Direct and novel regulation of cAMP-dependent protein kinase by Mck1p, a yeast glycogen synthase kinase-3.
The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.
Rayner
Timothy F
TF
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA. t.rayner@bio.gla.ac.uk
Gray
Joseph V
JV
Thorner
Jeremy W
JW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D038341:Glycogen Synthase Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; C068124:MCK1 protein, S cerevisiae; C085710:mcm2 protein, S pombe