Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.
Huang
D
D
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202, USA.
Farkas
I
I
Roach
P J
PJ
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D038341:Glycogen Synthase Kinases; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D017931:DNA Primers; D005656:Fungal Proteins; D010710:Phosphates; D029701:Saccharomyces cerevisiae Proteins; D006003:Glycogen; C071570:SNF1-related protein kinases; D038341:Glycogen Synthase Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D005947:Glucose