Isolation of phosphorylated calmodulin from rat liver and identification of the in vivo phosphorylation sites.
A procedure is described for the isolation of calmodulin (CaM) from rat liver which produces a fraction containing non-phosphorylated, mono-, di-, and triphosphocalmodulin as determined by mass spectrometric analysis. The distribution of CaM between the various phospho-species varies from preparation to preparation even though the isolation procedure is rigidly defined, suggesting that CaM phosphorylation may be a very labile phenomenon dependent on the state of the liver as it is removed from the animal. Approximately 15% of CaM in the cell is phosphorylated. The in vivo phosphorylation sites were determined by mass spectrometric analysis of a combined CNBr and trypsin digestion of the phosphocalmodulin (phospho-CaM)-containing fractions. Phosphorylated peptides were sequenced using two mass scanning devices linked together for collisionally activated fragmentation studies to determine peptide sequences, and the phosphorylation sites were determined as Thr-79, Ser-81, and Ser-101. These correspond to three of the four in vitro target sites of calmodulin phosphorylation by casein kinase II, which indicates that this may be the enzyme responsible for the phosphorylation in vivo. A preliminary study on the modulatory activity of phosphorylated calmodulin using a sample extensively phosphorylated in vitro with casein kinase II confirmed that phospho-CaM has an altered biological activity, i.e. reduced activation of the erythrocyte plasma membrane Ca2+ pump.
Quadroni
M
M
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
James
P
P
Carafoli
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D047390:Casein Kinase II; D002462:Cell Membrane / Q000201:enzymology; D003488:Cyanogen Bromide; D004789:Enzyme Activation; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D003488:Cyanogen Bromide