Limits...
Deacetylation of α-tubulin and cortactin is required for HDAC6 to trigger ciliary disassembly.

Ran J, Yang Y, Li D, Liu M, Zhou J - Sci Rep (2015)

Bottom Line: Ciliary dysfunction is associated with a variety of diseases known as ciliopathies.Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators.These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

No MeSH data available.


Mutation of the deacetylase domain of HDAC6 abolishes its activities to deacetylate α-tubulin and cortactin and to induce ciliary disassembly.(A) Western blot analysis of acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6 wild-type (WT), H216A, H611A, or H216/611A. (B) RPE1 cells were transfected with Flag-cortactin and GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A. The anti-Flag immunoprecipitate was then probed with acetylated lysine and Flag antibodies, and the cell lysate was probed with GFP antibody. (C–E) Immunofluorescence images (C), percentage of ciliated cells (D), and ciliary length (E) in RPE1 cells transfected with GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526867&req=5

f4: Mutation of the deacetylase domain of HDAC6 abolishes its activities to deacetylate α-tubulin and cortactin and to induce ciliary disassembly.(A) Western blot analysis of acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6 wild-type (WT), H216A, H611A, or H216/611A. (B) RPE1 cells were transfected with Flag-cortactin and GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A. The anti-Flag immunoprecipitate was then probed with acetylated lysine and Flag antibodies, and the cell lysate was probed with GFP antibody. (C–E) Immunofluorescence images (C), percentage of ciliated cells (D), and ciliary length (E) in RPE1 cells transfected with GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.

Mentions: To corroborate the above findings, RPE1 cells were transfected with various mutants of HDAC6. The mutants that we used include H216A (mutation of histidine 216 in the first deacetylase domain to alanine), H611A (mutation of histidine 611 in the second deacetylase domain to alanine), and H216/611A (mutation of both histidine 216 and histidine 611 to alanines). We found that H611A and H216/611A, but not H216A, remarkably abolished HDAC6-mediated deacetylation of α-tubulin in RPE1 cells (Fig. 4A). In addition, H216A and H611A partially inhibited HDAC6-mediated deacetylation of cortactin, whereas H216/611A entirely abolished the deacetylation of cortactin (Fig. 4B). We further found that while wild-type HDAC6 and H216A resulted in significant ciliary disassembly, H611A and H216/611A did not (Fig. 4C–E). Collectively, the above data suggest that the deacetylase activity is critical for HDAC6 to induce ciliary disassembly.


Deacetylation of α-tubulin and cortactin is required for HDAC6 to trigger ciliary disassembly.

Ran J, Yang Y, Li D, Liu M, Zhou J - Sci Rep (2015)

Mutation of the deacetylase domain of HDAC6 abolishes its activities to deacetylate α-tubulin and cortactin and to induce ciliary disassembly.(A) Western blot analysis of acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6 wild-type (WT), H216A, H611A, or H216/611A. (B) RPE1 cells were transfected with Flag-cortactin and GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A. The anti-Flag immunoprecipitate was then probed with acetylated lysine and Flag antibodies, and the cell lysate was probed with GFP antibody. (C–E) Immunofluorescence images (C), percentage of ciliated cells (D), and ciliary length (E) in RPE1 cells transfected with GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526867&req=5

f4: Mutation of the deacetylase domain of HDAC6 abolishes its activities to deacetylate α-tubulin and cortactin and to induce ciliary disassembly.(A) Western blot analysis of acetylated α-tubulin, α-tubulin, and GFP in RPE1 cells transfected with GFP vector or GFP-HDAC6 wild-type (WT), H216A, H611A, or H216/611A. (B) RPE1 cells were transfected with Flag-cortactin and GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A. The anti-Flag immunoprecipitate was then probed with acetylated lysine and Flag antibodies, and the cell lysate was probed with GFP antibody. (C–E) Immunofluorescence images (C), percentage of ciliated cells (D), and ciliary length (E) in RPE1 cells transfected with GFP vector or GFP-HDAC6 WT, H216A, H611A, or H216/611A, serum-starved for 24 hours, and stained with acetylated α-tubulin antibody and DAPI. Scale bar, 5 μm. *P < 0.05, ***P < 0.001; ns, not significant. Error bars indicate SEM.
Mentions: To corroborate the above findings, RPE1 cells were transfected with various mutants of HDAC6. The mutants that we used include H216A (mutation of histidine 216 in the first deacetylase domain to alanine), H611A (mutation of histidine 611 in the second deacetylase domain to alanine), and H216/611A (mutation of both histidine 216 and histidine 611 to alanines). We found that H611A and H216/611A, but not H216A, remarkably abolished HDAC6-mediated deacetylation of α-tubulin in RPE1 cells (Fig. 4A). In addition, H216A and H611A partially inhibited HDAC6-mediated deacetylation of cortactin, whereas H216/611A entirely abolished the deacetylation of cortactin (Fig. 4B). We further found that while wild-type HDAC6 and H216A resulted in significant ciliary disassembly, H611A and H216/611A did not (Fig. 4C–E). Collectively, the above data suggest that the deacetylase activity is critical for HDAC6 to induce ciliary disassembly.

Bottom Line: Ciliary dysfunction is associated with a variety of diseases known as ciliopathies.Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators.These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis.

No MeSH data available.