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Identification of ECM3 interactors by affinity purification followed by mass spectrometry analysis.

ABSTRACT: Identification of ECM3 interactors by affinity purification followed by mass spectrometry analysis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Ernesto Nakayasu

LAB HEAD: Ernesto Nakayasu

PROVIDER: PXD017773 | Pride | 2022-11-03

REPOSITORIES: Pride

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			Action	DRS
	EMC3_3T3_Control_1_12Sep18_Polaroid_18-08-05.raw	Raw
	EMC3_3T3_Control_2_12Sep18_Polaroid_18-08-05.raw	Raw
	EMC3_3T3_Control_3_12Sep18_Polaroid_18-08-05.raw	Raw
	EMC3_3T3_Control_4_12Sep18_Polaroid_18-08-05.raw	Raw
	EMC3_3T3_Control_5_12Sep18_Polaroid_18-08-05.raw	Raw

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Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.

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Identification of ECM3 interactors by affinity purification followed by mass spectrometry analysis.

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