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Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase
A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation...
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Published in: | Biochemistry (Easton) 1986-03, Vol.25 (6), p.1458-1464 |
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creator | Lukas, Thomas J Burgess, Wilson H Prendergast, Franklyn G Lau, Wai Watterson, D. Martin |
description | A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified. |
doi_str_mv | 10.1021/bi00354a041 |
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Martin</creator><creatorcontrib>Lukas, Thomas J ; Burgess, Wilson H ; Prendergast, Franklyn G ; Lau, Wai ; Watterson, D. Martin</creatorcontrib><description>A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00354a041</identifier><identifier>PMID: 3754463</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Amino Acids - analysis ; Analytical, structural and metabolic biochemistry ; Animals ; Binding Sites ; Biological and medical sciences ; calmodulin ; Calmodulin - metabolism ; Chickens ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; gizzard ; Gizzard, Avian - enzymology ; Kinetics ; Macromolecular Substances ; Molecular Weight ; Muscle, Smooth - enzymology ; Myosin-Light-Chain Kinase ; Peptide Fragments - analysis ; Phosphopeptides - analysis ; Phosphorylation ; Protein Binding ; Protein Conformation ; Protein Kinases - metabolism ; Transferases</subject><ispartof>Biochemistry (Easton), 1986-03, Vol.25 (6), p.1458-1464</ispartof><rights>1987 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a480t-afbd9b9bc81be4ebdec53c3e1bd190099e5f8f352547778eed8172152fc0e06c3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00354a041$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00354a041$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,27063,27923,27924,56765,56815</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7960994$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3754463$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lukas, Thomas J</creatorcontrib><creatorcontrib>Burgess, Wilson H</creatorcontrib><creatorcontrib>Prendergast, Franklyn G</creatorcontrib><creatorcontrib>Lau, Wai</creatorcontrib><creatorcontrib>Watterson, D. Martin</creatorcontrib><title>Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.</description><subject>Amino Acids - analysis</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>calmodulin</subject><subject>Calmodulin - metabolism</subject><subject>Chickens</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gizzard</subject><subject>Gizzard, Avian - enzymology</subject><subject>Kinetics</subject><subject>Macromolecular Substances</subject><subject>Molecular Weight</subject><subject>Muscle, Smooth - enzymology</subject><subject>Myosin-Light-Chain Kinase</subject><subject>Peptide Fragments - analysis</subject><subject>Phosphopeptides - analysis</subject><subject>Phosphorylation</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Protein Kinases - metabolism</subject><subject>Transferases</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><recordid>eNqFkcFrFDEYxYNY6lo9eRbmIHqQ0S-TZDLxpovVQkHB9eIlJJmkm3YmWZMZcKV_vCmzLIJCD-FLvvfjEd5D6BmGNxga_FZ7AMKoAoofoBVmDdRUCPYQrQCgrRvRwiP0OOfr8qTA6Sk6JZxR2pIVul2rYYz9PPhQaR96H66qPo7Kh_yuMluVlJls8r_V5GOooqtUtdvGXE7aD8tShb4y_7pkP9nKpThW4z7mIgz-ajvdeZb7jQ8q2yfoxKkh26eHeYa-n3_crD_Xl18-XazfX9aKdjDVyuleaKFNh7WlVvfWMGKIxbrHAkAIy1znCGsY5Zx31vYd5k3JwRmw0Bpyhl4uvrsUf842T3L02dhhUMHGOUve8k4AJfeCmFICrGsK-HoBTYo5J-vkLvlRpb3EIO9KkX-VUujnB9tZj7Y_socWiv7ioKtcknRJBePzEeOlQCFoweoF83myv46ySjey5cVLbr5-k-3mA_zAm0aeF_7VwiuT5XWcUygh__eDfwDvyrHu</recordid><startdate>19860301</startdate><enddate>19860301</enddate><creator>Lukas, Thomas J</creator><creator>Burgess, Wilson H</creator><creator>Prendergast, Franklyn G</creator><creator>Lau, Wai</creator><creator>Watterson, D. Martin</creator><general>American Chemical Society</general><scope>BSCLL</scope><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>7QP</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>19860301</creationdate><title>Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase</title><author>Lukas, Thomas J ; Burgess, Wilson H ; Prendergast, Franklyn G ; Lau, Wai ; Watterson, D. Martin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a480t-afbd9b9bc81be4ebdec53c3e1bd190099e5f8f352547778eed8172152fc0e06c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Amino Acids - analysis</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>calmodulin</topic><topic>Calmodulin - metabolism</topic><topic>Chickens</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gizzard</topic><topic>Gizzard, Avian - enzymology</topic><topic>Kinetics</topic><topic>Macromolecular Substances</topic><topic>Molecular Weight</topic><topic>Muscle, Smooth - enzymology</topic><topic>Myosin-Light-Chain Kinase</topic><topic>Peptide Fragments - analysis</topic><topic>Phosphopeptides - analysis</topic><topic>Phosphorylation</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Protein Kinases - metabolism</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lukas, Thomas J</creatorcontrib><creatorcontrib>Burgess, Wilson H</creatorcontrib><creatorcontrib>Prendergast, Franklyn G</creatorcontrib><creatorcontrib>Lau, Wai</creatorcontrib><creatorcontrib>Watterson, D. Martin</creatorcontrib><collection>Istex</collection><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>Calcium & Calcified Tissue Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lukas, Thomas J</au><au>Burgess, Wilson H</au><au>Prendergast, Franklyn G</au><au>Lau, Wai</au><au>Watterson, D. Martin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1986-03-01</date><risdate>1986</risdate><volume>25</volume><issue>6</issue><spage>1458</spage><epage>1464</epage><pages>1458-1464</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>3754463</pmid><doi>10.1021/bi00354a041</doi><tpages>7</tpages></addata></record> |
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subjects | Amino Acids - analysis Analytical, structural and metabolic biochemistry Animals Binding Sites Biological and medical sciences calmodulin Calmodulin - metabolism Chickens Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology gizzard Gizzard, Avian - enzymology Kinetics Macromolecular Substances Molecular Weight Muscle, Smooth - enzymology Myosin-Light-Chain Kinase Peptide Fragments - analysis Phosphopeptides - analysis Phosphorylation Protein Binding Protein Conformation Protein Kinases - metabolism Transferases |
title | Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase |
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