Autoinhibition and autoactivation of the DNA replication checkpoint kinase Cds1.
Cds1 is the ortholog of Chk2 and the major effector of the DNA replication checkpoint in Schizosaccharomyces pombe. Previous studies have shown that Cds1 is activated by a two-stage mechanism. In the priming stage, the sensor kinase Rad3 and the mediator Mrc1 function to phosphorylate a threonine residue, Thr(11), in the SQ/TQ domain of Cds1. In the autoactivation stage, primed Cds1 molecules dimerize via intermolecular interactions between the phosphorylated Thr(11) in one Cds1 and the forkhead-associated domain of the other. Dimerization activates Cds1, probably by promoting autophosphorylation. To define the mechanisms for the autoactivation of primed Cds1 and the regulation of this process, we carried out genetic and biochemical studies to identify phosphorylatable residues required for checkpoint activation. Our data indicate that dimerization of Cds1 promotes trans-autophosphorylation of a number of residues in the catalytic domain, but phosphorylation of a highly conserved threonine residue (Thr(328)) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. Autophosphorylation of Thr(328) and kinase activation in unprimed, monomeric Cds1 are strongly inhibited by the C-terminal 27-amino acid tail of the enzyme. This autoinhibitory effect may play an important role in preventing spontaneous activation of the replication checkpoint during normal cell cycles. The two-stage activation pathway and the autoinhibition mechanism, which are probably shared by other members of the Chk2 family, provide sensitivity, specificity, and noise immunity, properties required for the replication checkpoint.
Xu
Yong-Jie
YJ
Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021, USA. yong-jie.xu@wright.edu
Kelly
Thomas J
TJ
D000906:Antibodies; D020134:Catalytic Domain; D064447:Checkpoint Kinase 2; D003470:Culture Media; D004261:DNA Replication / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology; D010748:Phosphopeptides / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D017384:Sequence Deletion
D000906:Antibodies; D003470:Culture Media; D010748:Phosphopeptides; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases