Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

Abstract

In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

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

“…These functions appear to be exerted through the binding of different cyclins (Huang et al 1998). Some cyclins are synthesized periodically while the others are constitutively present (Measday et al 1997;Aerne et al 1998).…”

New Structural Insights into Phosphorylation-free Mechanism for Full Cyclin-dependent Kinase (CDK)-Cyclin Activity and Substrate Recognition

“…Pcl10/Pcl8 targets Pho85 to negatively regulate the substrate, Gsy2. Gsy2 is the predominant nutritionally regulated isoform of glycogen synthase in yeast whose activity is inhibited by Pho85-Pcl8/10 mediated phosphorylation, preventing the hyper-accumulation of glycogen (11,17). Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATP␥S.…”

Combining chemical genetics and proteomics to identify protein kinase substrates

“…However, cells lacking PHO85 have additional phenotypes unrelated to the phosphate starvation pathway (10,11). Ten different Pho85 cyclins, termed Pcls, have been shown to associate with Pho85 and are thought to direct it to phosphorylate distinct substrates involved in different cellular processes (12,13). To better understand the role of the Pho85-Pcl1 cyclin-dependent kinase, we undertook a systematic screen of the yeast proteome for substrates, making use of an AS version of Pho85, Pho85(F82G), which functionally substitutes for wild-type Pho85 in vivo, and which is able to use the ATP analog N 6 -benzyl-ATP in vitro (14).…”