Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA.
Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.
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Institut de Biologie Structurale, J.-P. Ebel, 41 rue Jules Horowitz, F-38027 Grenoble Cedex 1, France.
Munshi
Anil
A
Cannella
Dominique
D
Pennaneach
Vincent
V
Koundrioukoff
Stephane
S
Jaquinod
Michel
M
Forest
Eric
E
Podust
Vladimir
V
Fotedar
Arun
A
Fotedar
Rati
R
Jacquinod
Michel
M
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D019556:COS Cells; D002522:Chlorocebus aethiops; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D013912:Threonine / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D013912:Threonine; D004247:DNA; D018844:Cyclin-Dependent Kinases; D051818:Replication Protein C