New roles for the LKB1→AMPK pathway
Introduction
Genes encoding the subunits of the AMP-activated protein kinase (AMPK) complex can be recognized in all eukaryotes for which genome sequences have been completed, including fungi, plants, and animals ranging from the primitive protozoon Giardia lamblia to humans [1]. Genetic studies show that in the yeast Saccharomyces cerevisiae the AMPK homologue (the SNF1 complex) is required for the response to glucose starvation [2], while in the moss Physcomitrella patens it is required to allow the plant to survive periods of darkness, the equivalent of carbon-source starvation in a photosynthetic organism [3••]. In mammalian cells AMPK is activated by glucose deprivation, as in yeast, but it is also switched on by other stresses that cause depletion of ATP either by inhibiting its synthesis (e.g. hypoxia), or by accelerating its consumption (e.g. contraction in muscle). Once activated, the system switches on catabolic pathways that generate ATP, such as the uptake and oxidation by cells of glucose and fatty acids, while switching off ATP-requiring processes that are not essential to the short-term survival of the cell, including most biosynthetic pathways. In this short review I will focus on selected papers published within the last two years, and readers should refer to earlier reviews for more comprehensive bibliographies [1, 2, 4, 5, 6].
AMPK exists as heterotrimeric complexes comprised of the catalytic α and regulatory β and γ subunits, with isoforms of all three subunits encoded by distinct genes (α1, α2; β1, β2; γ1, γ2, γ3). These give rise to at least 12 possible heterotrimeric combinations, with alternative splicing and/or alternative transcription start sites adding to the diversity. AMPK complexes are activated by phosphorylation at a specific threonine residue (Thr-172) on the α subunit, catalyzed by an upstream kinase that has recently been identified as LKB1 (see below). This is triggered by cellular stresses that cause a fall in the ATP:ADP ratio, which is amplified by adenylate kinase into a much larger rise in the AMP:ATP ratio [6]. Binding of AMP to the γ subunit causes increased phosphorylation by the upstream kinase and decreased dephosphorylation by protein phosphatases, while also causing further allosteric activation of the phosphorylated kinase. This triple mechanism (Figure 1) ensures an extremely sensitive response to a small rise in AMP over the appropriate concentration range. The three activating effects of AMP are also antagonized by high concentrations of ATP, so that the system responds not just to an increase in AMP but to an increase in the cellular AMP:ATP ratio.
Section snippets
Functions of the β subunit – the glycogen-binding domain
The β subunit in all eukaryotes contains a conserved C-terminal domain, and a stable, active complex can be formed using a truncated β subunit that only contains this domain [7••]. There is a second conserved region that is now recognized to be a glycogen-binding domain (GBD). The Protein Families database (www.sanger.ac.uk/pfam) classifies it as an N-isoamylase domain, which is a non-catalytic domain also found in enzymes that synthesize or degrade the α1 → 6 branches in α1 → 4-linked glucans,
Functions of the γ subunit – the AMP-binding Bateman domains
The γ subunits from all species contain four tandem repeats of a sequence termed a CBS motif, first recognized by Bateman [10]. CBS motifs always occur in tandem pairs that together form a protein module that Kemp [11] has termed a ‘Bateman domain’. The γ subunits of AMPK contain two tandem Bateman domains, and each of these binds one molecule of AMP [12••]. Constructs containing both domains bind two molecules of AMP with strong positive co-operativity, suggesting that the second AMP-binding
Identification of LKB1 as the upstream kinase
Although the upstream kinase had been partially purified from rat liver and shown to phosphorylate the Thr-172 site, attempts to identify it had failed, leading several groups to focus on the S. cerevisiae system, where the α and γ subunit orthologues are called Snf1 and Snf4. Three kinases that act upstream were identified simultaneously by different groups. Pak1 [19] and Tos3 [20] were found to interact with Snf1 and Snf4, respectively, in genome-wide protein interaction screens, while Elm1
Regulation of the TOR pathway and cell growth by AMPK
The TOR (target of rapamycin) pathway is activated by amino acids and by growth factors like insulin-like growth factor 1 (IGF1), and stimulates protein synthesis and cell growth via multiple mechanisms including phosphorylation of the protein kinase S6K1 and the translation factor 4E-BP1. Genetic evidence in Drosophila suggests that this pathway plays a critical role in determining cell size (e.g. [34, 35]; see article ‘The expanding TOR signalling network’ by Martin and Hall in this issue).
Regulation of AMPK by adipokines
AMPK was originally perceived as a regulator of cellular energy balance that acted in a cell-autonomous matter. However, that view has recently been modified by findings that this protein kinase is regulated by hormones, particularly the cytokines secreted by fat cells known as ‘adipokines’ that regulate energy balance at the whole body level. The first adipokine to be defined was leptin, which can be regarded as a signal that body fat stores are adequate (Figure 4). The main action of leptin
Conclusions
While the AMPK system was traditionally viewed as a regulator of metabolism, the recent discoveries that the tumour suppressors LKB1 and TSC2 lie upstream and downstream, respectively, indicate that it may be equally important in the regulation of cell growth, proliferation and apoptosis, areas that are ripe for future development. The recent discoveries that it is regulated by adipokines also show that it has acquired new roles during the evolution of multicellular animals, and another key
Update
Bettencourt-Dias et al [53] recently screened for cell cycle defects caused by double-stranded RNAs targeted at protein kinases in S2 cells of Drosophila melanogaster. They found that down-regulation of either the α (SNF1A) or γ (SNF4γ) subunit of Drosophila AMPK caused increases in the proportion of cells in both S and G2/M phases, indicating that AMPK is involved at multiple steps in the cell cycle. Down-regulation of SNF1A and the Drosophila homologues of LKB1 and the AMPK-related protein
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Studies in this laboratory were supported by the Wellcome Trust.
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