Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system

TY - JOUR

T1 - Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system

AU - Panina, Svetlana

AU - Stephan, Alexander

AU - la Cour, Jonas Marstrand

AU - Jacobsen, Kivin

AU - Kallerup, Line K.

AU - Bumbuleviciute, Rasita

AU - Knudsen, Kristoffer Vitting Klinkby

AU - Sanchez-Gonzalez, Pablo

AU - Villalobo, Antonio

AU - Olesen, Uffe Høgh

AU - Berchtold, Martin Werner

PY - 2012/5/25

Y1 - 2012/5/25

N2 - Calmodulin (CaM) was shown to be essential for survival oflower eukaryotes by gene deletion experiments. So far, no CaMgene deletion was reported in higher eukaryotes. In vertebrates,CaM is expressed from several genes, which encode an identicalprotein, making it difficult to generate a model system to studythe effect ofCaMgene deletion. Here, we present a novel geneticsystem based on the chicken DT40 cell line, in which the twofunctional CaM genes were deleted and one allele replaced witha CaM transgene that can be artificially regulated.Weshow thatCaM is essential for survival of vertebrate cells as they die in theabsence of CaM expression. Reversal of CaM repression orectopic expression of HA-tagged CaM rescued the cells. Cellsexclusively expressing HA-CaM with impaired individual calciumbinding domains as well as HA-CaM lacking the ability tobe phosphorylated at residues Tyr99/Tyr138 or trimethylated atLys115 survived and grew well. CaM mutated at both Ca2 bindingsites 3 and 4 as well as at both sites 1 and 2, but to a lesserdegree, showed decreased ability to support cell growth. Cellsexpressing CaM with all calcium binding sites impaired diedwith kinetics similar to that of cells expressing no CaM. Thissystem offers a unique opportunity to analyze CaM structurefunctionrelationships in vivo without the use of pharmacologicalinhibitors and to analyze the function of wild type andmutated CaM in modulating the activity of different target systemswithout interference of endogenous CaM.

AB - Calmodulin (CaM) was shown to be essential for survival oflower eukaryotes by gene deletion experiments. So far, no CaMgene deletion was reported in higher eukaryotes. In vertebrates,CaM is expressed from several genes, which encode an identicalprotein, making it difficult to generate a model system to studythe effect ofCaMgene deletion. Here, we present a novel geneticsystem based on the chicken DT40 cell line, in which the twofunctional CaM genes were deleted and one allele replaced witha CaM transgene that can be artificially regulated.Weshow thatCaM is essential for survival of vertebrate cells as they die in theabsence of CaM expression. Reversal of CaM repression orectopic expression of HA-tagged CaM rescued the cells. Cellsexclusively expressing HA-CaM with impaired individual calciumbinding domains as well as HA-CaM lacking the ability tobe phosphorylated at residues Tyr99/Tyr138 or trimethylated atLys115 survived and grew well. CaM mutated at both Ca2 bindingsites 3 and 4 as well as at both sites 1 and 2, but to a lesserdegree, showed decreased ability to support cell growth. Cellsexpressing CaM with all calcium binding sites impaired diedwith kinetics similar to that of cells expressing no CaM. Thissystem offers a unique opportunity to analyze CaM structurefunctionrelationships in vivo without the use of pharmacologicalinhibitors and to analyze the function of wild type andmutated CaM in modulating the activity of different target systemswithout interference of endogenous CaM.

U2 - 10.1074/jbc.m112.339382

DO - 10.1074/jbc.m112.339382

M3 - Journal article

SN - 0021-9258

VL - 287

SP - 18173

EP - 18181

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 22

ER -