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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*

S. Panina,A. Stephan,Jonas M. la Cour,K. Jacobsen,Line K. Kallerup,Rasita Bumbuleviciute,Kristoffer V. K. Knudsen,Pablo Sánchez-González,A. Villalobo,U. H. Olesen,M. Berchtold

Published 2012 in Journal of Biological Chemistry

ABSTRACT

Background: Calmodulin is a Ca2+ binding protein and a major regulator of multiple signaling pathways. Results: Inactivation of the Ca2+ binding sites in the N- and C-terminal lobe of CaM affects cell viability differentially. Conclusion: Ca2+ binding to CaM is required for vertebrate cell survival. Significance: A novel vertebrate knock-out/knock-in system for studying the function of CaM is described. Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr99/Tyr138 or trimethylated at Lys115 survived and grew well. CaM mutated at both Ca2+ binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.

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