Project description:To study the consequences of MAK-2 activity modulation during vegetative cell fusion, we took advantage of a previously constructed allele of MAK-2 (MAK-2Q100G) to specifically perturb kinase signaling during germling vegetative cell fusion (inhibition of MAK-2Q100G activity by addition of the ATP analog 1NM-PP1 results in a phenotype indistinguishable from mak-2 deletion strains). Whole genome microarrays of mak-2Q100G cells following 20 min 1NM-PP1 treatment were performed. Two-condition experiment, Neurospora crassa cells containing MAK2Q100G allele treated with 1NM-PP1 inhibitor vs untreated control. Cy3 and Cy5 dye swaps were performed.
Project description:To study the consequences of MAK-2 activity modulation during vegetative cell fusion, we took advantage of a previously constructed allele of MAK-2 (MAK-2Q100G) to specifically perturb kinase signaling during germling vegetative cell fusion (inhibition of MAK-2Q100G activity by addition of the ATP analog 1NM-PP1 results in a phenotype indistinguishable from mak-2 deletion strains). Whole genome microarrays of mak-2Q100G cells following 20 min 1NM-PP1 treatment were performed.
Project description:General impact of Jak1 and Jak2 inhibition on IFNg-mediated target gene expression. U4C-Jak1AS and g2A-Jak2AS cells were stimulated with IFNg and treated with either 1NM-PP1 (to inhibit the activity only of the analog-sensitive mutant) or JI1 (to suppress both wild-type and analog-sensitive Jaks) for 24h.
Project description:The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. On major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, the well-established substrate of pUL97, the retinoblastoma protein (Rb) was no longer phosphorylated. This effect was detectable as early as 4 hours post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.
Project description:The goal of the study was to compare the response to Protien Kinase A (PKA) inhibition between Saccharomyces cerevisiae and Kluyveromyces lactis. The ancestor of K. lactis did not undergo the Whole Genome Duplication (or Whole Genome Hybridization) event that S. cerevisiae experienced. We found that many paralog pairs in S. cerevisiae were differentially induced in response to PKA inhibition, and that the shared ortholog for these paralog paris in K. lactis was typically not induced. To inhibit PKA, strains containing point mutations rendering PKA sensitive to inhibition by the ATP analog 1-NM-PP1 were generated. The transcription factors Msn2/4 and Rph1/Gis1 in S. cerevisiae and their shared orthologs in K. lactis were deleted in both species to quantify and compare the effect of those transcription factors on the response to PKA inhibition in each species.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis. mRNA-Seq of 8 S. cerevisiae strains treated with either DMSO alone or 1NM-PP1, a small molecule inhibitor for analog-sensitive kinases such as sch9-as.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis. ChIP-Seq of 8 S. cerevisiae strains treated with 1NM-PP1, a small molecule inhibitor for analog-sensitive kinases such as sch9-as.
Project description:To determine the impact of CDK8 kinase activity on steady-state mRNA levels, we performed RNA-seq analysis of colorectal carcinoma cell line HCT116 (CDK8wt/wt and CDK8as/as) in the presence of absence of 3MB-PP1 to specifically inhibit analog senstitive (as) CDK8.
Project description:A strain of M. oryzae was constructed in which the native PKC1 gene was replaced with an analogue-sensitive PKC1 allele (pkc1AS). This allele could be inhbited by the analogue 1NA-PP1. Global transcriptional profiling of mycelium following selective PKC inhibition identified significant changes in gene expression associated with cell wall re-modelling, autophagy, signal transduction and secondary metabolism. Mycelium was grown in the presence and absence (control) of the inhibitor 1NA-PP1 for a number of different times up to 24 hours.