Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits


Research Report

J RNAi Gene Silencing (2012), 8, 470-478

doi: jrgsxx

Published online: 04 October 2012

Full Text: (pdf ~1121kb)

Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits

Larissa Tangeman, Christopher N Wyatt* and Thomas L Brown*

Program in Microbiology and Immunology and Department of Neuroscience, Cell Biology and Physiology, Wright State University Boonshoft School of Medicine, Dayton, Ohio 45435, USA

*Correspondence toThomas Brown, Email: thomas.L.brown@wright.edu, Tel: +937 7753809, Fax: +937 7753391, or Christopher Wyatt, Email: christopher.wyatt@wright.edu

Received: 08 May 2012, Revised: 25 September 2012, Accepted: 03 October 2012

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ABSTRACT

AMP-activated protein kinase (AMPK) is a master metabolic regulator that responds to the AMP: ATP ratio and promotes ATP production when the cell is low on energy. There are two isoforms of the catalytic alpha subunit, AMPKa1 and AMPKa2. Here, we describe the production of a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) targeting both catalytic isoforms of AMPK in human, mouse, and rat. Multiple loop sequences were tested to generate the most effective shRNA. The shRNA causes significant knockdown of both isoforms of AMPKa in mouse and human cells. The shRNA effectively knocked down AMPKa1 and AMPKa2 protein levels, compared to a five basepair mismatch-control shRNA in mouse fibroblast NIH3T3 cells and significantly knocked down AMPKa1 (63%) and AMPKa2 (72%) levels compared to control in human embryonic kidney cells, HEK293s. The shRNA also causes a significant reduction in AMPK activity, measured as phosphorylation of acetyl-CoA carboxylase (ACC), a direct phosphorylation target. While the protein levels of total ACC remained the same between the AMPKa1&2 shRNA and control shRNA-treated cells, there was a 41% reduction in phospho-ACC protein levels. The generation of this AMPKa1&2 shRNA can be used to stably knock down protein levels and activity of both catalytic isoforms of AMPK in different species to assess function.

Keywords: AMPK, PRKAA, AMPKa1, AMPKa2, shRNA, siRNA

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