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Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.

Zhang L, Liu S, Liu N, Zhang Y, Liu M, Li D, Seto E, Yao TP, Shui W, Zhou J - Protein Cell (2014)

Bottom Line: However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates.Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1.These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

View Article: PubMed Central - PubMed

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

ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

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HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction. (A and B) Lysates from wild-type and HDAC6 knockout MEFs were immunoprecipitated with anti-Hsc70 (A) or anti-DNAJA1 (B) antibodies. The immunoprecipitates and cell lysates were then subjected to immunoblotting with anti-DNAJA1 or anti-Hsc70 antibodies. (C and D) 293T cells were treated with DMSO (−) or tubacin (+) for 8 h, and the lysates were immunoprecipitated with anti-Hsc70 (C) or anti-DNAJA1 (D) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1 or anti-Hsc70 antibodies. (E and F) 293T cells were transfected with GFP-HDAC6 wild-type (WT), the catalytically inactive mutant (MT), or GFP alone, and the lysates were immunoprecipitated with anti-Hsc70 (E) or anti-DNAJA1 (F) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1, anti-Hsc70, or anti-GFP antibodies
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Fig5: HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction. (A and B) Lysates from wild-type and HDAC6 knockout MEFs were immunoprecipitated with anti-Hsc70 (A) or anti-DNAJA1 (B) antibodies. The immunoprecipitates and cell lysates were then subjected to immunoblotting with anti-DNAJA1 or anti-Hsc70 antibodies. (C and D) 293T cells were treated with DMSO (−) or tubacin (+) for 8 h, and the lysates were immunoprecipitated with anti-Hsc70 (C) or anti-DNAJA1 (D) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1 or anti-Hsc70 antibodies. (E and F) 293T cells were transfected with GFP-HDAC6 wild-type (WT), the catalytically inactive mutant (MT), or GFP alone, and the lysates were immunoprecipitated with anti-Hsc70 (E) or anti-DNAJA1 (F) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1, anti-Hsc70, or anti-GFP antibodies

Mentions: Hsc70 and DNAJA1 are known to function together to promote proper folding of proteins (Meacham et al., 1999). Our findings that HDAC6 deacetylates both Hsc70 and DNAJA1 prompted us to determine whether the association between these two proteins is influenced by HDAC6. By immunoprecipitation assays, we found that the interaction between Hsc70 and DNAJA1 was significantly decreased in HDAC6 knockout MEFs as compared to wild-type MEFs (Fig. 5A and 5B). In addition, treatment of cells with the HDAC6 inhibitor tubacin remarkably inhibited the interaction between these two proteins (Fig. 5C and 5D). Furthermore, overexpression of wild-type HDAC6, but not the catalytically inactive mutant that harbors H216A and H611A mutations (Grozinger et al., 1999), enhanced the interaction between Hsc70 and DNAJA1 (Fig. 5E and 5F). These data suggest that HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction.Figure 5


Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity.

Zhang L, Liu S, Liu N, Zhang Y, Liu M, Li D, Seto E, Yao TP, Shui W, Zhou J - Protein Cell (2014)

HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction. (A and B) Lysates from wild-type and HDAC6 knockout MEFs were immunoprecipitated with anti-Hsc70 (A) or anti-DNAJA1 (B) antibodies. The immunoprecipitates and cell lysates were then subjected to immunoblotting with anti-DNAJA1 or anti-Hsc70 antibodies. (C and D) 293T cells were treated with DMSO (−) or tubacin (+) for 8 h, and the lysates were immunoprecipitated with anti-Hsc70 (C) or anti-DNAJA1 (D) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1 or anti-Hsc70 antibodies. (E and F) 293T cells were transfected with GFP-HDAC6 wild-type (WT), the catalytically inactive mutant (MT), or GFP alone, and the lysates were immunoprecipitated with anti-Hsc70 (E) or anti-DNAJA1 (F) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1, anti-Hsc70, or anti-GFP antibodies
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4286133&req=5

Fig5: HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction. (A and B) Lysates from wild-type and HDAC6 knockout MEFs were immunoprecipitated with anti-Hsc70 (A) or anti-DNAJA1 (B) antibodies. The immunoprecipitates and cell lysates were then subjected to immunoblotting with anti-DNAJA1 or anti-Hsc70 antibodies. (C and D) 293T cells were treated with DMSO (−) or tubacin (+) for 8 h, and the lysates were immunoprecipitated with anti-Hsc70 (C) or anti-DNAJA1 (D) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1 or anti-Hsc70 antibodies. (E and F) 293T cells were transfected with GFP-HDAC6 wild-type (WT), the catalytically inactive mutant (MT), or GFP alone, and the lysates were immunoprecipitated with anti-Hsc70 (E) or anti-DNAJA1 (F) antibodies. The immunoprecipitates and cell lysates were then immunoblotted with anti-DNAJA1, anti-Hsc70, or anti-GFP antibodies
Mentions: Hsc70 and DNAJA1 are known to function together to promote proper folding of proteins (Meacham et al., 1999). Our findings that HDAC6 deacetylates both Hsc70 and DNAJA1 prompted us to determine whether the association between these two proteins is influenced by HDAC6. By immunoprecipitation assays, we found that the interaction between Hsc70 and DNAJA1 was significantly decreased in HDAC6 knockout MEFs as compared to wild-type MEFs (Fig. 5A and 5B). In addition, treatment of cells with the HDAC6 inhibitor tubacin remarkably inhibited the interaction between these two proteins (Fig. 5C and 5D). Furthermore, overexpression of wild-type HDAC6, but not the catalytically inactive mutant that harbors H216A and H611A mutations (Grozinger et al., 1999), enhanced the interaction between Hsc70 and DNAJA1 (Fig. 5E and 5F). These data suggest that HDAC6-mediated deacetylation of Hsc70 and DNAJA1 are critical for their interaction.Figure 5

Bottom Line: However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates.Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1.These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

View Article: PubMed Central - PubMed

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

ABSTRACT
Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

Show MeSH
Related in: MedlinePlus