Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain.
The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported (Hashimoto et al., 1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released [32P]phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN (Kincaid et al., 1988) allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding site. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM.
Martensen
T M
TM
Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852.
Martin
B M
BM
Kincaid
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D007700:Kinetics; D008715:Methionine / Q000032:analysis; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010768:Phosphoserine; D008715:Methionine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D003488:Cyanogen Bromide