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Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase 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.


MEKK4ΔN, a constitutively active Gadd45a-independent MEKK4 construct.A, schematic of the MEKK4ΔN and MEKK4ΔN-T1483A constructs. B, TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, TA muscles were harvested for immunoblot analysis using monoclonal anti-FLAG IgG. C, one TA per mouse was transfected with 5 μg of empty pcDNA plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies. D, one TA per mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies.
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Figure 6: MEKK4ΔN, a constitutively active Gadd45a-independent MEKK4 construct.A, schematic of the MEKK4ΔN and MEKK4ΔN-T1483A constructs. B, TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, TA muscles were harvested for immunoblot analysis using monoclonal anti-FLAG IgG. C, one TA per mouse was transfected with 5 μg of empty pcDNA plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies. D, one TA per mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies.

Mentions: To investigate whether MEKK4 activity might be sufficient to induce skeletal muscle atrophy, we generated a constitutively active Gadd45a-independent MEKK4 construct that retains the C-terminal kinase domain but lacks the N-terminal autoinhibitory and Gadd45a binding domains (26, 29). This construct, termed MEKK4ΔN, is illustrated in Fig. 6, A and B. As a control for potential kinase-independent effects of MEKK4ΔN, we also generated an inactive MEKK4ΔN construct in which the autophosphorylation site in the kinase activation loop, threonine 1483, is mutated to alanine (MEKK4ΔN-T1483A; Fig. 6, A and B). When transfected into mouse skeletal muscle in vivo, the MEKK4ΔN plasmid strongly induced MKK3/6, MKK4, and p38 phosphorylation in a Gadd45a-independent manner (Fig. 6C), and these effects were abolished by the T1483A mutation (Fig. 6D). Thus, MEKK4ΔN is constitutively active in mouse skeletal muscle fibers, whereas MEKK4ΔN-T1483A is inactive.


Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4 * ♦
MEKK4ΔN, a constitutively active Gadd45a-independent MEKK4 construct.A, schematic of the MEKK4ΔN and MEKK4ΔN-T1483A constructs. B, TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, TA muscles were harvested for immunoblot analysis using monoclonal anti-FLAG IgG. C, one TA per mouse was transfected with 5 μg of empty pcDNA plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies. D, one TA per mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies.
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Figure 6: MEKK4ΔN, a constitutively active Gadd45a-independent MEKK4 construct.A, schematic of the MEKK4ΔN and MEKK4ΔN-T1483A constructs. B, TA muscles were transfected with 5 μg of empty pcDNA plasmid, 5 μg of MEKK4ΔN-FLAG plasmid, or 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, TA muscles were harvested for immunoblot analysis using monoclonal anti-FLAG IgG. C, one TA per mouse was transfected with 5 μg of empty pcDNA plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies. D, one TA per mouse was transfected with 5 μg of MEKK4ΔN-FLAG plasmid, and the contralateral TA in each mouse was transfected with 5 μg of MEKK4ΔN-T1483A-FLAG plasmid, as indicated. Three days post-transfection, bilateral TA muscles were harvested for immunoblot analysis using the indicated antibodies.
Mentions: To investigate whether MEKK4 activity might be sufficient to induce skeletal muscle atrophy, we generated a constitutively active Gadd45a-independent MEKK4 construct that retains the C-terminal kinase domain but lacks the N-terminal autoinhibitory and Gadd45a binding domains (26, 29). This construct, termed MEKK4ΔN, is illustrated in Fig. 6, A and B. As a control for potential kinase-independent effects of MEKK4ΔN, we also generated an inactive MEKK4ΔN construct in which the autophosphorylation site in the kinase activation loop, threonine 1483, is mutated to alanine (MEKK4ΔN-T1483A; Fig. 6, A and B). When transfected into mouse skeletal muscle in vivo, the MEKK4ΔN plasmid strongly induced MKK3/6, MKK4, and p38 phosphorylation in a Gadd45a-independent manner (Fig. 6C), and these effects were abolished by the T1483A mutation (Fig. 6D). Thus, MEKK4ΔN is constitutively active in mouse skeletal muscle fibers, whereas MEKK4ΔN-T1483A is inactive.

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.