Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast<i>Schizosaccharomyces pombe</i>

Čipák, Luboš; Zhang, Chao; Kovacikova, Ines; Rumpf, Cornelia; Miadoková, Eva; Shokat, Kevan M.; Gregáň, Juraj

doi:10.4161/cc.10.20.17792

Cited by 40 publications

(54 citation statements)

References 25 publications

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“…Previously reported sid2-as alleles were not sensitive to most of the small-molecule inhibitors, being only partially sensitive to 40 mm of 3-BrB-PP1 (Cipak et al 2011). To create a new sid2-as mutant strain, we employed site-directed mutagenesis (QuikChangeII, Site Directed Mutagenesis Kit) and mutated the methionine M285 gatekeeper residue to a glycine (Gly) as described by Cipak et al (2011).…”

Section: Media Strains and General Methodsmentioning

confidence: 99%

“…To create a new sid2-as mutant strain, we employed site-directed mutagenesis (QuikChangeII, Site Directed Mutagenesis Kit) and mutated the methionine M285 gatekeeper residue to a glycine (Gly) as described by Cipak et al (2011). However, instead of using a hygromycin-resistant sid2-as construct in a sid2D/ sid2 + heterozygous strain (Cipak et al 2011), we PCR-amplified the sid2-as mutant allele, and the resulting DNA fragment was transformed in the sid2-150 strain. Growing colonies were selected at 35°(including revertants, suppressors, and carrying-sid2-as allele colonies).…”

Section: Media Strains and General Methodsmentioning

confidence: 99%

“…This latter strain was used to produce etd1D mutants with the desired genetic background, removing the nmt81x-etd1 marker by tetrad dissection. cdr1-191 block was achieved by temperature shift to 36° (Liu et al 2000), except in Etd1-depletion experiments, in which 34°was used.Previously reported sid2-as alleles were not sensitive to most of the small-molecule inhibitors, being only partially sensitive to 40 mm of 3-BrB-PP1 (Cipak et al 2011). To create a new sid2-as mutant strain, we employed site-directed mutagenesis (QuikChangeII, Site Directed Mutagenesis Kit) and mutated the methionine M285 gatekeeper residue to a glycine (Gly) as described by Cipak et al (2011).…”

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Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

et al. 2014

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In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedbackactivate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.F ISSION yeast cells grow by elongating at their cell ends before dividing by forming a medially placed division septum during cytokinesis. New-born cells initiate growth in a monopolar manner from the old cell end that existed before division. During G2, cells initiate growth from the new end in a process known as NETO (new end take-off). These cells then grow in a bipolar mode until mitosis, when they stop growth and initiate cell division by septation (Mitchison and Nurse 1985;Hayles and Nurse 2001). Both polar growth and septation require proper cell membrane and cell-wall synthesis to ensure cell integrity. In Schizosaccharomyces pombe, the cell wall is composed of b-glucan, a-glucan, and mannoproteins. Synthesis of the major cell wall and primary septum polymer, 1,3-b-glucan, requires the activity of Drc1/Cps1/Bgs1 1,3-b-glucan synthase, which in turn is regulated by the guanosine triphosphate (GTP)-binding protein Rho1 (Garcia et al. 2006;Cortes et al. 2007). As with all GTPases, Rho1 acts as a binary switch, cycling between inactive GDP-bound and active GTPbound conformational states (Yang et al. 2003). This transition is controlled by three types of proteins: GEFs (guanine exchange factors), which catalyze the exchange of GDP for GTP, rendering the protein active; GAPs (GTPase activating proteins), which enhance nucleotide hydrolysis, inactivating the GTPase; and guanine nucleotide dissociation inhibitors, which appear to block spontaneous activation (Garcia et al. 2006).Upon the onset of mitosis, growth ceases at the cell ends and is re-established in the middle of the cell, where the actomyosin ring forms. At the end of mitosis, once the two sets of chromosomes have segregated, actomyosin ring constriction and septum formation is triggered by the septation initiation network (SIN) from the spindle pole body (SPB) (Krapp et al. 2004;Wolfe and Gould 2005). The nucl...

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Section: Media Strains and General Methodsmentioning

confidence: 99%

Section: Media Strains and General Methodsmentioning

confidence: 99%

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confidence: 99%

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Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

et al. 2014

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“…These include the cell cycle regulator Cdc2 as well as several kinases which coordinate cell growth, cell polarity and cell morphogenesis within the cell cycle [1]. Another one of these essential kinases is Prp4.…”

Section: Introductionmentioning

confidence: 99%

“…Adding the inhibitor 1-NM-PP1 to a growing cell culture expressing Prp4-as2 leads to growth arrest [1]. We performed semi-quantitative RT-PCR analyses to detect mRNA and pre-mRNA of different intron containing genes, using RNA of a growing culture prepared 10, 20 and 30 minutes after adding the kinase inhibitor.…”

Section: Inhibition Of Prp4 Kinase Activity Affects Splicing Of Intromentioning

confidence: 99%

The Prp4 Kinase: Its Substrates, Function and Regulation in Pre-mRNA Splicing

Lützelberger¹,

Käufer²

2012

Protein Phosphorylation in Human Health

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Identification of two forms of the Eso1 protein in Schizosaccharomyces pombe

Chen

Cao

Guo

et al. 2014

Cell Biology International

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In Schizosaccharomyces pombe, Eso1p is a protein fusion. Two-thirds of its N-terminus is conserved to budding yeast Rad30, which functions in error-free replication of UV-damaged DNA. A third of the C-terminus is highly conserved to budding yeast Eco1, a lysine acetyltransferase, which is essential for the establishment of cohesion. Both Rad30p and Eco1p need to be finely tuned in budding yeast. Given the distinct function existed in Rad30p and Eco1p, it is enigmatic how the Eso1p, the protein fusion regulated in S. pombe, works. We have identified two forms of the Eso1 protein by Western blot, and detected the Eco1-homology fragment by M/S analysis following TAP purification of Eso1 protein. The result raises the possibility that Eso1 might be processed in vivo to release the Eco1-homology fragment, which allows the independent regulation of Rad30-homology and Eco1-homology fragments.

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Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeastSchizosaccharomyces pombe

Cited by 40 publications

References 25 publications

Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

The Prp4 Kinase: Its Substrates, Function and Regulation in Pre-mRNA Splicing

Identification of two forms of the Eso1 protein in Schizosaccharomyces pombe

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