PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae

Abstract

Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression.

Investigation of Proteomic and Phosphoproteomic Responses to Signaling Network Perturbations Reveals Functional Pathway Organizations in Yeast

“…With enrichment analysis of regulators, we found 36 proteins that were distinctly regulated by the HOG pathway ( Figure 3C ). Among them, 35 are known osmolality-responsive genes or showed consistent changes at mRNA level in cognate deletion strains previously ( Baccarini et al, 2015 ; García et al, 2009 ; Greatrix and van Vuuren, 2006 ; Nadal-Ribelles et al, 2012 ; Norbeck and Blomberg, 1997 ; Norbeck et al, 1996 ; van Wageningen et al, 2010 ), demonstrating the robustness and reliability of our analysis. Similarly, we detected 39 phosphosites (on 33 proteins) that were significantly modulated by the HOG pathway ( Figure 3D ).…”

RNA Sequencing Reveals Specific Transcriptomic Signatures Distinguishing Effects of the [SWI+] Prion and SWI1 Deletion in Yeast Saccharomyces cerevisiae

“…The recent findings suggest that SWI/SNF plays an important role in the stress response. Indeed, the Snf2 catalytic subunit of SWI/SNF was shown to be recruited to response of the genes under osmotic stress by means of the Cyc8-Tup1 regulatory network [75], and this recruitment is positively affected by kinases of the PKA family [76]. In addition, the sets of genes misregulated by deletions of SWI2 and TAF14 that encode the transcriptional regulator involved in the stress alleviation overlap significantly [77].…”

The role of PKA in the translational response to heat stress in Saccharomyces cerevisiae

“…Regulatory and catalytic subunits of PKA localize to the nucleus, cytoplasm or SGs/PBs depending on a range of external factors including the carbon source, nutrient availability, osmotic conditions or entry into stationary phase [ 27 ]; [ 18 ]; [ 19 ]; [ 28 ]. Here we study how PKA subunits localization is affected by moderate and severe thermal stress.…”

“…Different PKA subunits have been shown to display diverse subcellular localizations depending on the quality of the carbon source, level of glucose, degree of osmotic stress and in stationary phase [ 27 ]; [ 18 ]; [ 28 ]. Moreover, we have shown previously that two of the catalytic subunits of PKA, Tpk2 and Tpk3, accumulate in PBs and SGs in response to glucose starvation and in stationary phase [ 18 ]; [ 19 ].…”

“…In terms of their physical interactions, data available on the Saccharomyces Genome Database [ 53 ] indicates that Tpk1, Tpk2 and Tpk3 physically interact with 320, 98 and 114 proteins, respectively, while 87.6% of these proteins interact with only one of the Tpks. In terms of the varying isoform-specific functions of PKA, the relative localization of the catalytic subunits appears to play an important role: distinct nuclear-cytoplasmic localization or PB/SG localizations have been observed for each Tpk in response to different stimuli such as nutrient availability and hyperosmotic stress [ 18 , 19 , 28 ]. Also, differential phosphorylation status of each Tpk could contribute to divergence in their catalytic activity and cAMP-dependent regulation [ 25 , 54 ].…”