Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit M(r) of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit M(r) of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the K(m) value for fructose 6-phosphate from 87 to 42 μM without affecting the V(max), whereas the phosphorylation by protein kinase C increased the V(max) value from 55 to 85 milliunits/mg without altering the K(m) value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Ar -Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phorphorylated by protein kinase C.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1988|
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