Autoinhibition and Autoactivation of the DNA Replication Checkpoint Kinase Cds1

Abstract

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 twostage 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.

The Phosphorylation Network for Efficient Activation of the DNA Replication Checkpoint in Fission Yeast

“…Previous studies showed that activation of Cds1 requires two sequential phosphorylations of this kinase; one is the Rad3-dependent phosphorylation of Thr 11 in the SQ/TQ domain, and the other is the autophosphorylation of Thr 328 in the kinase domain (31). Phosphorylation of the two sites in Cds1 has been confirmed by mutations, phosphospecific antibodies, two-dimensional phosphopeptide mapping, and MS (23,31).…”

“…Previous studies showed that activation of Cds1 requires two sequential phosphorylations of this kinase; one is the Rad3-dependent phosphorylation of Thr 11 in the SQ/TQ domain, and the other is the autophosphorylation of Thr 328 in the kinase domain (31). Phosphorylation of the two sites in Cds1 has been confirmed by mutations, phosphospecific antibodies, two-dimensional phosphopeptide mapping, and MS (23,31). In this study, we investigated the potential Rad3 phosphorylation in the sensor proteins, confirmed the phosphorylation of the SQ cluster and TQ motifs in Mrc1 and the C terminus of Rad9, and discovered that the three phosphorylations on Mrc1 and Rad9 are probably the only Rad3-dependent phosphorylations required for efficient phosphorylation and subsequent activation of Cds1.…”

“…The phosphospecific antibodies for phosphorylated Rad9-Ser 423 and Rad9-Thr 225 , also prepared by Bethyl Laboratories, Inc., were kindly provided by Furuya et al (22). The antibodies against phosphorylated Cds1-Thr 11 and Cds1-Thr 328 have been described previously (31). Western Blotting-The phosphorylation of Rad9 and Cds1, tagged with a hemagglutinin (HA) epitope, was assayed in proteins immunoprecipitated with anti-HA antibody (Santa Cruz Biotechnology).…”

“…Phosphorylation of Thr 11 in Cds1 by Rad3 is essential for activation of the effector kinase in vivo that primes Cds1 for autoactivation by trans-autophosphorylation of Thr 328 in the kinase domain (31). To assay the Cds1 phosphorylation, antibodies against phosphorylated Thr 11 and Thr 328 of Cds1 were generated (31). After the specificity of the antibody against phosphorylated Cds1-Thr 11 was confirmed by immunoblottings (supplemental Figs.…”

“…In response to replication stress, Cds1 is activated by two sequential phosphorylations at Thr 11 and Thr 328 (31). Phosphorylation of Thr 11 in Cds1 by Rad3 is essential for activation of the effector kinase in vivo that primes Cds1 for autoactivation by trans-autophosphorylation of Thr 328 in the kinase domain (31). To assay the Cds1 phosphorylation, antibodies against phosphorylated Thr 11 and Thr 328 of Cds1 were generated (31).…”