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

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 C 4 HC 3 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 chk1⌬ 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 rhp6⌬ mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage (1). The fission yeast gene msc1 was found in a genetic screen as a multicopy suppressor of chk1 (2). Msc1 is similar to the mammalian protein RBP2 that binds the tumor suppressor retinoblastoma (3) as well as to PLU-1, encoded by a gene that is up-regulated in breast cancer cells (4). Database searches indicate that there are also homologues of Msc1 in Caenorhabditis elegans and Drosophila, but no apparent homologue with the same domain structure exists in Saccharomyces cerevisiae. Both RBP2 and PLU-1 associate with proteins that have roles in regulating gene transcription and chromatin structure (5-8).Msc1 contains Jumonji N and Jumonji C domains, three plant homeodomain (PHD) 2 fingers, and a C 2 C 5 zinc finger. The PHD finger is a motif defined by seven cysteines and a histidine arranged in a C 4 HC 3 consensus. The PHD domain was first identified in a homeodomain protein involved in plant root development (9). Since then more than 400 eukaryotic proteins have been identified containing PHD fingers. Most of these proteins are nuclear and are thought to play roles in gene expression (10).The solution structure of the PHD finger of the KAP-1 corepressor shows that the zinc ligation scheme and cross-brace topology of the anti-parallel ␤-sheets are very similar to the structure of the RING (really interesting new gene) finger (11). Many proteins with RING fingers can act as E3 ubiquitin ligases in t...

Camptothecin-induced downregulation of MLL5 contributes to the activation of tumor suppressor p53

“…However, it is still unclear whether MLL5 can modify p53 upon interaction, or MLL5 merely serves as a chromatin-anchored adapter for a larger protein complex that negatively regulates p53. Better characterization of the function of conserved domains on MLL5, such as the PHD zinc finger (Scheel and Hofmann, 2003;Dul and Walworth, 2007;Kim and Buratowski, 2009) and SET domain (Berger, 2002), will be useful to understand the nature of the negative regulation of p53 by MLL5 in more details.…”

The H3K4me3 Histone Demethylase Fbxl10 Is a Regulator of Chemokine Expression, Cellular Morphology, and the Metabolome of Fibroblasts

“…PHD domains were shown to bind different methylated lysines on the histone tail of H3 functioning as reader domains (7). Due to the high similarity to RING domains which are well known E3 ligase domains, the PHD domains were recently also analyzed for E3 ligase function (7,8). Fbox domains function most likely as an anchor between a target protein and an E3 ubiquitin ligase (9).…”

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe

“…Additionally, while the three PHD domains of Msc1 suggest that it may behave as a reader of H3 lysine methyl marks, we were unable to detect binding of the Msc1 PHD domains to histone proteins (Dul and Walworth 2007) or methyl-modified histone peptides (C. T. Bernard and N. C. Walworth, unpublished data). We could, however, demonstrate association of Msc1 with the ubiquitin-conjugating enzyme Rhp6 and found that the PHD domains of Msc1 possess E3 ubiquitin protein ligase activity, a property shared with PHD domains of several other proteins (Dul and Walworth 2007).…”

“…A role for Msc1 in chromosome segregation has become apparent as a result of both gain-and loss-of-function genetic interactions with various centromere and kinetochore mutants (Ahmed et al 2004(Ahmed et al , 2007Dul and Walworth 2007;Qiu et al 2010). A recent study suggests a role for a Msc1-mediated pathway in establishing neocentromeres in fission yeast (Ogiyama et al 2013).…”

“…Msc1 shares with members of the mammalian KDM5 family of proteins a similar organization of multiple conserved domains including JmjN and JmjC domains, three PHD fingers, and a C 2 C 5 zinc finger (Ahmed et al 2004;Blair et al 2011). The PHD fingers of Msc1 possess E3 ubiquitin ligase activity (Dul and Walworth 2007). Cells with a deletion of the msc1 gene (msc1D) exhibit increased levels of bulk histone H3 K9 and/or K14 acetylation (Ahmed et al 2004).…”