Opposing HDAC4 nuclear fluxes due to phosphorylation by β-adrenergic activated protein kinase A or by activity or Epac activated CaMKII in skeletal muscle fibres

Key points • Application of either the beta-adrenergic agonist isoproterenol, dibutyryl cAMP or specific PKA activator N6 benzoyl cAMP caused nuclear influx of wild-type (wt) HDAC4-GFP expressed in cultured adult skeletal muscle fibres, but caused no change in nuclear/cytoplasmic distribution of expressed ‘mut’ HDAC4-GFP mutated (S 265 and 266 to A) at the protein kinase A (PKA) phosphorylation site(s), demonstrating that PKA promotes HDAC4 nuclear influx by phosphorylation of HDAC4 at the PKA sites. • In non-transfected muscle fibres, myocyte enhancer factor 2 (MEF2)-driven luc reporter activity was decreased by application of isoproterenol, indicating that endogenous HDAC4 increased in fibre nuclei and suppressed MEF2 transcriptional activity. Levels of phosphorylated (i.e. active) PKA were elevated by exposure to dibutyryl (Db) cAMP. • Fibre repetitive electrical stimulation with 10 Hz trains caused a CaMKII dependent nuclear efflux of wt and mut HDAC4, which was partially decreased by Db cAMP for wt but not for mut HDAC4-GFP. • The specific activator 8-CPT of Epac caused efflux of both wt and mut HDAC4-GFP, which was eliminated by the CaMK inhibitor KN-93 or by buffering cytosolic Ca2+ using BAPTA-AM loading, both of which also eliminated a slow elevation of cytosolic Ca2+ during 8-CPT application. • Using a submaximally effective stimulus frequency of 4 Hz trains of electrical stimulation, Db cAMP increased the rate of nuclear influx of mut HDAC4-GFP, which cannot be phosphorylated by PKA but can be phosphorylated by CaMKII, which is here activated via the cAMP/Epac pathway. • Immunostain for active PKA or for GTP-bound RAP1, which is an indicator of Epac activation, showed responses consistent with the functional results above. Abstract Class IIa histone deacetylases (HDACs) move between skeletal muscle fibre cytoplasm and nuclei in response to various stimuli, suppressing activity of the exclusively nuclear transcription factor Mef2. Protein kinase A (PKA) phosphorylates class IIa HDACs in cardiac muscle, resulting in HDAC nuclear accumulation, but this has not been examined in skeletal muscle. Using HDAC4–green fluorescent protein (HDAC4-GFP) expressed in isolated skeletal muscle fibres, we now show that activation of PKA by the beta-receptor agonist isoproterenol or dibutyryl (Db) cAMP causes a steady HDAC4-GFP nuclear influx. The beta-receptor blocker propranolol or PKA inhibitor Rp-cAMPS blocks the effects of isoproterenol on the nuclear influx of HDAC4-GFP, and Rp-cAMPS blocks the effects of Db cAMP. The HDAC4-GFP construct having serines 265 and 266 replaced with alanines, HDAC4 (S265/266A)-GFP, did not respond to beta-receptor or PKA activation. Immunoprecipitation results show that HDAC4-GFP is a substrate of PKA, but HDAC4 (S265/266A)-GFP is not, implicating HDAC4 serines 265/266 as the site(s) phosphorylated by PKA. During 10 Hz trains of muscle fibre electrical stimulation, the nuclear efflux rate of HDAC4-GFP, but not of HDAC4 (S265/266)-GFP, was decreased by PKA activation, directly demonstrating antagonism between the effects of fibre stimulation and beta-adrenergic activation of PKA on HDAC4 nuclear fluxes. 8-CPT, a specific activator of Epac, caused nuclear efflux of HDAC4-GFP, opposite to the effect of PKA. Db cAMP increased both phosphorylated PKA and GTP-bound Rap1. Our results demonstrate that the PKA and CaMKII pathways play important opposing roles in skeletal muscle gene expression by oppositely affecting the subcellular localization of HDAC4.