The Plant Homeodomain Fingers of Fission Yeast Msc1 Exhibit E3 Ubiquitin Ligase Activity

Barbara E Dul,Nancy C Walworth

doi:10.1074/jbc.m700729200

Barbara E Dul, Nancy C Walworth

Open Access

https://doi.org/10.1074/jbc.m700729200

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Abstract

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains. PHD finger domains in viral proteins and in the cellular protein kinase MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1) have been implicated as ubiquitin E3 protein ligases that affect protein stability. The close structural relationship of PHD fingers to RING fingers suggests that other PHD domain-containing proteins might share this activity. We show that each of the three PHD fingers of Msc1 can act as ubiquitin E3 ligases, reporting for the first time that PHD fingers from a nuclear protein exhibit E3 ubiquitin ligase activity. The function of the PHD fingers of Msc1 is needed to rescue the DNA damage sensitivity of a chk1Delta strain. Msc1 co-precipitates Rhp6, the S. pombe homologue of the human ubiquitin-conjugating enzyme Ubc2. Strikingly, deletion of msc1 confers complete suppression of the slow growth phenotype, UV and hydroxyurea sensitivities of an rhp6 deletion strain and restores deficient histone H3 methylation observed in the rhp6Delta mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.

Highlights

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage [1]
The sample was Msc1 plant homeodomain (PHD) fingers was detectable, the control protein centrifuged at 15,000 ϫ g for 1 min, and the supernatant consisting of the PHD finger of ING2 fused to GST pulled down transferred to a fresh tube. 100 ␮l of HA antibody, F7 (Santa histones as described [22] (Fig. 1A)
There has been disagreement in the literature over whether PHD fingers can act as E3 ubiquitin ligases [37,38,39]

Summary

EXPERIMENTAL PROCEDURES

Yeast Plasmids, Strains, and Manipulation—Standard Schizosaccharomyces pombe media and genetic manipulations were used as described by Moreno et al [24]. The same methodology was used to generate point mutations in the GST-PHD protein fusions. All strains were grown to mid-log phase (0.5 ϫ 107 cells/ml) at 30 °C. To assay UV sensitivity, cells were grown to mid-log phase at 30 °C. Ubiquitylation Assays—GST-PHD fusion proteins were incubated for 2 h at 30 °C in a final volume of 180 ␮l with 4 mM ATP, 10 mM MgCl2, 86 mM ubiquitin (Sigma), 10 ␮M yeast E1 (Boston Biochem), 80 ␮M human E2 (Boston Biochem), and 200 ␮M histones extracted from S. pombe or purchased from Sigma. Histone in vitro binding assays were performed as described by Shi et al [22], except that GST-PHD finger fusions were incubated with either 15 or 50 ␮g of calf thymus total histones from Worthington. The histones used in the assay contain methylated histone H3 as confirmed by detection with antibody to monomethyl-, dimethyl-, and trimethylhistone H3 (supplemental Fig. 1)

RESULTS

Because the mutant forms of

DISCUSSION