A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11

Abstract

Phosphorylation often regulates protein-protein interactions to control biological reactions. The Sld2 and Dpb11 proteins of budding yeast form a phosphorylation-dependent complex that is essential for chromosomal DNA replication. The Sld2 protein has a cluster of 11 cyclin-dependent kinase (CDK) phosphorylation motifs (Ser/Thr-Pro), six of which match the canonical sequences Ser/Thr-Pro-X-Lys/Arg, Lys/Arg-Ser/Thr-Pro and Ser/Thr-Pro-Lys/Arg. Simultaneous alanine substitution for serine or threonine in all the canonical CDK-phosphorylation motifs severely reduces complex formation between Sld2 and Dpb11, and inhibits DNA replication. Here we show that phosphorylation of these canonical motifs does not play a direct role in complex formation, but rather regulates phosphorylation of another residue, Thr84. This constitutes a non-canonical CDK-phosphorylation motif within a 28-amino-acid sequence that is responsible, after phosphorylation, for binding of Sld2-Dpb11. We further suggest that CDK-catalysed phosphorylation of sites other than Thr84 renders Thr84 accessible to CDK. Finally, we argue that this novel mechanism sets a threshold of CDK activity for formation of the essential Sld2 to Dpb11 complex and therefore prevents premature DNA replication.

Figure 1

The interaction regions of Sld2 and Dpb11. (A) Sld2 family proteins in yeast and fungi. Vertical black and grey lines show canonical and non-canonical CDK-phosphorylation motifs, respectively. The shaded boxes indicate the regions containing a cluster of CDK-phosphorylation motifs. Asterisks on boxes indicate Thr84 and Ser100 in Sld2 (Sc) and corresponding residues in Sld2 homologues. Sc, Saccharomyces cerevisiae; Eg, Eremothecium gossypii; Ca, Candida albicans; Sp, Schizosaccharomyces pombe; Nc, Neurospora crassa. (B) Schematic representation of CDK-phosphorylation motifs in Sld2. The amino-acid sequence required for binding to Dpb11 is shown with the corresponding sequence of Sld2 homologues in different species. Identical amino acids (grey shading) and CDK-phosphorylation motifs (asterisks) are highlighted. (C) The region of Sld2 interacting with Dpb11 in a two-hybrid assay. Various fragments derived from SLD2 were cloned into pBTM116 (Bartel and Fields, 1995). The resultant plasmids and pACT2-DPB11 (Kamimura et al, 1998) were introduced into the yeast strain L40 and the interaction was detected by lacZ expression in colour. (D) The interaction region of Dpb11 with Sld2 in a two-hybrid assay. Various fragments of DPB11 were cloned into pACT2 (Bai and Elledge, 1997) and used for a two-hybrid assay with pBTM116-SLD2 (Kamimura et al, 1998) as described in (C).

Figure 2

The 28-amino-acid stretch with pThr84 in Sld2 interacts directly with Dpb11. (A) Complex formation between Flag-Sld2-P1 (residues 79–263) and GST-Dpb11-C (residues 291–631) in vitro. Flag-Sld2-P1 (18 pmol) was incubated with 36 units of rCdc28–Clb5 in the presence (+) or absence (−) of ATP at 30°C for 1 h and serial dilutions were further incubated with GST-Dpb11-C (30 pmol) or GST (30 pmol) proteins immobilized on GSH-Sepharose beads at 4°C for 1 h. Sld2-P1 bound to beads was detected by Western blotting with monoclonal anti-Flag antibody (M2, Sigma Aldrich) after SDS–PAGE. The asterisk indicates the slower migrating phosphorylated form of Sld2-P1. (B) Peptide sequence used for the assay. Thick and thin underlines of Sld2-P1 sequence indicate canonical and non-canonical CDK-phosphorylation motifs, respectively. The 28-amino-acid stretch required for binding to Dpb11 is shaded. ‘P' indicates phosphorylated residues. (C,F) Peptide competition for Sld2-P1 binding to Dpb11. Various peptides shown in (B) were mixed with phosphorylated Flag-Sld2-P1 (0.1 μM) and GST-Dpb11-C (30 pmol) immobilized beads and further incubated at 4°C for 1 h. (D) Biotinylated 28-2P or 28-NP peptides (250 pmol) in (B) immobilized on streptavidin beads were incubated with indicated concentrations of GST-Dpb11-C at 4°C for 30 min and the protein bound to the peptides was detected by Western blotting with anti-GST monoclonal antibody. (E) No binding was observed of Flag-Sld2-P1Δ28 (residues 107–263) lacking the 28-amino-acid binding stretch to the GST-Dpb11-C protein. Reactions were carried out as described in (A), except that Flag-Sld2-P1Δ28 was used instead of Flag-Sld2-P1.

Figure 3

Thr84 of Sld2 is phosphorylated in a CDK-dependent manner and this phosphorylation is essential for cell growth. (A) YYK3 (Δsld2) cells bearing YCpSLD2 (Kamimura et al, 1998) (−) or YCpSLD2-10FLAG (+) were arrested in S phase by 0.2 M hydroxyurea (HU) and disrupted by glass beads. The Sld2-10Flag protein was precipitated with anti-Flag antibody M2 from cell lysates. The precipitated proteins were separated using SDS–PAGE and subjected to Western blotting with anti-pThr84 and anti-Flag M2 antibodies. The asterisk indicates the phosphorylated Sld2-10Flag. (B) YS125 (a GAL-SIC1ΔNT) and its parental strain W303a harbouring YCp-Gal-SLD2-10FLAG were arrested at G1 phase by α factor and released in YPGal as described (Masumoto et al, 2002). The samples were withdrawn at the indicated time and budding cells were counted under a microscope. The Sld2-10Flag protein was precipitated from disrupted cells and analysed as described in (A). (C) YYK3 (Δsld2 (YEp195SLD2)) (Kamimura et al, 1998) cells were transformed with YCplac22 (Vector) or YCpSLD2 bearing the indicated mutation. The resulting transformants were cultivated in YPDA medium and spotted onto plates containing 5-fluoroorotic acid (FOA), as described (Masumoto et al, 2002), after serial dilutions. The numbers above the photograph indicate the estimated number of cells placed on a spot. (D) Y799 (drc1-1; drc1-1 is a temperature-sensitive allele of SLD2) (Wang and Elledge, 1999) cells bearing one of YCplac22 (Vector), YCp22SLD2 (WT) and YCp22sld2T84A (T84A) were arrested in G1 and released from G1 at 36°C. Cells were withdrawn at the indicated times and DNA content was measured by flow cytometry. The percentages of budded cells are also shown. (E) The Sld2-10Flag was precipitated from HU-arrested HMS65 (pep4Δ∷G418^rdrc1-1 DPB11-9myc) cells harbouring YCp22SLD2-10FLAG (WT) or YCp22SLD2-10FLAG bearing a T84A mutation (T84A) as described in (A). Co-precipitation of Dpb11-9myc was detected by anti-myc 9E10 antibody. Note that Sld2-10Flag (T84A) migrated slower than that arrested in G1 phase, as did the WT.

Figure 4

Phosphorylations other than Thr84 affect the phosphorylation of Thr84 in vivo. (A) The sld2 mutations at CDK-phosphorylation motifs used for two-hybrid assays and for plasmid-shuffling assays to examine the ability to support cell growth. For a two-hybrid assay, pACT2-Dpb11 (Figure 1D; Dpb11) (Kamimura et al, 1998) and pBTM116-SLD2-P1 (Figure 1C; P1) bearing the indicated mutation were used as described in the legend to Figure 1C. Plasmid-shuffling assays were performed as shown in (B) and Figure 3 (C). Brackets in the growth columns indicate results reported previously (Masumoto et al, 2002). Note that 6A was previously designated All-A. (B) YST387 (Δsld2∷TRP1 (YEp195SLD2)) were transformed with YCplac111 (Vector) or YCp111SLD2 bearing the indicated mutation. The resulting transformants were streaked onto an FOA plate. A few colonies carrying SLD2-6A on plasmid showed up in the plasmid-shuffling assay, probably because of the increased copy of mutant proteins. (C) The LexA-fused Sld2-P1 and its variant proteins were precipitated with an anti-LexA antibody (Santa Cruz) from HU-arrested cells carrying pBTM116-SLD2-P1 and its variants shown in (A). The precipitated proteins were analysed by Western blotting using anti-pThr84 and anti-LexA antibodies. The asterisk indicates the slower migrating form of the protein.

Figure 5

Phosphorylation of Thr84 is regulated by other phosphorylations in vitro. (A) Time course of phosphorylations at three CDK motifs. Sld2-P1 (45 pmol) was mixed with rCdc28–Clb5 (55 units) in 80 μl buffer and aliquots were withdrawn at 20-min intervals to examine the phosphorylation level of Sld2. Phosphorylation of a specific amino-acid residue was monitored by Western blotting with indicated anti-phospho antibodies. The phosphorylation level was measured using NIH image and normalized to the level at 120 min as 100%. (B) The Sld2-P1 protein (2 pmol) was incubated with the indicated activity of rCdc28–Clb5 at 25°C for 15 min. The phosphorylation level was measured as described in (A) and normalized to the level with 36 units of rCdc28–Clb5 as 100%.

Figure 6

Phosphorylations other than Thr84 render it accessible to DNA-PK. (A) YYK3 (Δsld2 (YEp195SLD2)) (Kamimura et al, 1998) cells were transformed with one of YCplac22 (Vector), YCpSLD2 (WT) and YCpsld2P85Q (P85Q). The resulting transformants were streaked onto an FOA plate. (B) Y799 (drc1-1; a temperature-sensitive allele of SLD2) (Wang and Elledge, 1999) cells harbouring YCp22sld2P85Q were arrested at the G1 phase, released at 36°C and the DNA content was measured as described in the legend to Figure 3D. The DNA content of WT strain (Figure 3D) is also shown as a control. (C) Schematic representation of phosphorylation in the Sld2-P1 protein with a P85Q mutation. (D) The Sld2-P1 protein (12 pmol) with indicated substitution was first incubated with or without rCdc28-Clb5 (4 units) at 30°C for 1 h. The same reactions were incubated in the presence of 10 μM CDK inhibitor purvalanol A (Gray et al, 1998) with or without DNA-PK (25 U: Promega) for further 1 h. Note that DNA-PK alone could not phosphorylate Thr84 of WT Sld2-P1 (Supplementary Figure 4). The asterisk indicates the slower migrating form of the protein.

Figure 7

Regulatory model of the interaction between Dpb11 and Sld2 phosphorylated by CDK. (A) The phosphorylation level of Sld2 is proportional to the level of CDK activity. However, phosphorylation of Thr84 in Sld2 requires prior phosphorylation of other CDK-phosphorylation motifs. When CDK activity increases beyond threshold, Sld2 may change its conformation by multiple phosphorylations and then CDK phosphorylates Thr84. When Thr84 is phosphorylated, Sld2 forms a complex with Dpb11 to initiate DNA replication. (B) When CDK activity increases, the pre-RC components and some other proteins are phosphorylated before Thr84 of Sld2 is phosphorylated. Thus, inactivation of the pre-RC formation and preceding origin association of some replication proteins are ensured.