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Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4 * ♦

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ABSTRACT

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

No MeSH data available.


MEKK4ΔN induces skeletal muscle fiber atrophy in a Gadd45a-independent manner.A–C, mouse TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4-FLAG plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, and/or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. All muscles were co-transfected with 2.5 μg of eGFP plasmid. TA muscles were harvested for histological analysis 7 days post-transfection. A, average diameters of skeletal muscle fibers. Each data point represents the mean of >400 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p values were determined with a one-way ANOVA and Dunnett's multiple comparison test. N.S., not significant or p > 0.05. B, size distribution of all muscle fibers from A. C, representative fluorescence microscopy images of muscle cross-sections.
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Figure 7: MEKK4ΔN induces skeletal muscle fiber atrophy in a Gadd45a-independent manner.A–C, mouse TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4-FLAG plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, and/or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. All muscles were co-transfected with 2.5 μg of eGFP plasmid. TA muscles were harvested for histological analysis 7 days post-transfection. A, average diameters of skeletal muscle fibers. Each data point represents the mean of >400 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p values were determined with a one-way ANOVA and Dunnett's multiple comparison test. N.S., not significant or p > 0.05. B, size distribution of all muscle fibers from A. C, representative fluorescence microscopy images of muscle cross-sections.

Mentions: To determine the effects of MEKK4 activity on skeletal muscle fiber size, we transfected mouse TA muscle fibers with plasmids encoding either MEKK4ΔN or MEKK4ΔN-T1483A. As additional controls, we transfected muscle fibers with empty plasmid or plasmid encoding full-length MEKK4 (an inactive kinase in the absence of Gadd45a). We found that MEKK4ΔN induced a dramatic loss of muscle fiber size (Fig. 7, A–C). In contrast, full-length MEKK4 and MEKK4ΔN-T1483A did not significantly alter muscle fiber size (Fig. 7, A–C). These data indicate that MEKK4 activity is sufficient to induce skeletal muscle fiber atrophy and provide further evidence that Gadd45a causes skeletal muscle atrophy at least in part by activating MEKK4.


Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4 * ♦
MEKK4ΔN induces skeletal muscle fiber atrophy in a Gadd45a-independent manner.A–C, mouse TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4-FLAG plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, and/or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. All muscles were co-transfected with 2.5 μg of eGFP plasmid. TA muscles were harvested for histological analysis 7 days post-transfection. A, average diameters of skeletal muscle fibers. Each data point represents the mean of >400 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p values were determined with a one-way ANOVA and Dunnett's multiple comparison test. N.S., not significant or p > 0.05. B, size distribution of all muscle fibers from A. C, representative fluorescence microscopy images of muscle cross-sections.
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Related In: Results  -  Collection

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Figure 7: MEKK4ΔN induces skeletal muscle fiber atrophy in a Gadd45a-independent manner.A–C, mouse TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4-FLAG plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, and/or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. All muscles were co-transfected with 2.5 μg of eGFP plasmid. TA muscles were harvested for histological analysis 7 days post-transfection. A, average diameters of skeletal muscle fibers. Each data point represents the mean of >400 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p values were determined with a one-way ANOVA and Dunnett's multiple comparison test. N.S., not significant or p > 0.05. B, size distribution of all muscle fibers from A. C, representative fluorescence microscopy images of muscle cross-sections.
Mentions: To determine the effects of MEKK4 activity on skeletal muscle fiber size, we transfected mouse TA muscle fibers with plasmids encoding either MEKK4ΔN or MEKK4ΔN-T1483A. As additional controls, we transfected muscle fibers with empty plasmid or plasmid encoding full-length MEKK4 (an inactive kinase in the absence of Gadd45a). We found that MEKK4ΔN induced a dramatic loss of muscle fiber size (Fig. 7, A–C). In contrast, full-length MEKK4 and MEKK4ΔN-T1483A did not significantly alter muscle fiber size (Fig. 7, A–C). These data indicate that MEKK4 activity is sufficient to induce skeletal muscle fiber atrophy and provide further evidence that Gadd45a causes skeletal muscle atrophy at least in part by activating MEKK4.

View Article: PubMed Central - PubMed

ABSTRACT

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

No MeSH data available.