Phosphorylation of Calmodulin Alters Its Potency as an Activator of Target Enzymes

Abstract

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

“…tinct from the reported effects on neuronal NOS (nNOS) kinetics: Quadroni et al (27) found that in vitro CK2 treatment of CaM increases the nNOS maximal activity. Although eNOS and nNOS have similar features, their amino acid sequences are only 60% identical.…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…Phosphorylation of CaM decreases its affinity for target substrates (378), and mutation of the conserved phosphorylation site in CaBP1 resulted in even greater [Ca 2ϩ ] i signaling inhibition (225). Although this site is not conserved in CIB1, it remains possible that phosphorylation, other covalent modifications, and other protein interactions could regulate the affinities of the interactions of CaBP1 and CIB1 with the InsP 3 R. Unbinding of the protein ligands could enable the channel to escape from inactivation, allowing the channel to again be activated by protein ligand rebinding, in a mechanism of repeated channel activation that is completely independent of InsP 3 .…”

Phosphorylation of the Ca2+-Binding Protein CaBP4 by Protein Kinase C ζ in Photoreceptors

“…Phosphorylation of Ca 2ϩ -binding proteins, including CaM, can affect their Ca 2ϩ -binding capability and interaction with target proteins (Benaim and Villalobo, 2002). Casein kinase 2 (CK2) phosphorylates several amino acid residues in CaM, including serine 101, which decreases the affinity of CaM for its substrates (Quadroni et al, 1998;Bildl et al, 2004;Kasri et al, 2004;Allen et al, 2007). Similar to CaM, an analogous residue (Serine 120) in CaBP1 is phosphorylated by casein kinase 2, which weakens the effect of CaBP1 in inhibiting Ca 2ϩ release by the IP 3 receptor (Kasri et al, 2004).…”