and day 3 more than 10-fold. At day 3 a half-life time of 2.5 to 3 h was determined for the 26 kDa protein by [35S]methionine incorporation and immunoprecipitation using affinity-purified anti-26 kDa. Incorporation of [32P]orthophosphate into the 26 kDa protein of cultured hepatocytes was found at serine residues (98%) and tyrosine residues (about 2%). The addition of dibutyryl cyclic adenosine monophosphate (db cAMP) to the culture medium at day 2 had two effects: After 15 min the extent of phosphorylation of the 26 kDa protein increased 2.7-fold whereas the total amount of the 26 kDa protein increased only 1.2-fold. After 3 h of incubation with db cAMP, a 2.5-fold increase of the 26 kDa protein was noticed which was accompanied by a 3.2-fold increase in phosphorylation of serine residues. The effects of db cAMP on phosphorylation of the 26 kDa protein could be augmented or mimicked by the addition of isoproterenol, theophylline or forskolin to the culture medium of hepatocytes. In extracts of rat hepatocarcinoma MH1C1 cells and dog kidney MDCK cells, a phosphorylated 26 kDa protein can be immunoprecipitated using anti-liver 26 kDa. These results demonstrate that the gap junction 26 kDa protein can be posttranslationally modified by cAMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liprin α3: a putative estrogen regulated acrosomal protein

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.     

Characterization of the cell-cycle-regulated protein calcyclin from Ehrlich ascites tumor cells. Identification of two binding proteins obtained by Ca2(+)-dependent affinity chromatography

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2(+)-dependent. Immunological criteria and partial sequence data identify the two calcyclin-binding proteins as the phospholipid-binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2(+)-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase.     

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.     

Human autoantibodies against a nucleolar protein

Autoimmune sera showing prominent immunofluorescence in nucleolus were selected and analysed by immunoblotting techniques. Immunoblots using a nucleolar extract as antigen source revealed sera recognizing a 38 kDa nucleolar protein. Low concentration of Actinomycin D, which inhibits the ribosomal RNA synthesis, caused a loss of fluorescence. This suggests that the nucleolar antigen may be associated with the assembly of packaging of the ribosomes. The present nucleolar antigen has properties similar to the previously described nucleolar phosphoprotein B23 of rat cells and the recently described nucleolar protein NO38 of mouse and Xenopus cells.     

Identification of blottin: a novel gastric trefoil factor family-2 binding protein

The trefoil factor family (TFF) peptides are important in gastro-intestinal mucosal protection and repair. Their mechanism of action remains unclear and receptors are sought. We aimed to identify and characterise proteins binding to TFF2. A fusion protein of mouse TFF2 with alkaline phosphatase was generated and used to probe 2-D protein blots of mouse stomach. The resulting spots were analysed by MS. The protein identified was characterised by bioinformatics, rapid amplification of cDNA ends, in situ hybridisation (ISH) and immunohistochemistry (IHC). Functional assays were performed in gastrointestinal cell lines. A single major murine protein was identified and named blottin. It was previously unknown as a translated product. Blottin is also present in rat and human; the latter gene is also known as GDDR. The predicted full-length proteins are 184 amino acids long (20 kDa), reducing to 164 amino acids (18 kDa) after signal peptide cleavage. ISH of gastrointestinal tissues shows abundant blottin mRNA in gastric surface and foveolar epithelium. IHC shows cytoplasmic staining for blottin protein, and by immunoelectron microscopy in mucus granules and Golgi stacks. Previous work showed that blottin is down-regulated in gastric cancers. Blottin contains a BRICHOS domain, and has 56% similarity with gastrokine-1. Cultured HT-29 cells express blottin and show increased DNA synthesis with antiblottin antibody; however, this effect is reversed by the immunising peptide. We have identified and characterised a TFF2-binding protein produced by gastric epithelium. Blottin may play a role in gastrointestinal mucosal protection and modulate gut epithelial cell proliferation.     

An accessory protein identical to mouse egasyn is complexed with rat microsomal beta-glucuronidase and is identical to rat esterase-3

We report biochemical, immunological, and genetic studies which demonstrate that an accessory protein with the essential features of mouse egasyn is complexed with and stabilizes a portion of beta-glucuronidase in microsomes of rat liver. The accessory protein exists as a complex with beta-glucuronidase since it coprecipitates with beta-glucuronidase after treatment of extracts with a specific beta-glucuronidase antibody. The two proteins are associated by noncovalent bonds since they are easily dissociated at elevated temperatures. Only 20-25% of total liver accessory protein is complexed with microsomal beta-glucuronidase. The remainder exists as a free form. The molecular weight of the accessory protein is 61 to 63 kDa depending upon the rat strain of origin. This protein, like mouse egasyn, has esterase catalytic activity and is concentrated in microsomes. The accessory protein is genetically polymorphic with at least four alleles. Combined biochemical and genetic evidence indicates it is identical with esterase-3 of the rat. Also, both mouse egasyn and rat esterase-3 react with antisera to egasyn and to rat esterase-3, indicating they are homologous proteins. Several inbred rat strains lack microsomal beta-glucuronidase. The same strains lack the accessory protein, suggesting that stabilization of beta-glucuronidase in rat microsomes requires egasyn.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

Presenilin-1 protein specifically expressed in Leydig cells with its expression level increased during rat testis development

Presenilin-1, mutations of which cause the early-onset of Alzheimer's disease, was shown to be abundantly expressed in the testis as well as the brain. In spite of the high expression level of this protein in the testis, no further analysis has been undertaken. We aimed to study the distribution and developmental changes in presenilin-1 protein, and to provide clues so as to elucidate the role of this protein in the rat testis. To evaluate the specificity of the anti presenilin-1 antibody, rat presenilin-1 protein was expressed in COS-7 cells and the recombinant protein was used for western blot analysis. A positive band of approximately 20 kDa corresponding to the C-terminal fragment of proteolyzed presenilin-1 protein was observed. Using testis and brain tissue samples, a 20 kDa band was detected in both tissues suggesting a similar proteolytic process, but the expression level in the testis was higher than that in the brain. The expression level increased significantly during postnatal testis development. By an immunohistochemical analysis of the rat testis, a strong signal was observed in interstitial cells and further study with cultured TM3 murine Leydig cells revealed an abundant expression of presenilin-1 in Leydig cells. Our study suggests that presenilin-1 expression in Leydig cells may play an important role in Leydig cell function and testis development.     

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.