AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform

Abstract

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric alphabetagamma complexes, where the catalytic subunit has two isoforms (alpha1 and alpha2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the alpha2 rather than the alpha1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein-alpha-subunit fusions. By all three methods a significant proportion of alpha2, but not alpha1, is localized in the nucleus. Like SNF1, AMPK-alpha2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of alpha1 and alpha2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

Functional Analysis of Mutations in the γ2 Subunit of AMP-activated Protein Kinase Associated with Cardiac Hypertrophy and Wolff-Parkinson-White Syndrome

“…The concentrations of AMP required for half-maximal stimulation of the ␥ 2 complexes are significantly higher than those previously reported for ␥ 1 -containing AMPK complexes. Immunoprecipitation of ␣ 1 and ␣ 2 complexes from rat liver, in which ␥1 accounts for over 90% of the total AMPK activity (9), yielded A 0.5 values for AMP of 12 Ϯ 3 and 22 Ϯ 3 M, respectively (30). We reported similar values for ␣1 (5.7 Ϯ 2 M) and ␣ 2 (16 Ϯ 3.5 M) complexes following co-expression with ␤ 1 ␥ 1 in mammalian cells (20).…”

Kinase activity-independent suppression of p73α by AMP-activated kinase α (AMPKα)

“…As reported by others (Salt et al, 1998;Turnley et al, 1999;da Silva Xavier et al, 2000), endogenous AMPKa1 was mainly located in the cytoplasm, whereas AMPKa2 were detected in the nucleus. When cells expressing GFP-p73a were stained with anti-AMPKa1 antibody and Texas Red-conjugated anti-rabbit IgG, AMPKa1 was found in the nucleus with p73a ( Figure 3a).…”

“…Direct regulation of gene transcription by AMPK is likely achieved through the phosphorylation of transcription factors (PPARa, PPARg, HNF4a, ChREBP, HIF-1 and p53), cofactors (p300, PGC-1 and TRIP6) and the basal transcriptional machinery (Pol I) in the nucleus (Lee et al, 2003;Leff, 2003;Bronner et al, 2004;Jones et al, 2005;Solaz-Fuster et al, 2006). Of the catalytic a subunits found in AMPK, a1 is located mainly in the cytoplasm, whereas a2 is detected preferentially in the nucleus (Salt et al, 1998;Turnley et al, 1999;da Silva Xavier et al, 2000), further indicating that the a subunit may modulate AMPK-regulated gene transcription. The suggested role of AMPK in transcriptional regulation also comes from the observation that Snf1, a yeast homolog of AMPK, acts as a histone kinase in cooperation with the histone acetyltransferase Gcn5 (Lo et al, 2001).…”

The Effects of Adiponectin and Metformin on Prostate and Colon Neoplasia Involve Activation of AMP-Activated Protein Kinase

“…complex consisting of a catalytic subunit and two regulatory subunits encoded by three genes, each of which has at least two isoforms (1). AMPK is a key regulator of the cellular response to energy stress (2,3).…”