Schilling - Chris Adams_Gadd45a

Schilling - Chris Adams_Gadd45a
Gadd45a Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4

  • Organism: Mouse
  • Instrument: TripleTOF 5600
  • SpikeIn: No
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, the MAP kinase kinase kinase MEKK4, which 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.
Experiment Description
To develop a Gadd45a construct suitable for tandem affinity purification (TAP), we placed two affinity tags (FLAG and S-tag) at the NH2-terminus of Gadd45a, generating a protein that we termed Gadd45a TAP. Gadd45a TAP served as a functional Gadd45a construct designed for tandem affinity purification in mouse skeletal muscle. Mouse tibialis anterior (TA) muscle fibers were transfected with 20 ug Gadd45a TAP plasmid plus 2.5 ug eGFP plasmid. In each mouse, the contralateral TA muscle fibers ("Control") were transfected with 20 ug empty TAP plasmid plus 2.5 ug eGFP plasmid. Bilateral TAs were harvested for analysis 7 days posttransfection.
Sample Description
Isolation and analysis of proteins that interact with Gadd45a TAP in mouse skeletal muscle fibers. TA skeletal muscle fibers of 48 mice were transfected with 20 μg empty TAP plasmid (one TA per mouse) or 20 μg Gadd45a TAP plasmid (the contralateral TA in each mouse). Ten days post-transfection, bilateral TA muscles were harvested and used to prepare pooled protein extracts from each of the two groups of skeletal muscles (control and Gadd45a). The pooled protein extracts were then subjected to sequential purification steps with anti-FLAG magnetic beads and S-protein affinity gel. A small aliquot of each final pulldown sample was visualized by SDS-PAGE and silver staining, as shown. The remaining portions of control and Gadd45a pulldown samples were subjected to mass spectrometry.
Created on 7/13/16, 4:38 PM
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Gadd45a_mouse_Suppl Spectral Viewer_v1_2016-05-22_15-58-51.sky.zip2016-07-131180982940