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.
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.
Ekdahl
K N
KN
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006597:Fructose-Bisphosphatase / Q000032:analysis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000032:analysis*; D013329:Structure-Activity Relationship; D013997:Time Factors
D012694:Serine; D011494:Protein Kinases; D006597:Fructose-Bisphosphatase