PMID- 10490639
OWN - NLM
STAT- MEDLINE
DCOM- 20000203
LR  - 20240407
IS  - 0270-7306 (Print)
IS  - 1098-5549 (Electronic)
IS  - 0270-7306 (Linking)
VI  - 19
IP  - 10
DP  - 1999 Oct
TI  - Substrate targeting of the yeast cyclin-dependent kinase Pho85p by the cyclin 
      Pcl10p.
PG  - 7020-30
AB  - 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.
FAU - Wilson, W A
AU  - Wilson WA
AD  - Department of Biochemistry and Molecular Biology, Indiana University School of 
      Medicine, Indianapolis, Indiana 46202, USA.
FAU - Mahrenholz, A M
AU  - Mahrenholz AM
FAU - Roach, P J
AU  - Roach PJ
LA  - eng
GR  - R01 DK042576/DK/NIDDK NIH HHS/United States
GR  - DK42576/DK/NIDDK NIH HHS/United States
GR  - R01 DK027221/DK/NIDDK NIH HHS/United States
GR  - DK27221/DK/NIDDK NIH HHS/United States
GR  - R37 DK027221/DK/NIDDK NIH HHS/United States
GR  - DK20542/DK/NIDDK NIH HHS/United States
GR  - R56 DK027221/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Mol Cell Biol
JT  - Molecular and cellular biology
JID - 8109087
RN  - 0 (Cyclins)
RN  - 0 (Fungal Proteins)
RN  - 0 (PHO80 protein, S cerevisiae)
RN  - 0 (Peptides)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Repressor Proteins)
RN  - 0 (Saccharomyces cerevisiae Proteins)
RN  - EC 2.4.1.11 (Glycogen Synthase)
RN  - EC 2.7.11.22 (Cyclin-Dependent Kinases)
RN  - EC 2.7.11.22 (PHO85 protein, S cerevisiae)
SB  - IM
MH  - Cyclin-Dependent Kinases/chemistry/genetics/*metabolism
MH  - Cyclins/genetics/*metabolism
MH  - Enzyme Activation
MH  - Escherichia coli/genetics
MH  - Fungal Proteins/genetics/*metabolism
MH  - Glycogen Synthase/*metabolism
MH  - Mass Spectrometry
MH  - Models, Biological
MH  - Peptides/metabolism
MH  - Phosphorylation
MH  - Protein Binding
MH  - Recombinant Proteins/metabolism
MH  - *Repressor Proteins
MH  - Saccharomyces cerevisiae/*physiology
MH  - *Saccharomyces cerevisiae Proteins
PMC - PMC84697
EDAT- 1999/09/22 00:00
MHDA- 1999/09/22 00:01
PMCR- 1999/10/01
CRDT- 1999/09/22 00:00
PHST- 1999/09/22 00:00 [pubmed]
PHST- 1999/09/22 00:01 [medline]
PHST- 1999/09/22 00:00 [entrez]
PHST- 1999/10/01 00:00 [pmc-release]
AID - 0635 [pii]
AID - 10.1128/MCB.19.10.7020 [doi]
PST - ppublish
SO  - Mol Cell Biol. 1999 Oct;19(10):7020-30. doi: 10.1128/MCB.19.10.7020.