Regulation by phosphorylation/dephosphorylation

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In addition to allosteric activation, AMPK is regulated by reversible phosphorylation (Figure 2). The key step in AMPK activation is its phosphorylation on threonine residue 172 (Thr-172), within the catalytic domain, by upstream kinases. The combination of the allosteric and phosphorylation effects causes >1000-fold increase in kinase activity (compared to up-to fivefold for allosteric activation alone), allowing high sensitivity in responses to small changes in cellular energy status (Suter et al., 2006). Three AMPK upstream kinases (AMPKKs) have been identified to date. The primary AMPKK is a complex between the tumor suppressor, LKB1, and two accessory subunits, STRAD and MO25 (Hawley et al., 2003; Woods et al., 2003a). LKB1 also functions upstream of 12 other kinases (AMPK-related kinases) situated on the same family as AMPK by phylogenetic analysis of kinase domain sequences (Lizcano et al., 2004). The LKB1 protein kinase activity appears to be constitutively active and is not regulated by AMP (Lizcano et al., 2004; Sakamoto et al., 2004). This view was recently challenged with recent studies showing that the subcellular localization of LKB1 and consequently, its activity may be modifiable. It has been suggested that SIRT1, one of the seven mammalian NAD(+)-dependent deacetylases silent mating type information regulator 2 ortholog (sirtuin) genes (Howitz et al., 2003), promotes LKB1-dependent AMPK stimulation through direct deacetylation and increased cytoplasmic/nuclear ratio of LKB1 (Lan et al., 2008). Also, recently it was shown that Fyn kinase phosphorylation of LKB1 on Tyr265 and Tyr365 residues results in cytoplasmic distribution of  LKB1 and increased AMPK phosphorylation (Yamada et al., 2010). Binding of AMP to AMPK promotes LKB1-dependent phosphorylation of Thr-172 through inhibition of dephosphorylation (by making AMPK complex a less efficient substrate for protein phosphatases) and produces a large effect on kinase activity by allosterically activating the phosphorylated form of AMPK. In addition, Ca2+/calmodulin-dependent protein kinase kinase b (CaMKKb) has also been identified as a separate AMPK kinase (Hawley et al., 2005; Hurley et al., 2005; Woods et al., 2005), that phosphorylates and activates AMPK in response to elevated intracellular Ca2+ concentrations, independent of any change in cellular AMP/ATP ratio. TGF-b-activated kinase 1 (TAK1) has also been recently implicated in the regulation of AMPK activity, although the physiological conditions during which TAK1 regulates AMPK are unclear (Momcilovic et al., 2006; Xie et al., 2006).

While a-Thr-172 is the major AMPK phosphorylation and activation site, a and b subunits are phosphorylated at multiple sites (Mitchelhill et al., 1997; Villen et al., 2007; Woods et al., 2003b), however, the physiological relevance of these sites remain unclear. Recent studies provide evidence that direct phosphorylation of AMPKa1/a2 at Ser485/491 antagonizes its activation and  correlates with inhibition of AMPK activity during insulin signaling in the heart (Horman et al., 2006) and cAMP-mediated signaling in an insulin-secreting cell line (Hurley et al., 2006). A hierarchical control by insulin was proposed for the reduction of AMPK activation in ischemic heart via PKB-induced phosphorylation of Ser485/491 (see below). The inhibitory effect of cAMP was linked to reduction in phosphorylation of AMPK at Thr172 and appears to be due, in part, to cAMP-dependent inhibition of the upstream AMPK kinase CaMKK, but not LKB1 (Hurley et al., 2006). On the other hand, cAMP-dependent attenuation of AMPK activity has also been correlated with increased phosphorylation of AMPKa1 Ser485/491 by PKA (Hurley et al., 2006). This is in contrast to studies in adipocytes, showing that agents stimulating PKA-mediated cAMP signaling (isoproterenol, isobutylmethylxanthine, forskolin, b-adrenergic agonist and adrenaline) result in increased AMPK activity (Daval et al., 2005; Koh et al., 2007; Moule and Denton, 1998; Omar et al., 2009; Yin et al., 2003). Since, cAMP-stimulated lipolysis in adipocytes was accompanied by an increase in oxidative stress (i.e., an increase AMP:ATP ratio), AMPK activation could be a consequence of lipolysis and the associated relative change in cellular energy balance rather than a direct effect of PKA (Gauthier et al., 2008). Similarly, it has been shown that IL-6 activates AMPK in skeletal muscle by increasing the concentration of cAMP and, secondarily, the AMP:ATP ratio (Kelly et al., 2009). However, potential crosstalk between PKA and AMPK signaling pathways underlying negative action of PKA on AMPK signaling has been recently reported in context of adipocytes. Central to this mechanism is the phosphorylation of AMPKa1 by PKA at Ser173 (Djouder et al., 2010). This site is highly conserved, located directly adjacent to the critical activation loop Thr172 and its phosphorylation may create constraints by steric hindrance or charge incompatible with subsequent phosphorylation at the Thr172 residue. This mechanism is critically important for the control of the lipolytic response. Stimulation of adipocyte lipolysis, via PKA activation, triggers a negative feedback mechanism involving AMPK to restrain the energy depletion and oxidative stress caused by lipolysis (Gauthier et al., 2008). By opposing the activity of AMPK-mediated negative feedback loop, PKA allows fine-tuning of lipolysis (Djouder et al., 2010).

Protein phosphatases have an important role in regulating AMPK phosphorylation at Thr-172  and consequently AMPK activity, although the exact mechanisms that modulate their action remains poorly understood. Their ability to dephosphorylate Thr172 on AMPK is inhibited by AMP binding to the g subunit (Davies et al., 1995; Sanders et al., 2007). Both protein phosphatases 2A (PP2A) and 2C were shown to dephosphorylate AMPK in vitro (Davies et al., 1995; Kudo et al., 1996). Recent findings revealed the important role of PPs activation in the suppression of AMPK activity by dephosphorylation in different species, organs, and nutrition types (Ravnskjaer et al., 2006; Wang and Unger, 2005; Wu et al., 2007). In support with these results, it has been reported that PP2A is involved in regulating the interaction between AMPK a2 and g1 (Gimeno-Alcaniz and Sanz, 2003).