Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.
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PMID:Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites. 137 44

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.
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PMID:HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells. 257 75

The structural proteins of mouse mammary tumor virus (MMTV) were analyzed by two-dimensional electrophoresis on isoelectric focusing and sodium dodecyl sulfate gels. Many of the viral proteins displayed heterogeneity in charge due to variable contents of carbohydrates (in particular, sialic acid) and phosphate residues. Neuraminidase treatment of the virions influenced the isoelectric pattern of the envelope glycoproteins. The glycoproteins of an MMTV variant which was attenuated by replication in feline kidney cells had different isoelectric points. This suggested that the acquisition of an altered carbohydrate configuration had changed the host range of the virus. The major MMTV structural core protein, p27, consisted of two species, which had identical iodinated tryptic peptide compositions but differed in phosphate contents. Another MMTV phosphoprotein, p21, was separated into four different phosphorylated species. Phosphorylation of p21 could be performed in vitro by the MMTV virion-associated protein kinase. This enzyme also has a high affinity for MMTV p30 as a substrate. Possible functions of this enzyme are discussed.
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PMID:Analysis of secondary modifications of mouse mammary tumor virus proteins by two-dimensional gel electrophoresis. 625 75

Purified proteoglycan subunits from human articular, bovine articular and nasal cartilages, and a rat chondrosarcoma were phosphorylated in vitro by beef heart cAMP-dependent protein kinase in the presence of gamma 32P-ATP. In these experiments, a maximum of 1.7 moles of 32P were incorporated per mole of proteoglycan from human cartilage. Phosphorylation was dependent on the presence of cAMP. Analysis by autoradiography revealed that serine residues in the core protein of the proteoglycan were the sites of phosphorylation. Treatment of proteoglycan subunits with chondroitinase ABC and alkaline phosphatase prior to reaction with cAMP-dependent protein kinase increased the incorporation of 32P by 12-30% when compared with untreated proteoglycans. These data indicate that proteoglycans in cartilage can be phosphorylated by cAMP-dependent protein kinase.
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PMID:Phosphorylation of proteoglycans from human articular cartilage by a cAMP-dependent protein kinase. 647 53

The nature of the protein kinase (PK) which phosphorylates the core protein of hepatitis B virus in vitro was studied. The PK copurified with the core particles during rate zonal centrifugation and gel chromatography. It showed the same size heterogeneity as the core particles, which consisted of a main fraction of 28-nm particles and a subfraction of 22- to 26-nm particles. DNA-containing heavy core particles with a density of 1.33 to 1.35 g/ml and less endogenous PK than did the light cores. The phosphorylation reaction had a rapid initial phase (several minutes) and a slow but long-lasting second phase (many hours). The PK had a high affinity for ATP (KM = 0.5 mumol/liter). Only few of the several hundred P21.9 subunits in one core particle were phosphorylated in vitro. The only amino acid which was phosphorylated in vitro was serine. The resistance of the introduced phospho group against alkaline phosphatase showed that the PK acceptor, and probably the enzyme itself, was located inside the core particle.
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PMID:Specificity and localization of the hepatitis B virus-associated protein kinase. 680 56

By moving chloride into epithelial cells, the Na-K-Cl cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III (1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5'-end of the human colonic cotransporter is rich in G + C content. Interestingly, a triple repeat (GCG)7 occurs within the 5'-coding region and contributes to a large alanine repeat (Ala15). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.
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PMID:Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. 762 5

We previously demonstrated that the core protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV core protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV core protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion protein expression system and in vitro transcription and translation system, the phosphorylation of HCV core protein was carried out by protein kinase A (PKA) and protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in core protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV core protein was essential for the suppressive activity of HCV core protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV core protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV core protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV core protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV core protein is a phospho-protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation. 781 94

A protein extract of mouse seminal-vesicle secretion was used to immunize mature mice (Balb/c) of both sexes. Results of Western-blot analyses for these secretory proteins indicated that only one minor protein component could be recognized by the autoantisera prepared from either autoimmunization of male mice or isoimmunization of female mice. The autoantigen was purified from seminal-vesicle secretion. The purified autoantigen retained the ability to induce autoantibody formation. The autoantigen has glycoprotein characteristics: the majority of the carbohydrate is N-linked and the remainder is O-linked. Rabbit antibodies to the autoantigen were used to isolate the corresponding cDNA from a mouse seminal-vesicle cDNA library. The primary structure deduced from the cDNA sequence was confirmed by direct amino acid sequence determination. The results indicate that the core protein consists of 131 amino acid residues. Analysis of the primary structure indicates that the autoantigen has two potential acceptor sites for the N-linked carbohydrate at Asn-12 and Asn-122, three potential phosphorylation sites for casein kinase II at Thr-55, Ser-68 and Thr-76, and three potential phosphorylation sites for protein kinase C at Thr-28, Thr-40 and Thr-124. The core protein and the carbohydrate portion together have a molecular mass of 19 kDa. Results from Western- and Northern-blot analyses for various tissues indicate that the seminal vesicle is the sole organ producing this autoantigen. Expression of this autoantigen gene was stimulated by testosterone.
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PMID:Primary structure and characterization of an androgen-stimulated autoantigen purified from mouse seminal-vesicle secretion. 828 54

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.
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PMID:Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers. 864 Nov 67

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.
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PMID:Phosphorylation of the core protein of hepatitis B virus by a 46-kilodalton serine kinase. 955 62


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