Identification of the site on calcineurin phosphorylated by Ca sup + /CaM-dependent kinase II: Modification of the CaM-binding domain

Journal Article · Tue Nov 28 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00450a002· OSTI ID:6855138

Martensen, T M; Kincaid, R L ^[1]; Martin, B M ^[2]

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1. National Institute on Alcohol Abuse and Alcoholism, Rockville, MD (USA)
2. National Institute of Mental Health, Bethesda, MD (USA)

The catalytic subunit of the Ca{sup 2+}/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca{sup 2+}/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. 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 ({sup 32}P)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 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 sites. 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.

• OSTI ID: 6855138
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:24; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACID SEQUENCE
ATP
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CALCIUM COMPOUNDS
CALMODULIN
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERASES
HYDROLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHATASES
PHOSPHOPROTEINS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
PURIFICATION
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
TRANSFERASES