Rat liver fructose-1,6-bisphosphatase. Identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro

• Published in: The Journal of biological chemistry Vol. 262; no. 34; pp. 16699 - 16703
• Main Author: Ekdahl, KN
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 05.12.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Enzymes and enzyme inhibitors; Fructose-Bisphosphatase - analysis; Fundamental and applied biological sciences. Psychology; Hydrolases; Immunologi; Immunology; Kinetics; Liver - enzymology; MEDICIN; MEDICINE; Microbiology, immunology, infectious diseases; Mikrobiologi, immunologi, infektionssjukdomar; Peptide Mapping; Phosphorylation; Protein Kinases - metabolism; Rats; Serine - analysis; Structure-Activity Relationship; Time Factors; Fructose-bisphosphatase; Vertebrata; Phosphorylation; Regulation(control); Mammalia; Rat; Structure activity relation; Protein kinase; Liver; Rodentia; Catalyst activity
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/S0021-9258(18)49311-8

Rat liver fructose-1,6-bisphosphatase was phosphorylated with [32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. After digestion with trypsin, two peptides were isolated containing 68 and 32% of the total radioactivity, respectively. The former was found to contain the sequence Ala-Lys-Ser(P)-Arg-Pro-Ser(P)-Leu-Pro. In this fragment, Ser-341, but not Ser-338, had earlier been reported to be a phosphorylation site. The other peptide contained phosphorylated Ser-356. It was demonstrated that all the protein-bound [32P]phosphate was distributed evenly between these three serines in the native enzyme regardless of the degree of phosphorylation. Preservation of the three-dimensional structure, however, was needed to obtain phosphorylation of Ser-356. Peptides containing each phosphorylatable serine residue were sequentially removed by digesting the enzyme with chymotrypsin which cleaved off Ser-356, denaturing it with urea, digesting it further with chymotrypsin, thus removing Ser-341, and finally treating it with trypsin which eliminated the rest of the radioactivity which was bound to Ser-338. Kinetic studies of fructose-1,6-bisphosphatase digested in this manner revealed that phosphorylation of Ser-338 decreased the apparent Km for fructose 1,6-bisphosphatase, whereas phosphorylation of Ser-341 decreased the inhibitory effect of AMP and fructose 2,6-bisphosphatase, Phosphorylation of Ser-356 did not affect these parameters.