Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase
Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of...
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| Veröffentlicht in: | The Journal of biological chemistry 1992-02, Vol.267 (5), p.3024-3029 |
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| description | Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa
light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849),
we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis
experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now
carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids
that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino
acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete
loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which
are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated
enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and
electrostatic interactions which appear to be conserved among various target enzymes of CaM. |
| doi_str_mv | 10.1016/s0021-9258(19)50689-5 |
| format | Article |
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light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849),
we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis
experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now
carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids
that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino
acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete
loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which
are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated
enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and
electrostatic interactions which appear to be conserved among various target enzymes of CaM.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/s0021-9258(19)50689-5</identifier><identifier>PMID: 1737757</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>activation ; Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Animals ; binding ; Binding Sites ; Biological and medical sciences ; calmodulin ; Calmodulin - metabolism ; Calmodulin - pharmacology ; Chickens ; Enzyme Activation ; Enzymes and enzyme inhibitors ; Escherichia coli - genetics ; Fundamental and applied biological sciences. Psychology ; Immunoblotting ; Kinetics ; Molecular Sequence Data ; Muscle, Smooth - enzymology ; Mutagenesis, Site-Directed ; myosin light chain kinase ; Myosin-Light-Chain Kinase - genetics ; Myosin-Light-Chain Kinase - isolation & purification ; Myosin-Light-Chain Kinase - metabolism ; Polymerase Chain Reaction - methods ; Protein Conformation ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sequence Homology, Nucleic Acid ; site-directed mutagenesis ; smooth muscles ; Transferases</subject><ispartof>The Journal of biological chemistry, 1992-02, Vol.267 (5), p.3024-3029</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c504t-b3b99089b20cc978e2c5b07aa89aa45854e7e2ac21db9216f1e659f842edde0f3</citedby><cites>FETCH-LOGICAL-c504t-b3b99089b20cc978e2c5b07aa89aa45854e7e2ac21db9216f1e659f842edde0f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5210545$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1737757$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bagchi, I C</creatorcontrib><creatorcontrib>Huang, Q H</creatorcontrib><creatorcontrib>Means, A R</creatorcontrib><title>Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa
light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849),
we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis
experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now
carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids
that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino
acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete
loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which
are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated
enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and
electrostatic interactions which appear to be conserved among various target enzymes of CaM.</description><subject>activation</subject><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>binding</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>calmodulin</subject><subject>Calmodulin - metabolism</subject><subject>Calmodulin - pharmacology</subject><subject>Chickens</subject><subject>Enzyme Activation</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Immunoblotting</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Muscle, Smooth - enzymology</subject><subject>Mutagenesis, Site-Directed</subject><subject>myosin light chain kinase</subject><subject>Myosin-Light-Chain Kinase - genetics</subject><subject>Myosin-Light-Chain Kinase - isolation & purification</subject><subject>Myosin-Light-Chain Kinase - metabolism</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Protein Conformation</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Homology, Nucleic Acid</subject><subject>site-directed mutagenesis</subject><subject>smooth muscles</subject><subject>Transferases</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkF9r1jAUh4Mo83X6EQZBRPSiM0mTtrkcwz-DwS6m4F1Ik9M12iSzp9Xt25v6vryemxz4Pb8TeAg54-ycM958QMYEr7RQ3Tuu3yvWdLpST8iOs66uasW_PyW7I_KcvED8wcpIzU_ICW_rtlXtjjxceUhLGIKzS8iJ5oHaGFKm1gWPFBC32E50yDN1dorZr1NItA_Jh3RHbfIFXcLvYx1jzstI44puAhofMxZ8CnfjQt1oy_4zJIvwkjwb7ITw6vCekm-fPn69_FJd33y-ury4rpxicqn6uteadboXzDnddiCc6llrbaetlapTEloQ1gnuey14M3BolB46KcB7YEN9St7u797P-dcKuJgY0ME02QR5RcMbIYXUuoBqD7o5I84wmPs5RDs_Gs7MZtzcbjrNptNwbf4ZN6r0zg4frH0E_7-1V1zyN4fcYhE4zDa5gEdMCc6U3M683mNjUfUnzGD6kN0I0YimNcrUTMj6L5bils0</recordid><startdate>19920215</startdate><enddate>19920215</enddate><creator>Bagchi, I C</creator><creator>Huang, Q H</creator><creator>Means, A R</creator><general>American Society for Biochemistry and Molecular Biology</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>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope></search><sort><creationdate>19920215</creationdate><title>Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase</title><author>Bagchi, I C ; Huang, Q H ; Means, A R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c504t-b3b99089b20cc978e2c5b07aa89aa45854e7e2ac21db9216f1e659f842edde0f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>activation</topic><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>binding</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>calmodulin</topic><topic>Calmodulin - metabolism</topic><topic>Calmodulin - pharmacology</topic><topic>Chickens</topic><topic>Enzyme Activation</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Immunoblotting</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Muscle, Smooth - enzymology</topic><topic>Mutagenesis, Site-Directed</topic><topic>myosin light chain kinase</topic><topic>Myosin-Light-Chain Kinase - genetics</topic><topic>Myosin-Light-Chain Kinase - isolation & purification</topic><topic>Myosin-Light-Chain Kinase - metabolism</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Protein Conformation</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Homology, Nucleic Acid</topic><topic>site-directed mutagenesis</topic><topic>smooth muscles</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bagchi, I C</creatorcontrib><creatorcontrib>Huang, Q H</creatorcontrib><creatorcontrib>Means, A R</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bagchi, I C</au><au>Huang, Q H</au><au>Means, A R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1992-02-15</date><risdate>1992</risdate><volume>267</volume><issue>5</issue><spage>3024</spage><epage>3029</epage><pages>3024-3029</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa
light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849),
we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis
experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now
carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids
that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino
acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete
loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which
are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated
enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and
electrostatic interactions which appear to be conserved among various target enzymes of CaM.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>1737757</pmid><doi>10.1016/s0021-9258(19)50689-5</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1992-02, Vol.267 (5), p.3024-3029 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | activation Amino Acid Sequence Analytical, structural and metabolic biochemistry Animals binding Binding Sites Biological and medical sciences calmodulin Calmodulin - metabolism Calmodulin - pharmacology Chickens Enzyme Activation Enzymes and enzyme inhibitors Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Immunoblotting Kinetics Molecular Sequence Data Muscle, Smooth - enzymology Mutagenesis, Site-Directed myosin light chain kinase Myosin-Light-Chain Kinase - genetics Myosin-Light-Chain Kinase - isolation & purification Myosin-Light-Chain Kinase - metabolism Polymerase Chain Reaction - methods Protein Conformation Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sequence Homology, Nucleic Acid site-directed mutagenesis smooth muscles Transferases |
| title | Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase |
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