In liver tissue, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of LKB1, MO25α/β, and STRADα/β proteins; however, this complex has yet to be characterized in skeletal muscle. In this report, we demonstrate the expression of the LKB1-MO25-STRAD AMPKK complex in adult skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these complex-forming proteins. To facilitate this characterization we used control (ctrl) and muscle-specific LKB1 knockout (LKB1-/-) mice. LKB1 detection in untreated ctrl and LKB1-/- muscle lysates revealed two protein bands at approximately 50 and 60 kDa; although, only the heavier band was significantly diminished in LKB1-/- samples (ctrl: 55±2.5 AU; LKB1-/-: 13±1.5 AU; p<0.01), suggesting that LKB1 is not represented at 50 kDa as cited previously. Detection of LKB1 at the higher molecular weight was further confirmed following purification of the AMPKK complex using polyethylene glycol (PEG) (ctrl: 43±5 AU; LKB1-/-: 8.4±4 AU; p<0.01). Following ion-exchange-fast protein liquid chromatography (FPLC) the low protein band was undetectable in ctrl and LKB1-/- fractions. Mass spectrometry of PEG-treated ctrl lysates confirmed LKB1 protein detection in the 60 kDa protein band while none was detected in the 50 kDa band. Co-immunoprecipitation assays demonstrated associations between all combinations of LKB1, MO25, and STRAD in LKB1-positive samples, confirming proper complex formation. Quantitative-PCR revealed significantly reduced expression of MO25α and STRADβ in LKB1-/- muscle. Lastly, detection of CaMKKα/β protein in ctrl and LKB1-/- muscle lysates confirmed the presence of another AMPKK in muscle. Interestingly, CaMKKβ protein is increased in LKB1-/- muscle (ctrl: 19±4.3 AU; LKB1-/-: 47±9.2 AU; p<0.05) without an increase in mRNA levels, suggesting compensation for null LKB1 expression. In all, these findings confirm the presence of the LKB1-MO25-STRAD complex in adult skeletal muscle, suggest a novel post-translational modification of LKB1, and identify a potential compensatory mechanism for loss of LKB1 protein in skeletal muscle.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-3802 |
| Date | 18 November 2010 |
| Creators | Smith, Cody Don |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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