Limits...
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.

Show MeSH

Related in: MedlinePlus

Movements of HDAC4-GFP in unstimulated fibers in the presence of leptomycin B, KN-62, and staurosporine. (A) A typical living fiber expressing HDAC4-GFP is shown in Ringer's solution without any treatment for 30 min (−30 and 0) and after 30 and 60 min in the presence of 40 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1. In the presence of leptomycin B, nuclear HDAC4-GFP fluorescence was significantly increased. No changes in cytoplasmic fluorescence were detected. Bar, 50 μm. (B) Time course of nuclear and cytoplasmic HDAC4-GFP fluorescence before and during exposure to 20 nM leptomycin B. Leptomycin B block of nuclear export of HDAC4-GFP caused an increase of HDAC4-GFP in the nucleus. (C) KN-62 was first added to culture dishes with fibers expressing HDAC4-GFP, and the fluorescence remained constant for 60 min. 1 μM staurosporine, a general kinase inhibitor, was then added to the same dishes without washing out of KN-62. The fluorescence signal was recorded for another 60 min. The same group of fibers that had no response to KN-62 subsequently responded to staurosporine, with a significant increase in nuclear HDAC4-GFP, indicating that staurosporine inhibition of a KN-62–insensitive kinase underlies the HDAC4 nuclear accumulation produced by staurosporine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171787&req=5

fig8: Movements of HDAC4-GFP in unstimulated fibers in the presence of leptomycin B, KN-62, and staurosporine. (A) A typical living fiber expressing HDAC4-GFP is shown in Ringer's solution without any treatment for 30 min (−30 and 0) and after 30 and 60 min in the presence of 40 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1. In the presence of leptomycin B, nuclear HDAC4-GFP fluorescence was significantly increased. No changes in cytoplasmic fluorescence were detected. Bar, 50 μm. (B) Time course of nuclear and cytoplasmic HDAC4-GFP fluorescence before and during exposure to 20 nM leptomycin B. Leptomycin B block of nuclear export of HDAC4-GFP caused an increase of HDAC4-GFP in the nucleus. (C) KN-62 was first added to culture dishes with fibers expressing HDAC4-GFP, and the fluorescence remained constant for 60 min. 1 μM staurosporine, a general kinase inhibitor, was then added to the same dishes without washing out of KN-62. The fluorescence signal was recorded for another 60 min. The same group of fibers that had no response to KN-62 subsequently responded to staurosporine, with a significant increase in nuclear HDAC4-GFP, indicating that staurosporine inhibition of a KN-62–insensitive kinase underlies the HDAC4 nuclear accumulation produced by staurosporine.

Mentions: Leptomycin B binds to the nuclear export mediator protein CRM1, thereby blocking the binding of CRM1 to proteins containing the nuclear export signal (Fukuda et al., 1997) and preventing their nuclear export. During exposure to leptomycin B (40 nM), nuclear HDAC4-GFP continuously increased over a 60-min experimental period, whereas the cytoplasmic fluorescence signal was stable (Fig. 8 A). Upon treatment with leptomycin B, HDAC4-GFP translocated into the nucleus at a constant rate (Fig. 8 B) of 0.88 ± 0.09%/min. Assuming that leptomycin B completely inhibited HDAC4 efflux from fiber nuclei, these results reveal a unidirectional influx of HDAC4 into nuclei at a rate of 0.88%/min. If influx was not altered by leptomycin B, then under resting conditions, before addition of leptomycin B and in the absence of muscle activity to activate protein kinases or protein phosphatases, there was balanced unidirectional nuclear influx and efflux of HDAC4 at this rate to give zero net flux of HDAC4 since nuclear HDAC4 was constant in resting fibers under control conditions.


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)

Movements of HDAC4-GFP in unstimulated fibers in the presence of leptomycin B, KN-62, and staurosporine. (A) A typical living fiber expressing HDAC4-GFP is shown in Ringer's solution without any treatment for 30 min (−30 and 0) and after 30 and 60 min in the presence of 40 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1. In the presence of leptomycin B, nuclear HDAC4-GFP fluorescence was significantly increased. No changes in cytoplasmic fluorescence were detected. Bar, 50 μm. (B) Time course of nuclear and cytoplasmic HDAC4-GFP fluorescence before and during exposure to 20 nM leptomycin B. Leptomycin B block of nuclear export of HDAC4-GFP caused an increase of HDAC4-GFP in the nucleus. (C) KN-62 was first added to culture dishes with fibers expressing HDAC4-GFP, and the fluorescence remained constant for 60 min. 1 μM staurosporine, a general kinase inhibitor, was then added to the same dishes without washing out of KN-62. The fluorescence signal was recorded for another 60 min. The same group of fibers that had no response to KN-62 subsequently responded to staurosporine, with a significant increase in nuclear HDAC4-GFP, indicating that staurosporine inhibition of a KN-62–insensitive kinase underlies the HDAC4 nuclear accumulation produced by staurosporine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171787&req=5

fig8: Movements of HDAC4-GFP in unstimulated fibers in the presence of leptomycin B, KN-62, and staurosporine. (A) A typical living fiber expressing HDAC4-GFP is shown in Ringer's solution without any treatment for 30 min (−30 and 0) and after 30 and 60 min in the presence of 40 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1. In the presence of leptomycin B, nuclear HDAC4-GFP fluorescence was significantly increased. No changes in cytoplasmic fluorescence were detected. Bar, 50 μm. (B) Time course of nuclear and cytoplasmic HDAC4-GFP fluorescence before and during exposure to 20 nM leptomycin B. Leptomycin B block of nuclear export of HDAC4-GFP caused an increase of HDAC4-GFP in the nucleus. (C) KN-62 was first added to culture dishes with fibers expressing HDAC4-GFP, and the fluorescence remained constant for 60 min. 1 μM staurosporine, a general kinase inhibitor, was then added to the same dishes without washing out of KN-62. The fluorescence signal was recorded for another 60 min. The same group of fibers that had no response to KN-62 subsequently responded to staurosporine, with a significant increase in nuclear HDAC4-GFP, indicating that staurosporine inhibition of a KN-62–insensitive kinase underlies the HDAC4 nuclear accumulation produced by staurosporine.
Mentions: Leptomycin B binds to the nuclear export mediator protein CRM1, thereby blocking the binding of CRM1 to proteins containing the nuclear export signal (Fukuda et al., 1997) and preventing their nuclear export. During exposure to leptomycin B (40 nM), nuclear HDAC4-GFP continuously increased over a 60-min experimental period, whereas the cytoplasmic fluorescence signal was stable (Fig. 8 A). Upon treatment with leptomycin B, HDAC4-GFP translocated into the nucleus at a constant rate (Fig. 8 B) of 0.88 ± 0.09%/min. Assuming that leptomycin B completely inhibited HDAC4 efflux from fiber nuclei, these results reveal a unidirectional influx of HDAC4 into nuclei at a rate of 0.88%/min. If influx was not altered by leptomycin B, then under resting conditions, before addition of leptomycin B and in the absence of muscle activity to activate protein kinases or protein phosphatases, there was balanced unidirectional nuclear influx and efflux of HDAC4 at this rate to give zero net flux of HDAC4 since nuclear HDAC4 was constant in resting fibers under control conditions.

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