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Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

de la Vega L, Hornung J, Kremmer E, Milanovic M, Schmitz ML - Nucleic Acids Res. (2013)

Bottom Line: Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes.Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein.This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, 35392 Giessen, Germany.

ABSTRACT
Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

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HIPK2 phosphorylates MEF2C. (A) Epitope-tagged versions of HIPK2 or HIPK2 K221A were coexpressed with MEF2C in 293T cells. Cell lysates were tested by western blotting for the electrophoretic behavior of MEF2C, and the position of the phosphorylated form is indicated. (B) Cells were transfected to express HIPK2 and MEF2C as shown, and lysates were either left untreated or incubated with λ phosphatase as shown. Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with the specified antibodies. (C) MEF2C was coexpressed with HDAC4, HIPK2 or increasing amounts of HIPK2 K221A. Extracts were further analyzed for MEF2C phosphorylation by western blotting, as revealed by the occurrence of the slower migrating phosphorylated form. (D) Cells transfected to express a HIPK2-specific shRNA were selected for 3 days in the presence of puromycin, followed by plating and retransfection to express MEF2C and HDAC4. Immunoblotting of cell extracts with specific antibodies ensured efficient HIPK2 knockdown, MEF2C phosphorylation was scored by the occurrence of the slower migrating form as shown.
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gkt262-F3: HIPK2 phosphorylates MEF2C. (A) Epitope-tagged versions of HIPK2 or HIPK2 K221A were coexpressed with MEF2C in 293T cells. Cell lysates were tested by western blotting for the electrophoretic behavior of MEF2C, and the position of the phosphorylated form is indicated. (B) Cells were transfected to express HIPK2 and MEF2C as shown, and lysates were either left untreated or incubated with λ phosphatase as shown. Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with the specified antibodies. (C) MEF2C was coexpressed with HDAC4, HIPK2 or increasing amounts of HIPK2 K221A. Extracts were further analyzed for MEF2C phosphorylation by western blotting, as revealed by the occurrence of the slower migrating phosphorylated form. (D) Cells transfected to express a HIPK2-specific shRNA were selected for 3 days in the presence of puromycin, followed by plating and retransfection to express MEF2C and HDAC4. Immunoblotting of cell extracts with specific antibodies ensured efficient HIPK2 knockdown, MEF2C phosphorylation was scored by the occurrence of the slower migrating form as shown.

Mentions: The gene-repressing effect of HIPK2 raises the question whether the kinase can bind to MEF2 and further components of the multi-protein complex controlling the expression of muscle-specific genes. To explore this issue, cells were transfected to express epitope-tagged versions of HIPK2 and MEF2C, followed by coimmunoprecipitation experiments. IP of Flag-tagged MEF2C allowed to detect binding of HIPK2 (Figure 2A). A similar approach was used to test the interaction between HIPK2 and HDAC4, which were also found to be associated (Figure 2B). Further coimmunoprecipitation experiments were performed with lysates from C2C12 cells to display the proteins that are constitutively associated with HIPK2 in undifferentiated myoblasts. Immunoprecipipitation of the endogenous kinase followed by immunoblotting revealed association with MEF2, HDAC3 and HDAC4 (Figure 2C). Indirect immunofluorescence showed colocalization between all proteins in the nucleus (Figure 2D). While HIPK2 and the nuclear fraction of HDAC4 show almost complete colocalization in nuclear speckles, only a fraction of HDAC3 binds to and colocalizes with HIPK2. This differential colocalization between HIPK2 and both HDACs is also reflected by the coimmunoprecipitation experiments that revealed preferential binding to HDAC4 (see Figure 2C), thus suggesting that the HIPK2-containing protein complex contains only substoichiometric amounts of HDAC3. In the course of these experiments, we noted that the coexpression of HIPK2 and MEF2C resulted in the occurrence of an upshifted MEF2C band in the presence of phosphatase inhibitors in the lysis buffer. To test the possible HIPK2-mediated phosphorylation of MEF2C, the transcription factor was coexpressed with the wild-type kinase or a kinase-inactive point mutant (HIPK2 K221A). Western blotting showed that the upshifted form of MEF2C occurred only in the presence of the wild-type kinase (Figure 3A). To investigate whether the slower electrophoretic migration of MEF2C is caused by phosphorylation, extracts from cells coexpressing HIPK2 and MEF2C were incubated with λ phosphatase. This treatment converted the slower migrating form of MEF2C into a band that migrated even slightly faster than the band that occurs on expression of MEF2C alone (Figure 3B), demonstrating that the upshifted band represents phosphorylated MEF2C. As published data report the association of HDAC4 with a kinase activity leading to MEF2 phosphorylation (42), it was interesting to investigate a possible contribution of HIPK2 to this enzymatic activity. Cells were transfected to express MEF2C along with HDAC4 and kinase-inactive HIPK2 that can associate with the endogenous kinase (3) and thus exert a trans-dominant negative function. Consistent with published data, the expression of HDAC4 was sufficient to trigger MEF2 phosphorylation (42). Expression of HIPK2 K221A dose-dependently diminished MEF2C phosphorylation (Figure 3C). Similarly, MEF2C phosphorylation by the HDAC4-associated kinase activity was significantly impaired on shRNA-mediated knockdown of HIPK2 (Figure 3D), thus revealing HIPK2 as another HDAC4-associated kinase.Figure 2.


Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

de la Vega L, Hornung J, Kremmer E, Milanovic M, Schmitz ML - Nucleic Acids Res. (2013)

HIPK2 phosphorylates MEF2C. (A) Epitope-tagged versions of HIPK2 or HIPK2 K221A were coexpressed with MEF2C in 293T cells. Cell lysates were tested by western blotting for the electrophoretic behavior of MEF2C, and the position of the phosphorylated form is indicated. (B) Cells were transfected to express HIPK2 and MEF2C as shown, and lysates were either left untreated or incubated with λ phosphatase as shown. Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with the specified antibodies. (C) MEF2C was coexpressed with HDAC4, HIPK2 or increasing amounts of HIPK2 K221A. Extracts were further analyzed for MEF2C phosphorylation by western blotting, as revealed by the occurrence of the slower migrating phosphorylated form. (D) Cells transfected to express a HIPK2-specific shRNA were selected for 3 days in the presence of puromycin, followed by plating and retransfection to express MEF2C and HDAC4. Immunoblotting of cell extracts with specific antibodies ensured efficient HIPK2 knockdown, MEF2C phosphorylation was scored by the occurrence of the slower migrating form as shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675480&req=5

gkt262-F3: HIPK2 phosphorylates MEF2C. (A) Epitope-tagged versions of HIPK2 or HIPK2 K221A were coexpressed with MEF2C in 293T cells. Cell lysates were tested by western blotting for the electrophoretic behavior of MEF2C, and the position of the phosphorylated form is indicated. (B) Cells were transfected to express HIPK2 and MEF2C as shown, and lysates were either left untreated or incubated with λ phosphatase as shown. Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting with the specified antibodies. (C) MEF2C was coexpressed with HDAC4, HIPK2 or increasing amounts of HIPK2 K221A. Extracts were further analyzed for MEF2C phosphorylation by western blotting, as revealed by the occurrence of the slower migrating phosphorylated form. (D) Cells transfected to express a HIPK2-specific shRNA were selected for 3 days in the presence of puromycin, followed by plating and retransfection to express MEF2C and HDAC4. Immunoblotting of cell extracts with specific antibodies ensured efficient HIPK2 knockdown, MEF2C phosphorylation was scored by the occurrence of the slower migrating form as shown.
Mentions: The gene-repressing effect of HIPK2 raises the question whether the kinase can bind to MEF2 and further components of the multi-protein complex controlling the expression of muscle-specific genes. To explore this issue, cells were transfected to express epitope-tagged versions of HIPK2 and MEF2C, followed by coimmunoprecipitation experiments. IP of Flag-tagged MEF2C allowed to detect binding of HIPK2 (Figure 2A). A similar approach was used to test the interaction between HIPK2 and HDAC4, which were also found to be associated (Figure 2B). Further coimmunoprecipitation experiments were performed with lysates from C2C12 cells to display the proteins that are constitutively associated with HIPK2 in undifferentiated myoblasts. Immunoprecipipitation of the endogenous kinase followed by immunoblotting revealed association with MEF2, HDAC3 and HDAC4 (Figure 2C). Indirect immunofluorescence showed colocalization between all proteins in the nucleus (Figure 2D). While HIPK2 and the nuclear fraction of HDAC4 show almost complete colocalization in nuclear speckles, only a fraction of HDAC3 binds to and colocalizes with HIPK2. This differential colocalization between HIPK2 and both HDACs is also reflected by the coimmunoprecipitation experiments that revealed preferential binding to HDAC4 (see Figure 2C), thus suggesting that the HIPK2-containing protein complex contains only substoichiometric amounts of HDAC3. In the course of these experiments, we noted that the coexpression of HIPK2 and MEF2C resulted in the occurrence of an upshifted MEF2C band in the presence of phosphatase inhibitors in the lysis buffer. To test the possible HIPK2-mediated phosphorylation of MEF2C, the transcription factor was coexpressed with the wild-type kinase or a kinase-inactive point mutant (HIPK2 K221A). Western blotting showed that the upshifted form of MEF2C occurred only in the presence of the wild-type kinase (Figure 3A). To investigate whether the slower electrophoretic migration of MEF2C is caused by phosphorylation, extracts from cells coexpressing HIPK2 and MEF2C were incubated with λ phosphatase. This treatment converted the slower migrating form of MEF2C into a band that migrated even slightly faster than the band that occurs on expression of MEF2C alone (Figure 3B), demonstrating that the upshifted band represents phosphorylated MEF2C. As published data report the association of HDAC4 with a kinase activity leading to MEF2 phosphorylation (42), it was interesting to investigate a possible contribution of HIPK2 to this enzymatic activity. Cells were transfected to express MEF2C along with HDAC4 and kinase-inactive HIPK2 that can associate with the endogenous kinase (3) and thus exert a trans-dominant negative function. Consistent with published data, the expression of HDAC4 was sufficient to trigger MEF2 phosphorylation (42). Expression of HIPK2 K221A dose-dependently diminished MEF2C phosphorylation (Figure 3C). Similarly, MEF2C phosphorylation by the HDAC4-associated kinase activity was significantly impaired on shRNA-mediated knockdown of HIPK2 (Figure 3D), thus revealing HIPK2 as another HDAC4-associated kinase.Figure 2.

Bottom Line: Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes.Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein.This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, 35392 Giessen, Germany.

ABSTRACT
Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

Show MeSH
Related in: MedlinePlus