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Next Generation Sequencing Facilitates Quantitative Analysis of wild typeHDAC1 and HDAC1_S434A Transcriptomes with or without PKA inhibitor H89 treatment

ABSTRACT: Cells need to reprogram their metabolism to adapt to extracellular nutrient changes. The histone acetyltransferase SAGA (Spt-Ada-Gcn5-acetyltransferase) complex has been reported to acetylate its subunit Ada3 and form homo-dimers to enhance its ability to acetylate nucleosomes and facilitate metabolic gene transcription. How cells transduce extracellular nutrient changes to SAGA structure and function changes remains unclear. Here, we found that SAGA is deacetylated by Rpd3L complex and uncovered how its deacetylase activity is repressed by nutrient sensor protein kinase A (PKA). When sucrose is used as the sole carbon source, PKA catalytic subunit Tpk2 is activated, which phosphorylates Rpd3L catalytic subunit Rpd3 at serine 50 and serine 354 to inhibit its ability to deacetylate Ada3. Moreover, Tpk2 phosphorylates Rpd3L subunit Ash1 at serine 149 and serine 388, which specifically reduces the interaction between Rpd3L and SAGA. By phosphorylating both Rpd3 and Ash1, Tpk2 inhibits Rpd3L-mediated Ada3 deacetylation, which promotes SAGA dimerization, nucleosome acetylation and transcription of genes involved in sucrose utilization and tricarboxylate (TCA) cycle, resulting in metabolic shift from glycolysis to TCA cycle. Most importantly, PKA phosphorylates HDAC1, the Rpd3 homolog in mammals to repress its deacetylase activity and promote TCA cycle gene transcription. Blocking PKA-catalyzed HDAC1 phosphorylation reduced TCA cycle and impaired cell growth. Our work hence reveals a conserved role of PKA in regulating Rpd3/HDAC1 and metabolism adaptation and provides a potential target for future cancer treatment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE284216 | GEO | 2025/03/12

REPOSITORIES: GEO

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Project description:The pattern of gene transcription in Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA), triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. To investigate the requirement of PKA for glucose control of gene expression, we have analyzed global transcription in strains devoid of PKA activity. In S. cerevisiae three genes, TPK1, TPK2, TPK3, encode catalytic subunits of PKA and the triple mutant tpk1 tpk2 tpk3 is unviable. We have worked, therefore, with two strains, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4, that bear suppressor mutations,. We have identified different classes of genes that can be induced, or repressed, by glucose in the absence of PKA. Among these genes, some are also controlled by a redundant signalling pathway involving PKA activation, while others do not respond to an increase in cAMP concentration. On the other hand, among genes which do not respond to glucose in the absence of PKA, some show a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. The goal of the present study was to investigate the occurrence of PKA-independent glucose signalling in S. cerevisiae. To this end, we have used global transcription analysis to study the effects of glucose on yeast strains completely devoid of PKA activity. In S. cerevisiae three genes TPK1, TPK2,and TPK3 encode catalytic subunits of PKA. While strains expressing only one of these genes grow normally, a triple null mutant (tpk1 tpk2 tpk3) is not viable (Toda et al 1987). Identification of different mutations able to suppress the growth defect of the triple mutant (Garrett and Broach 1989, Reinders et al 1998, Smith et al 1998) has allowed to determine what is the crucial function of PKA. As shown in Fig.1, PKA is needed to counteract the negative effect of the protein kinase Yak1 on yeast growth (Hartley et al 1994, Moriya et al 2001). In the presence of PKA the protein kinase Rim15 (Reinders et al 1998) and the transcription factors Msn2 and Msn4 (Görner et al 1998) can be phosphorylated and exported to the cytoplasm, transcription of the YAK1 gene, which is activated by Msn2/Msn4 (Smith et al 1998), is reduced, Yak1 levels remain low and growth is not hindered. In the absence of PKA, Rim15 remains in the nucleus where it can activate Msn2/Msn4 (Cameroni et al 2004) that turn on YAK1 transcription, thus blocking growth. This explains why strains lacking Rim15, Msn2/Msn4 or Yak1 no longer require PKA for growth. In this work we have used two isogenic strains lacking PKA and carrying the suppressor mutations msn2 msn4 or yak1. Two different suppressor mutants were used with the aim to enable a dissection of effects of the lack of PKA and effects of the suppressor mutations themselves.

Project description:The conserved cAMP-dependent protein kinase (PKA) holoenzyme is composed of two catalytic and two regulatory subunits. It plays critical roles in the regulation of many biological processes in eukaryotic organisms. In the human fungal pathogen Candida albicans, the PKA kinase has been extensively investigated for its importance in the regulation of morphological transitions and virulence. It has been long thought that the PKA catalytic subunit is essential for cell viability in C. albicans. Paradoxically, the single adenylyl cyclase-encoding gene, CRY1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. In this study, we successfully generated a null double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1 or t2t1), which encode two isoforms of the PKA catalytic subunit in C. albicans. We reevaluated the roles of the PKA catalytic subunit in cell growth and phenotypic transitions. Inactivation of the PKA catalytic subunit by deletion of both TPK1 and TPK2 blocked filamentation and dramatically attenuated the ability of white-to-opaque switching, but promoted sexual mating in C. albicans. Tpk2 plays a major role in these regulations, while Tpk1 generally functions as a negative regulator in morphological transitions and sexual mating. A comparative transcriptomic analysis demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared to the WT strain, the general transcriptional activity and expression of genes involved in metabolism, translation, biosynthesis, adhesion and filamentation are significantly decreased in both the t2t1 and cyr1/cyr1 mutants. And a portion of stress-response and cell wall-related genes were upregulated in these mutants, which is consistent with their increased ability of anti-stresses. To further explore the global regulatory role of the PKA kinase, we performed quantitative phosphoproteomics analysis. Combining with bioinformatics analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. Cell wall and membrane-related proteins (e.g. Ecm3, Bni1, and Smi1) were enriched in Tpk1-specific targets, while Tpk2-specific substrates include transporters, filamentation and cytoskeleton-related proteins (e.g. Smf3, Sep7, and Mhp1). There were also many Tpk1 and Tpk2 overlapped and coordinately regulated-substrates. Our study clarifies the essentiality of the PKA catalytic subunit and shed new insights into the global regulatory features of the cAMP/PKA pathway in C. ablicans. The t2t1 null mutant generated in this study would also be a new resource for the field to study this important pathway.

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Next Generation Sequencing Facilitates Quantitative Analysis of wild typeHDAC1 and HDAC1_S434A Transcriptomes with or without PKA inhibitor H89 treatment

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