Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase.
Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D003488:Cyanogen Bromide; D000242:Cyclic AMP / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D002147:Calmodulin; D010446:Peptide Fragments; D010710:Phosphates; D010750:Phosphoproteins; D010768:Phosphoserine; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000242:Cyclic AMP; D011494:Protein Kinases; D011770:Pyruvate Kinase; D003488:Cyanogen Bromide; D002118:Calcium