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

Wayne A. Wilson, Alan M. Mahrenholz, Peter Roach

Research output: Contribution to journalArticle

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Abstract

In Saccharomyces cerevisiae, PH085 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.

Original languageEnglish
Pages (from-to)7020-7030
Number of pages11
JournalMolecular and Cellular Biology
Volume19
Issue number10
StatePublished - Oct 1999

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Cyclins
Cyclin-Dependent Kinases
Yeasts
Phosphorylation
Gel Chromatography
Phosphotransferases
Glycogen Synthase
Enzymes
Cell Cycle Checkpoints
Saccharomyces cerevisiae
Catalytic Domain
Molecular Weight
Escherichia coli
Peptides

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p. / Wilson, Wayne A.; Mahrenholz, Alan M.; Roach, Peter.

In: Molecular and Cellular Biology, Vol. 19, No. 10, 10.1999, p. 7020-7030.

Research output: Contribution to journalArticle

Wilson, Wayne A. ; Mahrenholz, Alan M. ; Roach, Peter. / Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin Pcl10p. In: Molecular and Cellular Biology. 1999 ; Vol. 19, No. 10. pp. 7020-7030.
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