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Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Liu Y, Randall WR, Schneider MF - J. Cell Biol. (2005)

Bottom Line: Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2).Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4.Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca(2+) concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

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Effects of protein phosphatase inhibitors on the subcellular distribution of HDAC4-GFP. (A) The PP1 and PP2A inhibitor calyculin A dramatically decreased nuclear HDAC4-GFP in a 60-min period. HDAC4 nuclear and cytoplasmic fluorescence were constant during 30 min before calyculin A application. Calyculin A (final concentration 1 μM) caused a decline of nuclear fluorescence, but the cytoplasmic fluorescence remained constant. (B) 60-min exposure to the PP2B inhibitor CsA (1 μM) had no effect on nuclear fluorescence, whereas calyculin A subsequently decreased nuclear fluorescence in the same group of fibers.
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fig9: Effects of protein phosphatase inhibitors on the subcellular distribution of HDAC4-GFP. (A) The PP1 and PP2A inhibitor calyculin A dramatically decreased nuclear HDAC4-GFP in a 60-min period. HDAC4 nuclear and cytoplasmic fluorescence were constant during 30 min before calyculin A application. Calyculin A (final concentration 1 μM) caused a decline of nuclear fluorescence, but the cytoplasmic fluorescence remained constant. (B) 60-min exposure to the PP2B inhibitor CsA (1 μM) had no effect on nuclear fluorescence, whereas calyculin A subsequently decreased nuclear fluorescence in the same group of fibers.

Mentions: Next, we tested the role of phosphatases in HDAC4 shuttling in resting fibers. Over a 60-min period of treatment with 1 μM calyculin A (a PP1 and PP2A phosphatase inhibitor that does not affect PP2B; Ishihara et al., 1989), the nuclear HDAC4-GFP fluorescence decreased linearly with a mean net export rate of −0.71 ± 0.07%/min (Fig. 9 A, 13 nuclei from 5 fibers). This rate is a measure of the net efflux with the calyculin A–sensitive endogenous phosphatases (PP1 and 2A) inhibited in the absence of muscle fiber activity and the resulting Ca2+-dependent stimulation of kinases. Before addition of calyculin A, nuclear HDAC4 was constant, indicating that nuclear influx and efflux of HDAC4 were equal. The observed rate of efflux thus corresponds to the rate of PP1- and/or 2A-dependent HDAC4 influx into resting fibers. This rate is very similar to the rate of influx (0.88%/min) in the presence of leptomycin B, indicating that almost all of the influx of HDAC4 into nuclei during shuttling in resting fibers is mediated by PP1 or 2A dephosphorylation of HDAC4 in the cytoplasm. Interestingly, calyculin A also caused a similar rate of efflux of HDAC5-GFP (−0.62 ± 0.05, nine nuclei from six fibers) from nuclei as observed for HDAC4, establishing the ability of HDAC5 to move out of the nucleus even though it did not translocate in response to electrical stimulation.


Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Liu Y, Randall WR, Schneider MF - J. Cell Biol. (2005)

Effects of protein phosphatase inhibitors on the subcellular distribution of HDAC4-GFP. (A) The PP1 and PP2A inhibitor calyculin A dramatically decreased nuclear HDAC4-GFP in a 60-min period. HDAC4 nuclear and cytoplasmic fluorescence were constant during 30 min before calyculin A application. Calyculin A (final concentration 1 μM) caused a decline of nuclear fluorescence, but the cytoplasmic fluorescence remained constant. (B) 60-min exposure to the PP2B inhibitor CsA (1 μM) had no effect on nuclear fluorescence, whereas calyculin A subsequently decreased nuclear fluorescence in the same group of fibers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171787&req=5

fig9: Effects of protein phosphatase inhibitors on the subcellular distribution of HDAC4-GFP. (A) The PP1 and PP2A inhibitor calyculin A dramatically decreased nuclear HDAC4-GFP in a 60-min period. HDAC4 nuclear and cytoplasmic fluorescence were constant during 30 min before calyculin A application. Calyculin A (final concentration 1 μM) caused a decline of nuclear fluorescence, but the cytoplasmic fluorescence remained constant. (B) 60-min exposure to the PP2B inhibitor CsA (1 μM) had no effect on nuclear fluorescence, whereas calyculin A subsequently decreased nuclear fluorescence in the same group of fibers.
Mentions: Next, we tested the role of phosphatases in HDAC4 shuttling in resting fibers. Over a 60-min period of treatment with 1 μM calyculin A (a PP1 and PP2A phosphatase inhibitor that does not affect PP2B; Ishihara et al., 1989), the nuclear HDAC4-GFP fluorescence decreased linearly with a mean net export rate of −0.71 ± 0.07%/min (Fig. 9 A, 13 nuclei from 5 fibers). This rate is a measure of the net efflux with the calyculin A–sensitive endogenous phosphatases (PP1 and 2A) inhibited in the absence of muscle fiber activity and the resulting Ca2+-dependent stimulation of kinases. Before addition of calyculin A, nuclear HDAC4 was constant, indicating that nuclear influx and efflux of HDAC4 were equal. The observed rate of efflux thus corresponds to the rate of PP1- and/or 2A-dependent HDAC4 influx into resting fibers. This rate is very similar to the rate of influx (0.88%/min) in the presence of leptomycin B, indicating that almost all of the influx of HDAC4 into nuclei during shuttling in resting fibers is mediated by PP1 or 2A dephosphorylation of HDAC4 in the cytoplasm. Interestingly, calyculin A also caused a similar rate of efflux of HDAC5-GFP (−0.62 ± 0.05, nine nuclei from six fibers) from nuclei as observed for HDAC4, establishing the ability of HDAC5 to move out of the nucleus even though it did not translocate in response to electrical stimulation.

Bottom Line: Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2).Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4.Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

ABSTRACT
Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca(2+) concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca(2+)-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

Show MeSH
Related in: MedlinePlus