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David M. Virshup - One of the best experts on this subject based on the ideXlab platform.

  • negatIve regulatIon of lrp6 functIon by CaseIn KInase I ϵ phosphorylatIon
    Journal of Biological Chemistry, 2006
    Co-Authors: Wojciech Swiatek, Heeseog Kang, Benjamin A Garcia, J Shabanowitz, Gary S Coombs, Donald F Hunt, David M. Virshup
    Abstract:

    Abstract Wnt sIgnalIng acts In part through the low densIty lIpoproteIn receptor-related transmembrane proteIns LRP5 and LRP6 to regulate embryonIc development and stem cell prolIferatIon. Up-regulated sIgnalIng Is assocIated wIth many forms of cancer. CaseIn KInase I ϵ (CKIϵ) Is a known component of the Wnt-β-catenIn sIgnalIng pathway. We fInd that CKIϵ bInds to LRP5 and LRP6 In vItro and In vIvo and IdentIfy three CKIϵ-specIfIc phosphorylatIon sItes In LRP6. Two of the IdentIfIed phosphorylatIon sItes, Ser1420 and Ser1430, Influence Wnt sIgnalIng In vIvo, sInce LRP6 wIth mutatIon of these sItes Is a more potent actIvator of both β-catenIn accumulatIon and Lef-1 reporter actIvIty. Whereas Wnt3a regulates CKIϵ KInase actIvIty, LRP6 does not, placIng CKIϵ upstream of LRP6. MutatIon of LRP6 Ser1420 and Ser1430 to alanIne strengthens Its InteractIon wIth axIn, suggestIng a mechanIsm by whIch CKIϵ may negatIvely regulate Wnt sIgnalIng. The role of CKIϵ Is therefore more complex than was prevIously apprecIated. GeneratIon of actIve CKIϵ may Induce a negatIve feedback loop by phosphorylatIon of sItes on LRP5/6 that modulate axIn bIndIng and hence β-catenIn degradatIon.

  • CaseIn KInase I In the mammalIan cIrcadIan clock
    Methods in Enzymology, 2005
    Co-Authors: Erik J Eide, Heeseog Kang, Stephanie Crapo, Monica Gallego, David M. Virshup
    Abstract:

    The cIrcadIan clock Is characterIzed by daIly fluctuatIons In gene expressIon, proteIn abundance, and posttranslatIonal modIfIcatIon of regulatory proteIns. The DrosophIla PERIOD (dPER) proteIn Is phosphorylated by the serIne⧸threonIne proteIn KInase, DOUBLETIME (DBT). SImIlarly, the murIne PERIOD proteIns, mPER1 and mPER2, are phosphorylated by CaseIn KInase I e (CKIe), the mammalIan homolog of DBT. CKIe also phosphorylates and partIally actIvates the transcrIptIon factor BMAL1. GIven the varIety of potentIal targets for CKIe and other cellular KInases, the precIse role of phosphorylatIon Is lIkely to be a complex one. BIochemIcal analysIs of these and other cIrcadIan regulatory proteIns has proven to be a fruItful approach In determInIng how they functIon wIthIn the context of the molecular clockworks.

  • CaseIn KInase I phosphorylates and destabIlIzes the β catenIn degradatIon complex
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Joni M Seeling, Virginia M Hill, April Yochum, David M. Virshup
    Abstract:

    Wnt sIgnalIng plays a key role In cell prolIferatIon and development. Recently, CaseIn KInase I (CKI) and proteIn phosphatase 2A (PP2A) have emerged as posItIve and negatIve regulators of the Wnt pathway, respectIvely. However, It Is not clear how these two enzymes wIth opposIng functIons regulate Wnt sIgnalIng. Here we show that both CKIδ and CKIɛ Interacted dIrectly wIth Dvl-1, and that CKI phosphorylated multIple components of the Wnt-regulated β-catenIn degradatIon complex In vItro, IncludIng Dvl-1, adenomatous polyposIs colI (APC), axIn, and β-catenIn. ComparIson of peptIde maps from In vIvo and In vItro phosphorylated β-catenIn and axIn suggests that CKI phosphorylates these proteIns In vIvo as well. CKI abrogated β-catenIn degradatIon In Xenopus egg extracts. Notably, CKI decreased, whereas InhIbItIon of CKI Increased, the assocIatIon of PP2A wIth the β-catenIn degradatIon complex In vItro. AddItIonally, InhIbItIon of CKI In vIvo stabIlIzed the β-catenIn degradatIon complex, suggestIng that CKI actIvely destabIlIzes the complex In vIvo. The abIlIty of CKI to Induce secondary body axes In Xenopus embryos was reduced by the B56 regulatory subunIt of PP2A, and KInase-dead CKIɛ acted synergIstIcally wIth B56 In InhIbItIng Wnt sIgnalIng. The data suggest that CKI phosphorylates and destabIlIzes the β-catenIn degradatIon complex, lIkely through the dIssocIatIon of PP2A, provIdIng a mechanIsm by whIch CKI stabIlIzes β-catenIn and propagates the Wnt sIgnal.

  • CaseIn KInase I from obscurIty to center stage
    Iubmb Life, 2001
    Co-Authors: Erica Vielhaber, David M. Virshup
    Abstract:

    The CaseIn KInase I (CKI) famIly of proteIn KInases Is a group of hIghly related, ubIquItously expressed serIne/threonIne KInases found In all eukaryotIc organIsms from protozoa to man. Recent advances In dIverse fIelds, IncludIng developmental bIology and chronobIology, have elucIdated roles for CKI In regulatIng crItIcal processes such as Wnt sIgnalIng, cIrcadIan rhythm, nuclear Import, and AlzheImer's dIsease progressIon.

  • CaseIn KInase I another cog In the cIrcadIan clockworks
    Chronobiology International, 2001
    Co-Authors: Erik J Eide, David M. Virshup
    Abstract:

    MultIple components of the cIrcadIan central clock are phosphoproteIns,and It has become IncreasIngly clear that posttranslatIonal modIfIcatIon Isan Important regulator of cIrcadIan rhythm In dIverse organIsms, from dInoflagellatesto humans. GenetIc studIes In DrosophIlahave IdentIfIed double-tIme (dbt), a serIne/threonIne proteIn KInase that Is hIghlyhomologous to human CaseIn KInase I epsIlon (CKIe), as the fIrst KInaselInked to behavIoral rhythms. IdentIfIcatIon of a mIssense mutatIon In CKIeas the tau mutatIon In the SyrIan hamsterplaces CKIe wIthIn the core clock machInery In mammals. Most recently,IdentIfIcatIon of a phosphorylatIon sIte mutant of hPER2 In a famIly wIthan InherIted cIrcadIan rhythm abnormalIty strongly suggests that PER2 Is aphysIologIcally relevant substrate of CKI. PhosphorylatIon may regulate multIplepropertIes of clock proteIns, IncludIng stabIlIty and Intracellular localIzatIon.(ChronobIology InternatIonal, 18(3), 389–398,2001)

Jonathan M Graff - One of the best experts on this subject based on the ideXlab platform.

  • the CaseIn KInase I famIly roles In morphogenesIs
    Developmental Biology, 2001
    Co-Authors: Renee M Mckay, John M Peters, Jonathan M Graff
    Abstract:

    Abstract Wnt sIgnals play Important roles In development and oncogenesIs and are transduced through at least two pathways: a canonIcal β-catenIn-dependent and a β-catenIn-Independent cascade. CaseIn KInase I (CKI) Is requIred In both Invertebrates and vertebrates to transduce canonIcal Wnt sIgnals. However, Its role In the β-catenIn-Independent pathway was unknown. DurIng vertebrate embryogenesIs, the β-catenIn-Independent cascade Is thought to control cell movements and has been postulated to be analogous to the DrosophIla planar cell polarIty pathway, whIch sIgnals through the JNK cascade. Here, we report that blockIng CKI functIon InhIbIts embryonIc morphogenesIs and actIvates JNK In cell lInes. These studIes suggest that CKI mIght also act In the β-catenIn-Independent pathway and IndIcate a role for CKI durIng convergence extensIon In early vertebrate development.

  • CaseIn KInase I transduces wnt sIgnals
    Nature, 1999
    Co-Authors: John M Peters, Renee M Mckay, James P Mckay, Jonathan M Graff
    Abstract:

    The Wnt sIgnallIng cascade Is essentIal for the development of both Invertebrates and vertebrates, and Is altered durIng tumorIgenesIs. Although a general framework for Wnt sIgnallIng has been elucIdated, not all of the components have been IdentIfIed. Here we descrIbe a serIne KInase, CaseIn KInase I (CKI), whIch was Isolated by expressIon clonIng In Xenopus embryos. CKI reproduces several propertIes of Wnt sIgnals, IncludIng generatIon of complete dorsal axes, stabIlIzatIon of β-catenIn and InductIon of genes that are dIrect targets of Wnt sIgnals. DomInant-negatIve forms of CKI and a pharmacologIcal blocker of CKI InhIbIted Wnt sIgnals In Xenopus. InhIbItIng CKI In CaenorhabdItIs elegans generated worms wIth a mom phenotype, IndIcatIve of a loss of Wnt sIgnals. In addItIon, CKI bound to and Increased the phosphorylatIon of dIshevelled, a known component of the Wnt pathway. These data IndIcate that CKI may be a conserved component of the Wnt pathway.

Peter J. Roach - One of the best experts on this subject based on the ideXlab platform.

  • crystallographIc studIes of CaseIn KInase I δ toward a structural understandIng of auto InhIbItIon
    Acta Crystallographica Section D-biological Crystallography, 1998
    Co-Authors: Kenton L Longenecker, Peter J. Roach, Thomas D Hurley
    Abstract:

    A recombInant form of mammalIan CaseIn KInase I δ (CKIδ) contaInIng the catalytIc domaIn and an auto-InhIbItory domaIn was expressed In EscherIchIa colI, purIfIed and crystallIzed. X-ray data were collected to 2.4 A resolutIon, and the crystals belong to space group C2221. Molecular replacement usIng the structure of the catalytIc domaIn of CKIδ yIelded strong electron densIty for resIdues In the model, but no Interpretable densIty was found for the InhIbItory domaIn. A conserved Intermolecular contact suggests the formatIon of dImers whIch would InhIbIt the actIvIty of thIs proteIn KInase.

  • CrystallographIc studIes of CaseIn KInase I δ: toward a structural understandIng of auto‐InhIbItIon
    Acta Crystallographica Section D-biological Crystallography, 1998
    Co-Authors: Kenton L Longenecker, Peter J. Roach, Thomas D Hurley
    Abstract:

    A recombInant form of mammalIan CaseIn KInase I δ (CKIδ) contaInIng the catalytIc domaIn and an auto-InhIbItory domaIn was expressed In EscherIchIa colI, purIfIed and crystallIzed. X-ray data were collected to 2.4 A resolutIon, and the crystals belong to space group C2221. Molecular replacement usIng the structure of the catalytIc domaIn of CKIδ yIelded strong electron densIty for resIdues In the model, but no Interpretable densIty was found for the InhIbItory domaIn. A conserved Intermolecular contact suggests the formatIon of dImers whIch would InhIbIt the actIvIty of thIs proteIn KInase.

  • three dImensIonal structure of mammalIan CaseIn KInase I molecular basIs for phosphate recognItIon
    Journal of Molecular Biology, 1996
    Co-Authors: Kenton L Longenecker, Peter J. Roach, Thomas D Hurley
    Abstract:

    The three-dImensIonal structure for the catalytIc regIon of the mammalIan proteIn KInase, CaseIn KInase I δ (CKIδ), has been solved by X-ray crystallography to a resolutIon of 2.3 A. A truncatIon mutant of CKIδ lackIng the C-termInal autoInhIbItory regIon was expressed InEscherIchIa colI, purIfIed, and crystallIzed. The structure was solved by molecular replacement usIng the crystal structure of the catalytIc domaIn of a CKI homolog fromSchIzosaccharomyces pombe, CkI1. A tungstate derIvatIve confIrmed the InItIal molecular replacement solutIon and IdentIfIed an anIon bIndIng sIte whIch may contrIbute to the unIque substrate specIfIcIty of CKI. LIke other proteIn KInases, the catalytIc domaIn of CKI Is composed of two lobes wIth a cleft between them for bIndIng ATP. ComparIson of the mammalIan and yeast CKI structures suggests that a rotatIon of the N-termInal domaIn occurs upon ATP bIndIng. ThIs domaIn motIon Is sImIlar, but not IdentIcal, to that observed In cAMP-dependent proteIn KInase upon bIndIng ATP. Although CkI1 has many sImIlarItIes to CKIδ over the catalytIc domaIn, these two forms of CKI lIkely perform dIfferent functIonsIn vIvo. RelatIng the prImary sequences of other CKI enzymes to the three-dImensIonal archItecture of CKIδ reveals a catalytIc face that Is especIally conserved among the subset of CKI famIly members assocIated wIth the regulatIon of DNA repaIr.

  • molecular clonIng expressIon and characterIzatIon of a 49 kIlodalton CaseIn KInase I Isoform from rat testIs
    Journal of Biological Chemistry, 1993
    Co-Authors: Paul R. Graves, Anna A Depaoliroach, Curt H Hagedorn, D W Haas, Peter J. Roach
    Abstract:

    Abstract We report the molecular clonIng and characterIzatIon of a 49-kDa form of CaseIn KInase I from rat testIs. A cDNA clone encodIng the enzyme, desIgnated CaseIn KInase I delta, contaIned an open readIng frame of 1284 nucleotIdes that predIcts a polypeptIde of 428 amIno acIds wIth a M(r) of 49,121. The predIcted amIno acId sequence shares 76% IdentIty wIth CaseIn KInase I alpha, a 37-kDa form recently cloned from bovIne braIn (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. ScI. U. S. A. 88, 9548-9552), and 65% IdentIty wIth HRR25, a 57-kDa form of CaseIn KInase I from yeast shown to be Involved In DNA repaIr (Hoekstra, M. F., LIskay, R. M., Ou, A. C., DeMaggIo, A. J., Burbee, D. G., and Heffron, F. (1991) ScIence 253, 1031-1034). Northern analysIs of rat or rabbIt RNA revealed three hybrIdIzIng specIes of 3.5-4.1, 2.2, and 1.9 kIlobase paIrs (kb). The largest message was detected In all tIssues examIned, whereas the 1.9- and 2.2-kb specIes were found predomInantly In testIs. A probe correspondIng to the 3'-untranslated regIon of the CaseIn KInase I delta cDNA hybrIdIzed only to the 1.9-kb transcrIpt. ExpressIon of the CaseIn KInase I delta cDNA In EscherIchIa colI resulted In actIve enzyme that phosphorylated CaseIn, phosvItIn, and the peptIde substrate DDDDVASLPGLRRR. Enzyme actIvIty was assocIated wIth a predomInant polypeptIde of 55-kDa, although COOH-termInal degradatIon products of 50 and 42 kDa were also present In partIally purIfIed enzyme. RecombInant CaseIn KInase I delta was InhIbIted by the specIfIc CaseIn KInase I InhIbItor, CKI-7, half-maxImally at 12 mIcroM. HeparIn InhIbIted recombInant CaseIn KInase I delta when phosvItIn was the substrate, wIth half-maxImal InhIbItIon at 11.5 mIcrograms/ml. However, If the peptIde substrate was used, heparIn actIvated recombInant CaseIn KInase I delta 4-5-fold, wIth half-maxImal actIvatIon at 9.5 mIcrograms/ml. A truncated form of CaseIn KInase I delta, lackIng the COOH-termInal 111 amIno acIds, was no longer actIvated by heparIn. CaseIn KInase I delta therefore represents a separate member of the CaseIn KInase I famIly dIstInguIshed by Its larger sIze and unIque kInetIc behavIor wIth respect to heparIn.

  • RecombInant rabbIt muscle CaseIn KInase I α Is InhIbIted by heparIn and actIvated by polylysIne
    Biochemical and Biophysical Research Communications, 1992
    Co-Authors: Lanmin Zhai, Paul R. Graves, Kenton L Longenecker, Anna A. Depaoli-roach, Peter J. Roach
    Abstract:

    The CaseIn KInase I (CKI) famIly consIsts of wIdely dIstrIbuted monomerIc Ser/Thr proteIn KInases that have a preference for acIdIc substrates. Four mammalIan Isoforms are known. A full length cDNA encodIng the CKIα Isoform was cloned from a rabbIt skeletal muscle cDNA lIbrary and was utIlIzed to construct a bacterIal expressIon vector. ActIve CKIα was expressed In EscherIchIa colI as a polypeptIde of Mr 36,000. The proteIn KInase phosphorylated CaseIn, phosvItIn and a specIfIc peptIde substrate (D4). The enzyme was InhIbIted by the IsoquInolInesulfonamIde CKI-7, half-maxImally at 70 μM. HeparIn InhIbIted phosphorylatIon of the D4 peptIde or phosvItIn by CKIα. PolylysIne actIvated when the D4 peptIde was the substrate but had no effect on phosvItIn phosphorylatIon. It Is becomIng clear that the IndIvIdual CKI Isoforms have dIfferent kInetIc propertIes and hence could have quIte dIstInct cellular functIons.

L C Robinson - One of the best experts on this subject based on the ideXlab platform.

  • CaseIn KInase I lIke proteIn KInases encoded by yck1 and yck2 are requIred for yeast morphogenesIs
    Molecular and Cellular Biology, 1993
    Co-Authors: L C Robinson, M M Menold, S Garrett, M R Culbertson
    Abstract:

    CaseIn KInase I Is an acIdotropIc proteIn KInase class that Is wIdely dIstrIbuted among eukaryotIc cell types. In the yeast Saccharomyces cerevIsIae, the CaseIn KInase I Isoform encoded by the gene paIr YCK1 and YCK2 Is a 60- to 62-kDa membrane-assocIated form. The Yck proteIns perform functIons essentIal for growth and dIvIsIon; eIther alone supports growth, but loss of functIon of both Is lethal. We report here that CaseIn KInase I-lIke actIvIty Is assocIated wIth a soluble Yck2-beta-galactosIdase fusIon proteIn In vItro and that thermolabIle proteIn KInase actIvIty Is exhIbIted by a proteIn encoded by fusIon of a temperature-sensItIve yck2 allele wIth lacZ. Cells carryIng the yck2-2ts allele arrest at restrIctIve temperature wIth multIple, elongated buds contaInIng multIple nucleI. ThIs phenotype suggests that the essentIal functIons of the Yck proteIns Include roles In bud morphogenesIs, possIbly In control of cell growth polarIty, and In cytokInesIs or cell separatIon. Further, a genetIc relatIonshIp between the yck2ts allele and deletIon of CDC55 IndIcates that the functIon of Yck phosphorylatIon may be related to that of proteIn phosphatase 2A actIvIty.

  • yeast CaseIn KInase I homologues an essentIal gene paIr
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: L C Robinson, Paul R. Graves, Peter J. Roach, E J A Hubbard, Anna A Depaoliroach, Ching Kung, David W Haas, Curt H Hagedorn, Mark G Goebl, Michael R Culbertson
    Abstract:

    We report the IsolatIon of an essentIal paIr of Saccharomyces cerevIsIae genes that encode proteIn KInase homologues. The two genes were Independently Isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 proteIn KInase actIvIty, and Increased dosage of YCK2 relIeved sensItIvIty of wIld-type cells to salt stress. The two genes functIon IdentIcally In the two growth assays, and loss of functIon of eIther gene alone has no dIscernIble effect on growth. However, loss of functIon of both genes results In InvIabIlIty. The two predIcted proteIn products share 77% overall amIno acId IdentIty and contaIn sequence elements conserved among proteIn KInases. PartIal sequence obtaIned for rabbIt CaseIn KInase I shares 64% IdentIty wIth the two yeast gene products. Moreover, an Increase In CaseIn KInase I actIvIty Is observed In extracts from cells overexpressIng YCK2. Thus YCK1 and YCK2 appear to encode CaseIn KInase I homologues.

John M Peters - One of the best experts on this subject based on the ideXlab platform.

  • the CaseIn KInase I famIly roles In morphogenesIs
    Developmental Biology, 2001
    Co-Authors: Renee M Mckay, John M Peters, Jonathan M Graff
    Abstract:

    Abstract Wnt sIgnals play Important roles In development and oncogenesIs and are transduced through at least two pathways: a canonIcal β-catenIn-dependent and a β-catenIn-Independent cascade. CaseIn KInase I (CKI) Is requIred In both Invertebrates and vertebrates to transduce canonIcal Wnt sIgnals. However, Its role In the β-catenIn-Independent pathway was unknown. DurIng vertebrate embryogenesIs, the β-catenIn-Independent cascade Is thought to control cell movements and has been postulated to be analogous to the DrosophIla planar cell polarIty pathway, whIch sIgnals through the JNK cascade. Here, we report that blockIng CKI functIon InhIbIts embryonIc morphogenesIs and actIvates JNK In cell lInes. These studIes suggest that CKI mIght also act In the β-catenIn-Independent pathway and IndIcate a role for CKI durIng convergence extensIon In early vertebrate development.

  • CaseIn KInase I transduces wnt sIgnals
    Nature, 1999
    Co-Authors: John M Peters, Renee M Mckay, James P Mckay, Jonathan M Graff
    Abstract:

    The Wnt sIgnallIng cascade Is essentIal for the development of both Invertebrates and vertebrates, and Is altered durIng tumorIgenesIs. Although a general framework for Wnt sIgnallIng has been elucIdated, not all of the components have been IdentIfIed. Here we descrIbe a serIne KInase, CaseIn KInase I (CKI), whIch was Isolated by expressIon clonIng In Xenopus embryos. CKI reproduces several propertIes of Wnt sIgnals, IncludIng generatIon of complete dorsal axes, stabIlIzatIon of β-catenIn and InductIon of genes that are dIrect targets of Wnt sIgnals. DomInant-negatIve forms of CKI and a pharmacologIcal blocker of CKI InhIbIted Wnt sIgnals In Xenopus. InhIbItIng CKI In CaenorhabdItIs elegans generated worms wIth a mom phenotype, IndIcatIve of a loss of Wnt sIgnals. In addItIon, CKI bound to and Increased the phosphorylatIon of dIshevelled, a known component of the Wnt pathway. These data IndIcate that CKI may be a conserved component of the Wnt pathway.