Pho85p, a Cyclin-Dependent Protein Kinase, and the Snf1p Protein Kinase Act Antagonistically To Control Glycogen Accumulation in Saccharomyces cerevisiae

Abstract

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 ϳ36-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.

The Pho85 kinase, a member of the yeast cyclin‐ dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle

“…Suppression of growth arrest of a cln1 cln2 pho85 triple mutant was tested by growing MFY151 strain (Nishizawa et al 1998) harbouring Pho85 mutant on SD medium supplemented with methionine. Accumulation of glycogen was assayed as described (Huang et al 1996).…”

Swi5 Controls a Novel Wave of Cyclin Synthesis in Late Mitosis

“…Strains deleted for PHO85 grow slowly, are unable to use lactate or glycerol as a carbon source, and accumlate high levels of glycogen (Huang et al, 1996;Lenburg and O'Shea, 1996;Timblin et al, 1996). Thus, Pho85 may associate with other cyclins in addition to Pho80, Pcl1, and Pcl2, which play distinct roles.…”

A role for the Pcl9‐Pho85 cyclin–cdk complex at the M/G1 boundary in Saccharomyces cerevisiae

“…Glycogen synthase is required for glycogen biosynthesis and is inactive in its hyperphosphorylated form. In the absence of Pho85, glycogen hyperaccumulates, glycogen synthase kinase activity is decreased and glycogen synthase is activated (Huang et al, 1996;Timblin et al, 1996). Partial purification of yeast glycogen synthase kinase suggests that Pho85 is a major Gsy2 kinase in vivo (Huang et al, 1996).…”

“…In the absence of Pho85, glycogen hyperaccumulates, glycogen synthase kinase activity is decreased and glycogen synthase is activated (Huang et al, 1996;Timblin et al, 1996). Partial purification of yeast glycogen synthase kinase suggests that Pho85 is a major Gsy2 kinase in vivo (Huang et al, 1996). Recent studies have shown that two other Pho85 cyclins, Pcl8 and Pcl10, associate with Pho85 to convey specific kinase activity towards Gsy2 in vitro and in vivo (D. Huang and J. Moffat, unpublished).…”