Phosphorylation of calmodulin alters its potency as an activator of target enzymes.
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.
Quadroni
M
M
Protein Chemistry Laboratory, Swiss Federal Institute of Technology, Zürich, Switzerland.
L'Hostis
E L
EL
Corti
C
C
Myagkikh
I
I
Durussel
I
I
Cox
J
J
James
P
P
Carafoli
E
E
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D047388:Casein Kinases; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D015536:Down-Regulation; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008297:Male; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D013737:Testis / Q000201:enzymology
D002147:Calmodulin; D011494:Protein Kinases; D047388:Casein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium