Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p.
In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) catalytic subunit with multiple regulatory roles thought to be specified by association with different cyclin partners (Pcls). Pcl10p is one of four Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycogen synthase (Gsy2p). We report that recombinant Pho85p and Pcl10p produced in Escherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p phosphorylation required Pcl10p, occurred at physiologically relevant sites, and resulted in inactivation of Gsy2p. The activity of the reconstituted enzyme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclude that, unlike many Cdks, Pho85p does not require phosphorylation for activity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell lysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding the substrate. Synthetic peptides modeled on the sequences of known Pho85p sites were poor substrates with high K(m) values, and we propose that Pcl10p-Gsy2p interaction is important for substrate selection. Gel filtration of yeast cell lysates demonstrated that most Pho85p was present as a monomer, although a portion coeluted in high-molecular-weight fractions with Pcl10p and Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into association with Pcl10p. We suggest a model for Pho85p function in the cell whereby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activating the kinase and targeting it to substrate.
Wilson
W A
WA
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Mahrenholz
A M
AM
Roach
P J
PJ
D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins
D016213:Cyclins; D005656:Fungal Proteins; C066824:PHO80 protein, S cerevisiae; D010455:Peptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D006006:Glycogen Synthase; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae