Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe

Gregáň, Juraj; Zhang, Chao; Rumpf, Cornelia; Čipák, Luboš; Li, Zhang; Uluocak, Pelin; Nasmyth, Kim; Shokat, Kevan M.

doi:10.1038/nprot.2007.447

Cited by 44 publications

(51 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This strategy is broadly applicable to study protein kinase function and involves a single residue substitution in the conserved ATP-binding pocket of a kinase to confer chemical sensitivity to small molecule inhibitors (19). The strains used in our experiments either contain tpk1, tpk2, and tpk3 analog-sensitive mutations (designated pka) or an analog-sensitive sch9 mutation or combine all of these mutations (designated pka sch9).…”

Section: Regulation Of Maf1 Involves Phosphorylation On Multiplementioning

confidence: 99%

Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

Lee

Moir

Willis

2009

Journal of Biological Chemistry

104

138

View full text Add to dashboard Cite

Maf1 is a conserved repressor of transcription that functions at the downstream end of multiple nutrient and stress signaling pathways. How these different signaling pathways converge on Maf1 is not known. Previous work in yeast indicates that protein kinase A (PKA) regulates RNA polymerase (pol) III transcription, in part, by phosphorylating multiple sites in Maf1. Here we present additional evidence for this view and show that a parallel nutrient and stress-sensing pathway involving Sch9, an homologous kinase to metazoan S6 kinase, targets Maf1 at a subset of PKA sites. Using ATP analog-sensitive alleles of PKA and Sch9, we find that these two kinases account for the bulk of the phosphorylation on consensus PKA sites in Maf1. Deletion of Sch9 reduces RNA pol III transcription in a Maf1-dependent manner, yet the cells remain susceptible to further repression by rapamycin and other treatments. Because the rapamycin-sensitive kinase activity of the TORC1 complex is necessary for Sch9 function in vivo and in vitro, our results show that transcriptional regulation of RNA pol III and the coordinate control of ribosomal protein genes can be achieved by Sch9-dependent and -independent branches of the target of rapamycin (TOR) signaling pathway.Maf1 is essential for repressing transcription by RNA polymerase (pol) 2 III in yeast, and this function is conserved in mammals (1-4). In addition, human Maf1 is known to directly repress RNA pol II transcription of certain protein coding genes such as the TATA box-binding protein, TBP, and to indirectly repress transcription by RNA pol I (3). Repression by Maf1 occurs in response to a wide variety of nutritional and stress conditions, but the nature of the signaling pathways mediating these responses and how they converge on Maf1 is largely unknown (5).In budding yeast, Maf1 is phosphorylated on consensus PKA sites under optimal growth conditions and is rapidly dephosphorylated under starvation or stress conditions that repress transcription (5, 6). Dephosphorylation of these PKA sites correlates with the relocation of Maf1 from the cytoplasm to the nucleus and is thought to regulate its inhibitory interaction with RNA pol III (5-9). Nuclear import of Maf1 is directed by two redundant nuclear localization sequences (NtNLS and CtNLS) that are differentially sensitive to PKA site phosphorylation. Specifically, the NtNLS is inhibited by phosphorylation as acidic substitutions at the PKA sites, which are normally fully functional, can prevent nuclear import and repression when the CtNLS is disabled (6). The localization of Maf1 in the cytoplasm serves to fine-tune its regulation but is not essential for preventing repression at inappropriate times. The accumulation of Maf1 in the nucleus, either by mutation of the protein or by deletion of the exportin of the protein, Msn5, does not cause repression in the absence of additional changes triggered by cellular signaling pathways (6, 10).Numerous studies have implicated the RAS/PKA pathway and the rapamycin-sensitive TOR pathway as po...

show abstract

Section: Regulation Of Maf1 Involves Phosphorylation On Multiplementioning

confidence: 99%

Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

Lee

Moir

Willis

2009

Journal of Biological Chemistry

104

138

View full text Add to dashboard Cite

show abstract

“…24,25 To create plasmid pCloneBle1-hhp1, we cut out hhp1 homology regions from the plasmid pCloneNat1-hhp1, 26 using restriction enzymes HindIII and BamHI and inserted into HindIII/ BamHI sites of the plasmid pCloneBle1.…”

Section: Notementioning

confidence: 99%

S. pombegenome deletion project: An update

Špı́rek¹,

Benkő²,

Čarnecká³

et al. 2010

Cell Cycle

Self Cite

View full text Add to dashboard Cite

IntroductionAlthough forward genetic screens have provided many important insights into various aspects of cell biology, they are limited by the large numbers of mutants that must be analyzed and inherent biases in mutagenesis techniques. Systematic reverse genetic screens using genome-wide gene deletion collections or RNAi libraries provide a powerful alternative to forward genetic screens (reviewed in refs. 1-5). Studies with the budding yeast Saccharomyces cerevisiae have shown the usefulness of such gene deletion collection for numerous studies including drug discovery (reviewed in refs. 6-9). The fission yeast S. pombe is an important model organism sharing many features with cells of higher eukaryotes. The availability of the S. pombe genome sequence 10 and PCR-based gene deletion technology 11 allowed researchers involved in the S. pombe genome deletion project (KRIBB-Bioneer-CRUK consortium) to generate a set of 4.836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast open reading frames (Kim et al., in press). However, for some genes a deletion could either not be constructed or the analysis of mutant phenotypes gave ambiguous results. Here we use an efficient knock-out strategy to delete 29 of these genes. ResultsWe speculated that some of the genes which could not be deleted by the KRIBB-Bioneer-CRUK consortium may require longer regions of homology for efficient gene targeting 12,13 and may be the fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a pCr based strategy. However, deletion of some genes has not been possible using this methodology.Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. the collection of deletion mutants now covers 99% of the fission yeast open reading frames.

show abstract

“…The assays for the determination of IC 50 (μM) of PW12, TGX155, IC87114 and LY294002 were performed exactly as previously described (Gregan et al, 2007). …”

Section: Pi3k Activity Assaymentioning

confidence: 99%

Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages

Papakonstanti

Zwaenepoel

Bilancio

et al. 2008

Journal of Cell Science

View full text Add to dashboard Cite

The class IA isoforms of phosphoinositide 3-kinase (p110α, p110β and p110δ) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110δ selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110δ in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110α, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110δ. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110α also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110δ.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110δ isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110δ-selective PI3K inhibitors may be more effective in inflammation than in cancer.

show abstract

Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe

Cited by 44 publications

References 36 publications

Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

Regulation of RNA Polymerase III Transcription Involves SCH9-dependent and SCH9-independent Branches of the Target of Rapamycin (TOR) Pathway

S. pombegenome deletion project: An update

Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages

Contact Info

Product

Resources

About