Product Pathways - Kinases
AMPKα2β1γ1 Kinase #7471
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Description
Purified recombinant full-length human AMPK kinase (combination of α2/β1/γ1 subunits), supplied as a His tag fusion protein.
Source / Purification
The His-Kinase fusion protein complex was produced using a baculovirus expression system with a construct expressing full-length human AMPKα2 (Met1-Arg552) (GenBank Accession No. NM_006252), AMPKβ1 (Met1-Ile270) (GenBank Accession No. NM_006253), and AMPKγ1 (Met1-Pro331) (GenBank Accession No. NM_002733), all with carboxy-terminal His tags. The protein was purified by Immobilized Metal Affinity Chromatography (IMAC).
Gel Staining
Figure 1. The purity of the AMPKα2β1γ1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. AMPKα2β1γ1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, 0.1 mM AMP, Substrate: SAMS peptide, 200 ng/μl, and variable amounts of recombinant AMPKα2β1γ1.
Quality Control
The theoretical molecular weight of the 3 subunits of AMPK fusion protein is (α2/β1/γ1) 69, 38, and 40 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. AMPK α2β1γ1 kinase activity was determined using a radiometric assay [Fig.2].
Background
AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop and this phosphorylation is required for AMPK activation (3-5). AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinase and the biological significance of these phosphorylation events have yet to be elucidated (6). The β1 subunit is post-translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101, Ser108 and Ser182 (6,7). Phosphorylation at Ser108 of the β1 subunit seems to be required for the activation of AMPK enzyme, while phosphorylation at Ser24/25 and Ser182 affects AMPK localization (7). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (1,2). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1).
- Hardie, D.G. (2004) J. Cell Sci. 117, 5479-5487.
- Carling, D. (2004) Trends Biochem. Sci. 29, 18-24.
- Hawley, S.A. et al. (1996) J. Biol. Chem. 271, 27879-27887.
- Lizcano, J.M. et al. (2004) EMBO J. 23, 833-843.
- Shaw, R. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 3329-3335.
- Woods, A. et al. (2003) J. Biol. Chem. 278, 28434-28442.
- Warden, S.M. et al. (2001) Biochem. J. 354, 275-283.
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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
