Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p

Citation
Wa. Wilson et al., Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p, MOL CELL B, 19(10), 1999, pp. 7020-7030
Citations number
76
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MOLECULAR AND CELLULAR BIOLOGY
ISSN journal
02707306 → ACNP
Volume
19
Issue
10
Year of publication
1999
Pages
7020 - 7030
Database
ISI
SICI code
0270-7306(199910)19:10<7020:STOTYC>2.0.ZU;2-Q
Abstract
Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase ( Cdk) catalytic subunit with multiple regulatory roles thought to be specifi ed by association with different cyclin partners (Pcls), Pcl10p is one of f our Pcls with little sequence similarity to cyclins involved in cell cycle control. It has been implicated in specifying the phosphorylation of glycog en synthase (Gsy2p), We report that recombinant Pho85p and Pcl10p produced in Escherichia coli reconstitute an active Gsy2p kinase in vitro. Gsy2p pho sphorylation required Pcl10p, occurred at physiologically relevant sites, a nd resulted in inactivation of Gsy2p. The activity of the reconstituted enz yme was even greater than Pho85p-Pcl10p isolated from yeast, and we conclud e that, unlike many Cdks, Pho85p does not require phosphorylation for activ ity. Pcl10p formed complexes with Gsy2p, as judged by (i) gel filtration of recombinant Pcl10p and Gsy2p, (ii) coimmunoprecipitation from yeast cell l ysates, and (iii) enzyme kinetic behavior consistent with Pcl10p binding th e substrate, Synthetic peptides modeled on the sequences of known Pho85p si tes were poor substrates with high ii, values, and we propose that Pcl10p-G sy2p interaction is important for substrate selection, Gel filtration of ye ast cell lysates demonstrated that most Pho85p was present as a monomer, al though a portion coeluted in high-molecular-weight fractions with Pcl10p an d Gsy2p. Overexpression of Pcl10p sequestered most of the Pho85p into assoc iation with Pcl10p, We suggest a model for Pho85p function in the cell wher eby cyclins like Pcl10p recruit Pho85p from a pool of monomers, both activa ting the kinase and targeting it to substrate.