Identification of New Players in Cell Division, DNA Damage Response, and Morphogenesis Through Construction of <i>Schizosaccharomyces pombe</i> Deletion Strains

Chen, Jun‐Song; Beckley, Janel R.; McDonald, Nathan A.; Ren, Liping; Mangione, MariaSanta C.; Jang, Sylvia J; Elmore, Zachary C.; Rachfall, Nicole; Feoktistova, Anna; Jones, Christine M.; Willet, Alaina H.; Guillen, Rodrigo X.; Bitton, Danny A.; Bähler, Jürg; Jensen, Michael A.; Rhind, Nick; Gould, Kathleen L.

doi:10.1534/g3.114.015701

Cited by 21 publications

(30 citation statements)

References 57 publications

(63 reference statements)

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“…Because man1Δ mutant is not sensitive to HU (Fig S3), the specific nuclear localization of Lem2 may be required for its checkpoint function. Although another structurally unrelated INM protein Ima1 is also accumulated at SPB in S. pombe (34), the genome-wide studies suggest that Ima1Δ mutant is not sensitive to HU (28,29,53,54). Interestingly, we have recently found that the checkpoint function of Lem2 is specifically associated with its C-terminal domain (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast

2016

Cellular Signalling

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DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2+ sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling were seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside nucleus, a previously unknown mechanism.

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Section: Discussionmentioning

confidence: 99%

Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast

2016

Cellular Signalling

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“…Cellular locations using GO annotation are available for most of the unknown proteome based on a genome-wide localization study and inference from other models [19]. Viability data come from large-scale screens reported by Kim et al [20] and Chen et al [21].…”

Section: Supplementary Materialsmentioning

confidence: 99%

“…Kim et al [20] and Chen et al [21]. The fission yeast "conserved unknown" protein set [33] is reviewed continually for new functional data.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Hidden in plain sight: What remains to be discovered in the eukaryotic proteome?

Wood

Lock

Harris

et al. 2018

Preprint

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The first decade of genome sequencing stimulated an explosion in the characterization of unknown proteins. More recently, the pace of functional discovery has slowed, leaving around 20% of the proteins even in well-studied model organisms without informative descriptions of their biological roles. Remarkably, many uncharacterized proteins are conserved from yeasts to human, suggesting that they contribute to fundamental biological processes. To fully understand biological systems in health and disease, we need to account for every part of the system. Unstudied proteins thus represent a collective blind spot that limits the progress of both basic and applied biosciences.We use a simple yet powerful metric based on Gene Ontology (GO) biological process terms to define characterized and uncharacterized proteins for human, budding yeast, and fission yeast. We then identify a set of conserved but unstudied proteins in S. pombe, and classify them based on a combination of orthogonal attributes determined by large-scale experimental and comparative methods. Finally, we explore possible reasons why these proteins remain neglected, and propose courses of action to raise their profile and thereby reap the benefits of completing the catalog of proteins' biological roles.

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“…Deletion of hhp1 and hhp2 is known to cause several cellular phenotypes in yeast, including delayed repair of DNA double strand breaks ) that increases sensitivity to DNA damage (Bimbó et al, 2005;Chen et al, 2015;Dhillon and Hoekstra, 1994), defects in meiosis (Ishiguro et al, 2010;Phadnis et al, 2015), increased cell length , and increased monopolar growth (Koyano et al, 2010). However, the effects of overexpression or gain-of-function are poorly characterized.…”

Section: Ablating Ck1 Autophosphorylation Produces Gain-of-function Amentioning

confidence: 99%

Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

Cullati

Chen

Gould

2019

Preprint

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AbstractCK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. Here, we describe a new mechanism of CK1 autoregulation that is conserved in CK1 enzymes from yeast to human – the autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site. Phosphorylation at this site inhibits kinase activity, in contrast to well-characterized T-loop autophosphorylation in other kinase families. Consequently, yeast and human enzymes with phosphoablating mutations at this site are hyperactive. In S. pombe, hyperactive CK1 causes defects in cell growth and morphology at a high level but protection from heat shock at a low level, highlighting the necessity of regulated CK1 function. We propose that phosphorylation on the L-EF loop prevents substrate docking with the kinase domain by shielding the positively charged binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.Significance StatementKinases in the CK1 family are important signaling enzymes, and they function in multiple pathways within the same cell. Misregulation of CK1 activity contributes to human disease, including cancer, neurodegenerative disease, and sleep phase disorders, yet the mechanisms that control CK1 activity are not well understood. We have identified a conserved autophosphorylation site in the CK1 kinase domain that inhibits substrate phosphorylation. We hypothesize that by using kinase domain autophosphorylation in combination with other regulatory mechanisms, CK1 enzymes can coordinate the phosphorylation of their substrates in different pathways.

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Identification of New Players in Cell Division, DNA Damage Response, and Morphogenesis Through Construction of Schizosaccharomyces pombe Deletion Strains

Cited by 21 publications

References 57 publications

Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast

Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast

Hidden in plain sight: What remains to be discovered in the eukaryotic proteome?

Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

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