Negative regulation of Cdc18 DNA replication protein by Cdc2

Abstract

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

Figure 1

GST-Cdc18 is functional in vivo and induces rereplication when overexpressed. (A) Cells expressing GST (strain AL1853) or GST-Cdc18 (strain AL1854) from the thiamine-repressible nmt1 promoter were photographed after 2 d of incubation at 30°C on agar medium lacking thiamine. Cells expressing GST grew well, whereas cells expressing GST-Cdc18 underwent several divisions and then became cdc arrested. (B) DNA content was measured by FACS in cells grown in liquid medium lacking thiamine. Note that the nmt1 promoter becomes active approximately 10 h following depletion of thiamine from the growth medium, reaching maximum activity at approximately 16 h (Maundrell, 1993). 2C and 4C positions were determined using wild-type haploid and diploid strains growing exponentially in YES medium at 30°C.

Figure 2

GST-Cdc18 interacts with Orp2, Cdc2, and B-type cyclins. GST and GST-Cdc18 expression was induced for 18 h. Lysates from those cells, GST (lane 1) and GST-Cdc18 (lane 2), were incubated with GSH-Sepharose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for GST, Orp2, Cdc2, Cdc13, or Cig2, as indicated. GST-Cdc18 copurified specifically with Orp2, Cdc2, Cdc13, and Cig2.

Figure 3

GST-Cdc18 purified from fission yeast is phosphorylated by an associated kinase that is inhibited by Rum1. GST, GST-Cdc18, and GST-Atf1 expression was induced for 18 h in medium lacking thiamine. GST, GST-Cdc18, and GST-Atf1 were precipitated using GSH-Sepharose and incubated with Mg²⁺ and [γ-³²P]ATP in the presence of increasing concentrations of GST-Rum1 purified from bacteria. Autoradiography of samples subjected to gel electrophoresis revealed that GST-Cdc18 became phosphorylated by a kinase that was inhibited by GST-Rum1 (upper panel). As a positive control it was confirmed that phosphorylation of histone H1 by Cdc2-Cdc13 kinase, purified from S. pombe lysates by anti-Cdc13 immunoprecipitation, was inhibited by GST-Rum1 (middle panel). As a negative control it was found that phosphorylation of GST-Atf1 by associated Spc1 kinase was insensitive to GST-Rum1 (lower panel).

Figure 4

GST-Cdc18 is an in vitro substrate of Cdc2 kinase. (A) Precipitated GST-Cdc18 was treated with 1 mM FSBA to irreversibly inactivate the copurified protein kinases (compare lane 1, not treated GST-Cdc18, with lane 2, FSBA-treated GST-Cdc18). GST-Cdc18 treated with FSBA was mixed with active p13^suc1-Sepharose-purified Cdc2 kinase in [γ-³²P]ATP kinase assay buffer in the presence (lane 4) or absence of 250 nM GST-Rum1 (lane 3). GST-Cdc18 phosphorylation was restored by the addition of Cdc2 kinases in the reaction. The ability of Cdc2 kinases to phosphorylate GST-Cdc18 or histone H1 (right panel, lanes 5 and 6) was largely inhibited by GST-Rum1. (B) Two-dimensional tryptic phosphopeptide maps of GST-Cdc18 phosphorylated by associated kinases (left panel) or by Cdc2 kinase (right panel) were very similar. (C) Phospho-amino acid analysis of GST-Cdc18 phosphorylated by associated kinases detected only phosphothreonine.

Figure 5

GST-Cdc18 is activated by mutation of the threonine-104 phosphorylation site. (A) DNA content was examined in cells expressing GST-Cdc18 or GST-Cdc18-T104A constructs from the nmt1 promoter. FACS analysis revealed that the DNA content of cells expressing GST-Cdc18-T104A increased more rapidly following derepression of the nmt1 promoter (upper panels). The mean DNA content is plotted in the lower panel. (B) Serial dilutions of the strains cdc18-K9 (upper panel), cdc18-K9 having a single integrated copy of pAL27 (nmt1:GST:cdc18⁺, middle panel) or cdc18-K9 having a single integrated copy of pAL27-T104A (nmt1:GST:cdc18T104A, bottom panel). The cells were spotted on a growth medium (YES) that allows only low-level expression from the nmt1 promoter and then tested for their ability to form colonies at the restrictive temperature of 35.5°C. The cdc18-K9 mutation was rescued better by GST-Cdc18T104A than GST-Cdc18. (C) The T104A mutation eliminates phosphopeptide 2. GST-Cdc18 and GST-Cdc18-T104A were expressed in S. pombe, purified with GSH-Sepharose, and incubated with Mg²⁺ and [γ-³²P]ATP as described above. Phosphorylated GST-Cdc18 and GST-Cdc18-T104A were analyzed by two-dimensional tryptic phosphopeptide mapping. The maps of both proteins were very similar except that one of the major phosphopeptides detected within GST-Cdc18, labeled 2, was absent in GST-Cdc18-T104A. Note that these phosphopeptide patterns are less complex than those shown in Figure 2 due to more complete trypsin digestion achieved in this experiment. (D) The stability of GST-Cdc18 and GST-Cdc18-T104A proteins was determined by an nmt1 turn-off experiment. GST-Cdc18 or GST-Cdc18-T104A was expressed for 14 h before the nmt1 promoter was repressed by addition of thiamine. The levels of GST-Cdc18 and GST-Cdc18-T104A were analyzed by immunoblot analysis at the indicated time points and quantified using a cross-reacting band (*) as a loading control. The half-life of both proteins was very similar (∼50 min). Note that the half-life of GST-Cdc18 was quite long compared with HA epitope-tagged Cdc18 (∼5 min) (Muzi-Falconi et al., 1996b), presumably due to the stabilizing property of GST. (E) GST-Cdc18-T104A interacts with Cdc2-cyclin B complexes and Orp2 in vivo. S. pombe lysates derived from cells expressing GST-Cdc18 (lane 1) or GST-Cdc18-T104A (lane 2) were precipitated with GSH-Sepharose and subjected to immunoblot analysis. Orp2, Cdc2, Cdc13, and Cig2 were found in association with GST-Cdc18 and GST-Cdc18-T104A.

Figure 6

Cig2 B-type cyclin contributes to the inhibition of Cdc18 and DNA replication. (A) The cdc18-K9 temperature-sensitive mutation is rescued by deletion of the cig2⁺ gene. Strains harboring Δcig1, Δcig2, and cdc18-K9 mutations alone or in combinations were tested for their ability to form colonies in YES medium at the restrictive temperature of 35.5°C. The cig1⁺ gene encodes a B-type cyclin of unknown function. The Δcig1 and Δcig2 mutants grow as well as wild-type at 35.5°C. The Δcig1 mutation failed to rescue cdc18-K9. (B) Overexpression of Cig2 inhibits rereplication in cells lacking Cdc13. Cells lacking Cdc13 were obtained by selective germination of spores from strain PR1334 (cdc13::his7⁺/cdc13⁺ ura4⁺/ura4-D18, top) or JLP162 (cdc13:: his7⁺/cdc13⁺ nmt1:cig2⁺(ura4⁺)/ura4-D18, bottom). Rereplication was strongly inhibited by expression of cig2⁺ from the nmt1 promoter. Germinated cells were analyzed by FACS (left) or photographed after staining with DAPI (right). Representative data for cells after 16 h at 32°C are shown.