Abstract
Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.
Highlights
Calmodulin (CaM) is a highly conserved Ca2+-receptor protein, ubiquitous in all eukaryotic organisms, implicated in the regulation of many cellular systems by transducing changes in the concentration of free Ca2+ in the cytosol and other intracellular compartments and structures
We studied the fluorescence energy transfer from excited tyrosine residues to Tb3+, a surrogate ion that binds CaM at the Ca2+-binding sites, in wild type CaM and the different CaM mutants by measuring the fluorescence emitted by Tb3+ in the 535–550 nm region upon exciting at 280 nm the tyrosine residues located at sites III and IV of CaM
In contrast to the homogeneous electrophoretic migration ( 18 kDa) of the single or double Y/D and Y/E CaM mutants in the absence of Ca2+, and the near absence of Ca2+-induced electrophoretic mobility shift of the single or double CaM mutants where Y138 was substituted by acidic residues (D or E) observed in this work, the non-phosphorylatable Y/F CaM mutants previously described presented a different behavior [22]
Summary
Calmodulin (CaM) is a highly conserved Ca2+-receptor protein, ubiquitous in all eukaryotic organisms, implicated in the regulation of many cellular systems by transducing changes in the concentration of free Ca2+ in the cytosol and other intracellular compartments and structures (reviewed in [1]). CaM is multifunctional and controls a myriad of cellular processes ranging from egg fertilization, muscle contraction, osmotic control and ions transport, metabolism, gene expression, cell migration, proliferation, autophagy and programmed cell death, among. Phospho-(Tyrosine)-Mimetic Calmodulin Mutants supported by fellowship and grants (03-00-6057-2005 and PG-03-8728-2013) from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela
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