Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro
The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovir...
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| Veröffentlicht in: | Journal of virological methods 2001-06, Vol.95 (1), p.1-10 |
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| creator | Lee, Young I. Kim, Sun O. Kwon, Hyok J. Park, Jong G. Sohn, Mi J. Jeong, Soon S. |
| description | The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in
Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK. |
| doi_str_mv | 10.1016/S0166-0934(00)00282-2 |
| format | Article |
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Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.</description><identifier>ISSN: 0166-0934</identifier><identifier>EISSN: 1879-0984</identifier><identifier>DOI: 10.1016/S0166-0934(00)00282-2</identifier><identifier>PMID: 11377708</identifier><identifier>CODEN: JVMEDH</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Amino Acid Sequence ; Animal viral diseases ; Binding Sites ; Biological and medical sciences ; Circular dichroism ; Fundamental and applied biological sciences. Psychology ; HBx protein ; Hepatitis B virus ; Hepatitis B virus-X protein ; Human viral diseases ; Humans ; Hydrolysis ; Infectious diseases ; Medical sciences ; Microbiology ; Mitogen-activated protein kinase ; Mitogen-Activated Protein Kinases - metabolism ; Molecular Sequence Data ; Phosphoamino Acids - analysis ; Phosphorylation ; Protein kinase C ; Protein Kinase C - metabolism ; Protein Renaturation ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Recombinant Fusion Proteins - metabolism ; Recombinant Fusion Proteins - physiology ; Sequence Analysis, Protein ; Trans-Activators - genetics ; Trans-Activators - isolation & purification ; Trans-Activators - metabolism ; Trans-Activators - physiology ; Viral diseases ; Viral hepatitis</subject><ispartof>Journal of virological methods, 2001-06, Vol.95 (1), p.1-10</ispartof><rights>2001 Elsevier Science B.V.</rights><rights>2002 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c422t-264f9aad363986cb2c8bd9c59d231b31a4b926301a100b0803d5f020a036058b3</citedby><cites>FETCH-LOGICAL-c422t-264f9aad363986cb2c8bd9c59d231b31a4b926301a100b0803d5f020a036058b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0166093400002822$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14162390$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11377708$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lee, Young I.</creatorcontrib><creatorcontrib>Kim, Sun O.</creatorcontrib><creatorcontrib>Kwon, Hyok J.</creatorcontrib><creatorcontrib>Park, Jong G.</creatorcontrib><creatorcontrib>Sohn, Mi J.</creatorcontrib><creatorcontrib>Jeong, Soon S.</creatorcontrib><title>Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro</title><title>Journal of virological methods</title><addtitle>J Virol Methods</addtitle><description>The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in
Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.</description><subject>Amino Acid Sequence</subject><subject>Animal viral diseases</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>Circular dichroism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>HBx protein</subject><subject>Hepatitis B virus</subject><subject>Hepatitis B virus-X protein</subject><subject>Human viral diseases</subject><subject>Humans</subject><subject>Hydrolysis</subject><subject>Infectious diseases</subject><subject>Medical sciences</subject><subject>Microbiology</subject><subject>Mitogen-activated protein kinase</subject><subject>Mitogen-Activated Protein Kinases - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Phosphoamino Acids - analysis</subject><subject>Phosphorylation</subject><subject>Protein kinase C</subject><subject>Protein Kinase C - metabolism</subject><subject>Protein Renaturation</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Recombinant Fusion Proteins - physiology</subject><subject>Sequence Analysis, Protein</subject><subject>Trans-Activators - genetics</subject><subject>Trans-Activators - isolation & purification</subject><subject>Trans-Activators - metabolism</subject><subject>Trans-Activators - physiology</subject><subject>Viral diseases</subject><subject>Viral hepatitis</subject><issn>0166-0934</issn><issn>1879-0984</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc9uFSEUh4mxsdfWR9Cw0djF2APMH1gZvbHWpIlNqok7AgzjRWdgBOYm9wF8b2nv1S5cdAPk8P0OJ3wIPSfwhgBpz2_K0lYgWP0a4AyAclrRR2hFeCdKmdeP0eofcoyepvQDAJqOsSfomBDWdR3wFfp9vQlp3oS4G1V2weMw4HmJbnC2x9GaMGnnlc94Y-cCZJfwe7x1cUnVNzzHkK3zWO_w5HL4bn2lTHZblUv47-XPkk8WK_9faY3LeetyDKfoaFBjss8O-wn6evHhy_qyuvr88dP63VVlakpzRdt6EEr1rGWCt0ZTw3UvTCN6yohmRNVa0JYBUQRAAwfWNwNQUMBaaLhmJ-jVvm-Z5NdiU5aTS8aOo_I2LEl2IKBhNXkQJB3nlHe8gM0eNDGkFO0g5-gmFXeSgLwVJe9EyVsLEkDeiZK05F4cHlj0ZPv71MFMAV4eAJWMGoeovHHpnqtJS5mAwr3dc7b829bZKJNx1hvbu6Ivyz64B0b5AwxDsL4</recordid><startdate>20010601</startdate><enddate>20010601</enddate><creator>Lee, Young I.</creator><creator>Kim, Sun O.</creator><creator>Kwon, Hyok J.</creator><creator>Park, Jong G.</creator><creator>Sohn, Mi J.</creator><creator>Jeong, Soon S.</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U9</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20010601</creationdate><title>Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro</title><author>Lee, Young I. ; Kim, Sun O. ; Kwon, Hyok J. ; Park, Jong G. ; Sohn, Mi J. ; Jeong, Soon S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c422t-264f9aad363986cb2c8bd9c59d231b31a4b926301a100b0803d5f020a036058b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Amino Acid Sequence</topic><topic>Animal viral diseases</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>Circular dichroism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>HBx protein</topic><topic>Hepatitis B virus</topic><topic>Hepatitis B virus-X protein</topic><topic>Human viral diseases</topic><topic>Humans</topic><topic>Hydrolysis</topic><topic>Infectious diseases</topic><topic>Medical sciences</topic><topic>Microbiology</topic><topic>Mitogen-activated protein kinase</topic><topic>Mitogen-Activated Protein Kinases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Phosphoamino Acids - analysis</topic><topic>Phosphorylation</topic><topic>Protein kinase C</topic><topic>Protein Kinase C - metabolism</topic><topic>Protein Renaturation</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Recombinant Fusion Proteins - physiology</topic><topic>Sequence Analysis, Protein</topic><topic>Trans-Activators - genetics</topic><topic>Trans-Activators - isolation & purification</topic><topic>Trans-Activators - metabolism</topic><topic>Trans-Activators - physiology</topic><topic>Viral diseases</topic><topic>Viral hepatitis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lee, Young I.</creatorcontrib><creatorcontrib>Kim, Sun O.</creatorcontrib><creatorcontrib>Kwon, Hyok J.</creatorcontrib><creatorcontrib>Park, Jong G.</creatorcontrib><creatorcontrib>Sohn, Mi J.</creatorcontrib><creatorcontrib>Jeong, Soon S.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of virological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lee, Young I.</au><au>Kim, Sun O.</au><au>Kwon, Hyok J.</au><au>Park, Jong G.</au><au>Sohn, Mi J.</au><au>Jeong, Soon S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro</atitle><jtitle>Journal of virological methods</jtitle><addtitle>J Virol Methods</addtitle><date>2001-06-01</date><risdate>2001</risdate><volume>95</volume><issue>1</issue><spage>1</spage><epage>10</epage><pages>1-10</pages><issn>0166-0934</issn><eissn>1879-0984</eissn><coden>JVMEDH</coden><abstract>The recombinant human hepatitis B virus-X protein (rhHBx) has been expressed as inclusion bodies in
Escherichia coli and purified. By sequential dialysis of urea, rhHBx was folded into the native structure, which was demonstrated by both the efficacy of its transcriptional activation of the adenovirus major late promoter, fluorescence and circular dichroism (CD) analysis. The increase in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of HBx to refold in lower concentrations of urea to produce the active protein. After purification and renaturation, the rhHBx protein was found to be phosphorylated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). In vivo phosphorylation of HBx was also demonstrated. Although PKC and MAPK enhance the HBx phosphorylation in vitro, neither protein kinase A nor caseine kinase II (CKII) phosphorylate HBx protein, though there are possible substrate residues of both kinases in HBx protein. Phosphoamino acid analysis of the total acid hydrolyzed HBx showed that serine residues can be phosphorylated by PKC or MAPK.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>11377708</pmid><doi>10.1016/S0166-0934(00)00282-2</doi><tpages>10</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animal viral diseases Binding Sites Biological and medical sciences Circular dichroism Fundamental and applied biological sciences. Psychology HBx protein Hepatitis B virus Hepatitis B virus-X protein Human viral diseases Humans Hydrolysis Infectious diseases Medical sciences Microbiology Mitogen-activated protein kinase Mitogen-Activated Protein Kinases - metabolism Molecular Sequence Data Phosphoamino Acids - analysis Phosphorylation Protein kinase C Protein Kinase C - metabolism Protein Renaturation Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Recombinant Fusion Proteins - physiology Sequence Analysis, Protein Trans-Activators - genetics Trans-Activators - isolation & purification Trans-Activators - metabolism Trans-Activators - physiology Viral diseases Viral hepatitis |
| title | Phosphorylation of purified recombinant hepatitis B virus-X protein by mitogen-activated protein kinase and protein kinase C in vitro |
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