Oxidation of Met¹⁴⁴ and Met¹⁴⁵ in Calmodulin Blocks Calmodulin Dependent Activation of the Plasma Membrane Ca-ATPase

Abstract

Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.

Differential Modulation of Ca2+/Calmodulin-dependent Protein Kinase II Activity by Regulated Interactions with N-Methyl-D-aspartate Receptor NR2B Subunits and α-Actinin

“…Murine CaMKII␣ (wild-type and a double mutant of Thr 305 and Thr 306 autophosphorylation sites to Ala (T305A/T306A)) was purified from baculovirus-infected Sf9 cells, as described (27)(28)(29). Bovine brain calmodulin and recombinant chicken calmodulin expressed in E. coli were purified as described previously (30,31). All purified proteins were quantified using Bradford (Bio-Rad) or bicinchoninic acid (BCA, Pierce) assays.…”

Essential Role of Methionine Residues in Calmodulin Binding to Bordetella pertussis Adenylate Cyclase, as Probed by Selective Oxidation and Repair by the Peptide Methionine Sulfoxide Reductases

“…Oxidized CaM is unable to interact properly with and activate the plasma membrane Ca 2ϩ -ATPase (4). Similarly oxidized CaM exhibits a reduced affinity for nitric-oxide synthases (5).…”

Post-translational Modifications and Mass Spectrometry Detection

“…However, more recently evidence for a role of methionine residues in the redox regulation of enzyme activity has been found. A well-established case is that of calmodulin, a calcium regulatory protein, where the oxidation of two methionines has been reported to cause structural changes, altering its interaction with target proteins (185,186). The role of cysteine residues in redox regulation is determined by the chemistry of its thiol function.…”