Fig. 8: PKA phosphorylates HDAC1 to repress its deacetylase activity and promote TCA cycle. | Nature Communications

Fig. 8: PKA phosphorylates HDAC1 to repress its deacetylase activity and promote TCA cycle.

From: PKA plays a conserved role in regulating gene expression and metabolic adaptation by phosphorylating Rpd3/HDAC1

Fig. 8

a Immunoblot analysis of H3K9ac and H3K14ac in control, PRKACA knockdown and PRKACB knockdown HepG2 cells. b Immunoblot analysis of H3K9ac and H3K14ac in control, HDAC1 knockdown, HDAC2 knockdown, HDAC3 knockdown and HDAC8 knockdown HepG2 cells. c, d Co-IP and reciprocal IP showing HDAC1 interacted with PRKACA and PRKACB. e HDAC1 was phosphorylated at S434. Flag-HDAC1 and Flag-HDAC1-S434A were immunoprecipitated from HepG2 cells with anti-FLAG beads and the phosphorylation of HDAC1 was determined by phospho-PKA antibody. f An in vitro kinase assay showing purified Flag-PRKACA phosphorylated His-HDAC1 but not His-HDAC-S434A. g Ectopic expression of Flag-HDAC1-S434A in shHDAC1 HepG2 cells reduced global levels of H3K9ac and H3K14ac. h An in vitro deacetylase assay showing Flag-PRKACA-mediated HDAC1-S434 phosphorylation reduced the deacetylase activity of His-HDAC1 but not His-HDAC1-S434A. i Ectopic expression of HDAC1-S434A in shHDAC1 HepG2 cells reduced TCA cycle gene transcription. j Ectopic expression of HDAC1-S434A in shHDAC1 HepG2 cells reduced TCA cycle metabolites. k, l Ectopic expression of HDAC1-S434A in shHDAC1 HepG2 cells reduced cell growth (k) and colony formation (l). m–o Effect of PKA inhibitor H89 on HDAC1 phosphorylation, TCA cycle gene transcription, and TCA cycle metabolites in the liver of C57BL/6 J mice under fed and fasting conditions. Mice were injected with or without 5 mg/kg H89 followed by fed ad libitum or fasted for 12 h (5 mice per group). The live tissues were harvested. HDAC1 was immunoprecipitated with anti-HDAC1 antibody and its phosphorylation was probed with anti-phospho-PKA antibody. TCA cycle gene transcription and metabolites were analyzed by RT-qPCR and LC-MS, respectively. For (a, b, g–k, m, n), data represent means ± SE; n = 3 biological independent experiments; one-way ANOVA with Dunnett’s multiple comparisons tests were used for statistical analysis (a, b); two tailed unpaired t-tests were used for statistical analysis (g, i); two-way ANOVA with Šídák’s multiple comparisons tests (h, k) and Turkey’s multiple comparisons tests (m, n) were used for statistical analysis. For (o), n = 5 biological independent experiments. For (c–f, l), shown is the typical example of at least two biological independent experiments.

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