Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

1The yeast SNF1 (sucrose non-fermenting 1), mammalian AMPK (AMP-activated protein kinase), and plant SnRK1 (SNF1-related protein kinase 1) are metabolic sensors belonging to a highly conserved eukaryotic protein kinase family. In yeast, SNF1 plays a fundamental role in the shift from fermentative to oxidative metabolism in response to glucose deprivation, partly by releasing the repression of genes essential for the utilization of alternative carbon sources. AMPK, the mammalian counterpart, is activated by a raise in the “adenylate charge,” i.e., a raise in AMP and ADP levels relative to ATP upon glucose starvation caused by fasting, exercise, or stresses like heat shock and hypoxia. Once activated, AMPK implements an energy-saving program through direct enzyme regulation and transcriptional control. Anabolic processes such as the synthesis of fatty acids, cholesterol, and proteins are switched off, while catabolic pathways such as fatty acid oxidation, glycolysis, and autophagy are activated. AMPK plays also a broader role in regulating whole-body energy metabolism and glucose homeostasis through the regulation of processes like muscle glucose uptake, insulin production and secretion, management of body lipids, and appetite.

2The first piece of evidence on the functional conservation of the plant ortholog and the regulation of energy metabolism by SnRK1 came from the complementation of the yeast snf1Δ mutant with a rye Snf1-related cDNA. Complementation reestablished the utilization of non-fermentable carbon sources such as ethanol and glycerol, indicating that rye Snf1-related cDNA could substitute Snf1 in the sugar signaling pathway. Similar results were obtained in yeast complementation assays using SnRK1 from other plant species, such as tobacco, potato, and Arabidopsis.

3The large Arabidopsis SnRK super family is composed of three distinct subfamilies, SnRK1, SnRK2, and SnRK3. The SnRK2 and SnRK3 subfamilies include 35 more divergent protein kinases specific to plants and mostly known for their involvement in stress and abscisic acid (ABA) signaling. The SnRK1 subfamily comprises SnRK1α1/SnRK1α2/SnRK1α3 (also named SnRK1.1/SnRK1.2/SnRK1.3, AKINα1/AKINα2/AKINα3, KIN10/KIN11/KIN12, or AKIN10/AKIN11/AKIN12), the catalytic subunits of the SnRK1 complex and the closest relatives of Snf1 and AMPKα. Of these, only SnRK1α1/SnRK1α2 appear to be expressed. The gene family in cereals comprises two subgroups, of which SnRK1a is more closely related to the homolog present in dicots and SnRK1b is cereal-specific and mostly expressed in the seed.

4SnRK1 regulates metabolism and transcription in response to energy deprivation and ABA signals, and is inactivated by sugars that restore an energy balance. Mounting evidence indicates that SnRK1 plays a crucial role in the acclimation of plants to a wide range of biotic and abiotic stresses.

5Besides their role in metabolism and stress responses these kinases regulate virtually all aspects of cell function as well as multiple developmental processes. Consistent with the established role of SNF1 and AMPK in the control of cell growth and proliferation, SnRK1 was recently shown to regulate cell cycle progression. In addition, AMPK regulates cell polarity and apoptosis and SNF1 regulates yeast-specific processes like mating and sporulation. In plants, SnRK1 was shown to be crucial for seed filling and maturation, and to affect embryo development and cotyledon growth, as well as pollen development, lateral organ development and phase transition.

6Several aspects of the SnRK1 kinases are highly conserved, such as their core function as regulators of metabolism. Furthermore, plants also possess β and γ regulatory subunits that, together with the α catalytic subunits most probably form heterotrimeric complexes similar to the ones crystallized in other organisms. On the other hand, the SnRK family has largely diverged and expanded, and plants have also evolved unique regulatory subunits, presumably to perform plant-specific functions (;; Figure 1). In addition, although all Snf1/AMPKα/SnRK1α members require phosphorylation at a conserved activation loop threonine for their activity (Figure 1;;; ), the clear connection between such phosphorylation and differential kinase activity described for mammals and yeast is not well established in plants, suggesting additional regulatory mechanisms. Our aim is to provide a comprehensive review on the post-translational mechanisms that regulate SNF1/AMPK/SnRK1 kinases, some conserved across all eukaryotes and some specific for a particular member (Figure 2). These mechanisms most likely play a role in the swift regulation of kinase activity in response to stress. Less direct modes of regulation such as transcriptional control or alternative splicing are probably more important for SNF1/AMPK/SnRK1 complex composition in different tissues and developmental stages, and are beyond the scope of this review. Likewise, the detailed function of these kinases as well as the downstream mechanisms by which they regulate gene expression and protein function will not be covered here, as they have been extensively reviewed elsewhere.

7The SNF1/AMPK/SnRK1 protein kinases are conserved throughout all eukaryotes and share an αβγ heterotrimeric structure (Figure 1;;; ). The catalytic α-subunit is composed of two parts, the kinase domain and the regulatory domain. The kinase domain displays a canonical fold with 11 sub-domains and contains the activation loop (also called T-loop). The regulatory domain in yeast and mammals contains an auto-inhibitory sequence (AIS) which was shown to inhibit kinase activity. In plants, this region appears not to be inhibitory and harbors an ubiquitin-associated (UBA) domain that was proposed to mediate the interaction with ubiquitinated proteins. In addition, these kinases possess a kinase-associated 1 (KA1) domain responsible for the interaction with the regulatory subunits and the upstream phosphatases (Figure 1A;;;; ).

8The function, in yeast and mammals, of the γ regulatory subunit is to control the activity of the α-catalytic subunit. This occurs through binding of adenylates to the cystathionine-β-synthase (CBS) domains (; Figures 1A,B).

9The β-subunit acts as a scaffold keeping the α and the γ subunits together. This seems to be its primordial function, as the plant-specific β3 subunit, which contains only the scaffold part (the association with SNF1 complex, ASC domain; Figure 1), is able to complement the gal83Δsip1Δsip2Δ yeast mutant, devoid of all three β-subunits. Nevertheless, these subunits contain also a carbohydrate binding motif (CBM, previously called glycogen binding domain, GBD) that, in AMPK and SNF1 Gal83 and Sip2, binds glycogen in vitro. The β-subunits also contain an N-terminal extension that is believed to facilitate association with downstream targets and determines trimer localization in yeast.

10Even though SNF1/AMPK/SnRK1 subunits are globally conserved throughout eukaryotes, two atypical subunits exist in plants: the β3 subunit mentioned above and the βγ subunit (Figure 1A), a true γ-type subunit with an N-terminal extension containing a CBM. In addition to SnRK1βγ, plants have a large family of γ-like subunits, including SnRK1γ. However, in leaf mesophyll cells transiently overexpressing various subunit combinations, only SnRK1βγ interacts with β-subunits and assembles into a heterotrimeric complex. This observation, coupled to the fact that only SnRK1βγ complements snf4Δ (the SNF1 γ-subunit), suggests that SnRK1γ might not play a “canonical” γ-function.

11As illustrated in Figure 1, several heterotrimer compositions are possible in vivo in all eukaryotes (up to 12 in Arabidopsis). This is probably the first layer of regulation of these kinases, as for instance, different β-subunits within the SNF1 complex trigger differential localization.

12Phosphorylation of a conserved threonine in the T-loop of the catalytic subunit (SnRK1α1T175/SnRK1α2T176; AMPKα2T172; Snf1T210; Figure 1A) is essential for SNF1/AMPK/SnRK1 activity. Many protein kinases share this mode of regulation, which is presumably required for the proper alignment of ATP in order to allow its interaction with the catalytic lysine. When grown in high glucose concentrations Snf1T210 is predominantly in the dephosphorylated state and the SNF1 complex is inactive. Upon shifting the yeast cells to a medium depleted of glucose, Snf1T210 becomes strongly phosphorylated, rendering the complex active. Similarly to SNF1, the AMPK complex in mammals is activated by metabolic stresses that compromise ATP production or increase ATP consumption, and displays appreciable activity only when phosphorylated on the conserved activation loop threonine residue. In plants, analyses of total cell extracts reveal no differences in the phosphorylation levels of the activation loop between control and stress conditions. This may suggest the involvement of additional phosphoresidues or other mechanisms for controlling activity upon stress. However, recent analyses of SnRK1 phosphorylation following size fractionation revealed higher T-loop phosphorylation when the catalytic subunit was incorporated into a complex. It is hence possible that stress promotes only the phosphorylation of the catalytic subunits that are assembled into a complex, and that analyses of total cellular SnRK1 have missed this fine level of regulation.

13The first report on the existence of an SNF1/AMPK/SnRK1 upstream kinase was in mammals in 1987. However, the three yeast SNF1 activating kinases, Elm1, Tos3, and Sak1, were the first of the family to be identified by combining co-immunoprecipitation (Tos3 and Sak1 with Snf4), phylogenetic analyses (Elm1 clusters with Tos3 and Sak1), and genetics (triple sak1Δelm1Δtos3Δ shared the same phenotype than snf1Δ; ). To some extent SNF1 activating kinases are functionally redundant, since all three need to be knocked out to prevent growth on alternative carbon sources. Nevertheless, they present stress-dependent preferences toward specific β-subunits of the SNF1 complex with a differential impact on the phosphorylation of downstream targets such as the Mig1 transcription factor.

14Sak1 is the primary SNF1 upstream kinase as it is the only one that associates strongly with Snf1 and sak1Δ is more affected in SNF1 activity than the elm1Δ and tos3Δ mutants. Sak1 is the only upstream kinase controlling the SNF1 complexes containing Gal83 as a β-subunit under low glucose. In these conditions Gal83-SNF1 relocalizes from the cytoplasm to the nucleus, whilst Sip1-SNF1 relocalizes to the vacuolar membrane and Sip2-SNF1 remains cytoplasmic. Lack of Tos3 leads to a 20% decrease in SNF1 phosphorylation and activity during glucose starvation having a noticeable effect on growth only when cells are continuously maintained on non-fermentable carbon sources. Finally, Elm1 was already known to phosphorylate and regulate a number of proteins necessary for proper cell morphogenesis and cell-cycle progression before being identified as a SNF1 kinase.

15The tumor suppressor liver kinase B1 (LKB1) was the first AMPK upstream kinase to be identified in mammals based on its sequence similarity with the yeast SNF1 activating kinases. LKB1 associates in a constitutively active complex with the pseudokinase STRAD and the adaptor protein MO25/CAB39. LKB1 is expressed in virtually all human tissues, and it phosphorylates AMPK in response to an elevated AMP/ATP ratio. LKB1 has been implicated in numerous cellular processes including metabolism, cell cycle progression, cell polarity and embryogenesis, mostly due to its phosphorylation of AMPK and AMPK-related kinases.

16In certain cell types, like neurons, AMPKαT172 can also be phosphorylated by the Ca2+/calmodulin-dependent protein kinase kinase CaMKKβ, allowing the input of Ca2+-signals into the AMPK pathway, independently of the adenine nucleotide ratios. Consistent with the lack of regulation by adenylates, the CaMKKβ–AMPK complexes are devoid of AMPKγ and contain only AMPKα and β-subunits.

17Finally, the transforming growth factor-β-activated kinase (TAK1) was shown to complement the yeast elm1Δsak1Δtos3Δ triple mutant suggesting that it might also be an AMPK upstream kinase. Several lines of evidence support the role of TAK1 as an AMPK upstream kinase, in particular under conditions where reactive oxygen species and redox imbalance are generated.

18The similarity between LKB1 and the Arabidopsis proteins GRIK2/1 (geminivirus Rep-interacting kinase 2 and 1), prompted the hypothesis that they may be upstream kinases of SnRK1. Their identification as SnAK1/2 (SnRK1 activating kinase 1 and 2) in Arabidopsis occurred shortly after, through functional complementation of the yeast tos3Δpak1Δelm1Δ triple mutant. This was substantiated by the demonstration that SnAKs can autophosphorylate and subsequently phosphorylate and activate recombinant SnRK1α in vitro. Similarly to LKB1, SnAKs do not require Ca2+ and are insensitive to the CaMKK-specific inhibitor STO-609. They also appear to be constitutively active and insensitive to AMP when assayed in vitro on the recombinant kinase domain of the SnRK1α subunit. This is consistent with the indirect effect of AMP on LKB1 activity mediated by the AMPKγ subunit and AXIN (see regulation by adenylates;;; ). On the other hand, SnAKs autophosphorylate in vitro on AtSnAK1T153/AtSnAK2T154, and this phosphorylation is required for their activity, as the corresponding phospho-mutant (S153A) and phospho-mimetic (S153D) variants are constitutively inactive and active, respectively. In addition, they are phosphorylated and inhibited by SnRK1 on their T-loop (AtSnAK1S260/AtSnAK2S261) as part of a negative feedback loop to tightly control SnRK1 activity. Interestingly, SNF1 phosphorylates Sak1 in vitro, and Tos3 phosphorylation by another kinase was inhibited by SNF1, suggesting that this kinase cross-regulation might be conserved.

19The relevance of SnAKs as SnRK1 upstream kinases remains to be assessed in vivo, as the only evidence for their function in planta is an overlap between SnAK expression and SnRK1 phosphorylation in the shoot apical meristem. SnAKs/GRIKs are only detected in actively proliferating tissue and in geminivirus-infected mature leaves, whilst SnRK1 phosphorylation is readily detected in other tissues such as mature non-infected leaves. It is possible that SnAKs/GRIKs phosphorylate SnRK1 also in mature leaves, where they simply accumulate to undetectably low levels, as suggested by their reported proteasomal degradation. However, this may also indicate the existence of additional upstream kinases. A study on rice reported the interaction of SnRK1 with CIPK15 (calcineurin B-like-interacting protein kinase 15), and several lines of evidence were presented to support a role of CIPK15 as a SnRK1 upstream kinase. Nevertheless, biochemical evidence demonstrating direct SnRK1 phosphorylation and activation by CIPK15 is still required to substantiate this conclusion. As the mammalian CaMKK can phosphorylate purified spinach SnRK1 in vitro, the possibility that endogenous Ca2+-dependent kinases like CIPKs or calcium-dependent protein kinases can serve as SnAKs is still open.

20In addition to the phosphorylation of the conserved T-loop threonine, other phosphorylation events have been described in the AMPK α- and β-subunits (Figure 2;;;; ), although in most cases their functional relevance is still unclear. On AMPKα, phosphorylation of S485 represses T172 phosphorylation and AMPK activity in response to activation of the Akt/PKB kinase by insulin and in response to PKA-mediated cAMP signaling. PKA inhibits AMPKα by phosphorylating a second residue (S173) that also interferes with T172 phosphorylation. S485 does not seem to be conserved in SnRK1α. On the other hand, whilst the AGC protein kinase family, to which PKA and Akt/PKB belong, is also present in plants, there is no proof thus far for the existence of Akt/PKB or a cAMP activated kinase such as PKA in plants.

21All three AMPK subunits were reported to be phosphorylated by the autophagy kinase Atg1/ULK1, leading to AMPK inactivation, although the effect of individual phosphorylation events was not further explored. Given that AMPK controls autophagy by activating Atg1/ULK1 this was proposed to establish a negative feedback loop to reset AMPK after activation of autophagy.

22Interestingly, Adi3 (AvrPto-dependent Pto-interacting protein3), a known suppressor of cell death triggered by pathogens in tomato, was shown to interact with SnRK1α1 and to phosphorylate the Gal83 β-subunit, thereby inhibiting the activity of the SnRK1 complex. It would be interesting to test whether such a phosphorylation is specific to pathogen attack or whether it occurs in response to other environmental, metabolic, or hormonal cues.

23An increasing body of evidence indicates that the dephosphorylation step is crucial for regulating SNF1/AMPK/SnRK1 activity.

24To date, the best characterized protein phosphatases (PPs) of the SNF1/AMPK/SnRK1 family are those of yeast. The dephosphorylation of Snf1 catalytic subunit in response to the glucose signal is partly mediated by the PP1 phosphatase Glc7 that acts in a complex with the Reg1 regulatory subunit. Yeast cells lacking the Reg1 gene show a constitutively phosphorylated and active SNF1, even when glucose is available in the medium. On the other hand the glc7Δ mutation is lethal, partly because of excessive SNF1 activity. In addition to Glc7, recent studies demonstrate that Snf1 is also dephosphorylated by the type 2C phosphatase Ptc1 and the type 2A phosphatase Sit4.

25In low glucose conditions, Glc7-Reg1 is active toward the Mig1 transcription factor, whilst it is largely inactive toward the activation loop of Snf1. This indicates that glucose does not change Glc7-Reg1 activity, but rather controls SNF1 dephosphorylation indirectly by changing the ability of the activation loop to serve as a substrate for the phosphatase. This conclusion is in agreement with the fact that the activatory effect of ADP on SNF1 is due to a conformational change in the complex that renders it resistant to phosphatase action (Figure 2; see regulation by adenylates;; ). However, recent evidence also supports direct regulation at the level of the catalytic subunit and the phosphatase. Regulated activation loop phosphorylation/dephosphorylation was shown to occur also independently of the regulatory subunits and trimer formation. Consistent with this, mutagenesis of the yeast γ-subunit residues predicted to contact bound adenylates had no effect on SNF1 activity, suggesting that in contrast to mammals, adenylate binding to the γ-subunit in yeast is not required for its ability to protect Snf1 from dephosphorylation. The authors propose an alternative model in which phosphatase resistance is provided by ADP binding to the kinase active site, while incorporation of the phosphorylated catalytic subunit into the heterotrimer core is required for kinase activity. Finally, a recent study revealed that glucose exerts a more direct effect on phosphatase action, as it activates PP1 and PP2A post-translationally.

26AMPK has been shown to be dephosphorylated in vitro by PP1, PP2A, and the metal-dependent protein phosphatase PP2C, although PP1 and PP2C dephosphorylate AMPK more efficiently than PP2A. Both PP2C and PP1 phosphatases are able to dephosphorylate AMPK in vivo, suggesting that the type of regulation might depend on the tissue and conditions of cell stimulation. In mouse pancreatic β-cells, knockdown of the PP1α and PP1β catalytic subunits or R6, a regulatory subunit of PP1, caused a reduction in AMPKαT172 dephosphorylation following a low to high glucose switch. The R6 subunit was also reported to physically interact with the AMPKβ subunits in co-immunoprecipitation and yeast two-hybrid experiments. On the other hand, in human embryonic kidney cells, RNAi of Ppm1E, but not Ppm1A (both PP2C members) resulted in increased AMPKαT172 phosphorylation and in assays with lysates of cells stably depleted of Ppm1F Ppm1E, a threefold increase in AMPKαT172 phosphorylation was observed.

27In the case of plants, two PP2C phosphatases, ABI1 and PP2CA were recently reported to interact and dephosphorylate SnRK1α1, in agreement with earlier findings that human PP2C can dephosphorylate and inactivate spinach SnRK1α in vitro. These PP2Cs are well established negative regulators of the ABA pathway through their interaction with SnRK2s, and their repressive action is blocked by the ABA receptors upon ABA binding. Hence, the regulation of SnRK1 by these PP2Cs allows not only the inactivation of SnRK1 in response to sugars, but also its activation in response to ABA.

28Other PPs have also been reported to interact with SnRK1, although the functional relevance of those interactions is unknown. Namely, another PP2C, PP2C74, interacts with SnRK1α2 in vitro and in yeast two-hybrid. In addition, a dual-specificity protein tyrosine phosphatase, PTPKIS1, was reported to interact with SnRK1α2 in vitro and in yeast two-hybrid. This phosphatase was later on shown to harbor a CBM domain that allows binding to starch in vitro and in vivo and was identified as the component responsible for the starch overaccumulation of the sex4 mutant. SEX4/PTPKIS1 is chloroplastic and can bind and dephosphorylate phosphoglucans, suggesting that it regulates the initial steps of starch degradation at the granule surface.

29Although T-loop phosphorylation is generally considered the main mechanism for regulating SNF1/AMPK/SnRK1 activity, several other post-translational modifications have been described, including acetylation, ubiquitination, SUMOylation, and myristoylation and oxidation (Figure 2).

30Sip2, a β-regulatory subunit of SNF1, was identified as a non-chromatin substrate of the nucleosome acetyltransferase of H4 complex (NuA4) in a yeast proteome microarray, and its acetylation was validated by in vitro activity assays and confirmed in vivo. Sip2 acetylation stabilizes its interaction with the Snf1 catalytic subunit thereby inhibiting it. On the other hand, Sip2 acetylation decreases gradually with cell age and Sip2 acetylation mimetics live longer, altogether suggesting that Sip2 acetylation extends lifespan through inhibition of SNF1 activity.

31The AMPKα1 catalytic subunit was shown to be acetylated in vitro by the p300 acetyltransferase, but the in vivo confirmation of this modification awaits further studies. Additionally, a mass spectrometry analysis of AMPK subunits revealed that AMPKγ1 is acetylated on the N-terminus, with no other post-translational modifications detected in this subunit.

32Even though none of the SnRK1 subunits have been reported to be acetylated, studies in mammals and yeast suggest that all three subunits could be subjected to this modification.

33In yeast, ubiquitination negatively modulates Snf1 stability, phosphorylation, and catalytic activity during growth on alternative carbon sources. Ubp8, a subunit of the histone modifier SAGA complex, was shown to deubiquitinate Snf1. Accordingly, when grown on galactose medium, the ubp8Δ strain showed lower Snf1 levels and Snf1T210 T-loop phosphorylation than the wild-type (WT), due to enhanced proteasome-mediated protein degradation. On the other hand, Snf1 is SUMOylated (see below) and this modification promotes its proteasome-dependent degradation through the Slx5–Slx8 SUMO-targeted ubiquitin ligase.

34were the first to report the in vivo polyubiquitination of AMPKα1 and other AMPK-related kinases through unusual K29/K33-linked polyubiquitin chains. Although for the AMPK-related kinases ubiquitination was shown to interfere with T-loop phosphorylation and kinase activity, it remains to be determined whether AMPK activity is also similarly affected. Another study described the modulation of AMPK stability and activity by Cidea (cell death-inducing DFF45-like effector A)-mediated ubiquitination in brown adipose tissue. Cidea and AMPK form a complex in vivo, through a specific interaction with AMPKβ. Cidea-null mice accumulate higher levels of AMPK α-, β-, and γ-subunits with a consequent increment of AMPKαT172 phosphorylation and catalytic activity. Conversely, expression of Cidea promoted proteasomal degradation of the AMPK complex. On the other hand, mice deficient in the (UCH)-L3 deubiquitinating enzyme displayed increased AMPKαT172 phosphorylation and fatty acid oxidation, suggesting that ubiquitination might render AMPK more active. However, the effect might be indirect, since normal AMPK activity could not be restored by the short time replenishment of (UCH)-L3 expression within 4–6 days. Ubiquitination of AMPKβ with K63-linked chains, on the other hand, was shown to promote AMPKβ stability possibly through its allocation into inclusion bodies and subsequent protection from proteolytic turnover. Nevertheless, this modification did not induce detectable changes in AMPK activity.

35Interestingly, in plants, inactive kinase SnRK1α1K48M and T-loop phosphorylation SnRK1α1T175A mutant proteins accumulate to much higher levels than the WT SnRK1α1 protein, suggesting that activity and phosphorylation are connected with the stability of the protein. In agreement with this view, SnRK1α1 is targeted for proteasomal degradation under low nutrient conditions in a myoinositol polyphosphate 5-phosphatase (5PTase13)-dependent manner. SnRK1α1 degradation appears to be mediated also by the DDB1-CUL4-ROC1-PRL1 E3 ubiquitin ligase, in which PRL1 is the putative substrate receptor of the complex. SnRK1α1 interacts with PRL1 and its degradation via the 26S proteasome is slower in prl1 and cul4cs extracts than in the WT, accumulating to a higher extent in these mutants. In accordance, prl1 exhibits a higher activation of SnRK1 in comparison to the WT, and the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase, an enzyme inhibited by SnRK1 phosphorylation, is reduced in prl1 seedlings. On the other hand, PRL1 was reported to compete with SKP1/ASK1 for binding SnRK1α1 and SnRK1α2. SKP1/ASK1 is a component of the SCF E3 ubiquitin ligase and although SnRK1 participates in the assembly of a proteasomal complex with this E3 ligase, there is also the possibility that SnRK1 degradation is mediated either by the SCF complex or the CUL4-DDB1 machinery upon varied conditions.

36In yeast, a novel regulatory layer in SNF1 was uncovered through SUMOylation of its Snf1 catalytic subunit. In the presence of glucose, Snf1 is a target of the E3 SUMO ligase Mms21 which catalyzes the covalent attachment of SUMO at Snf1K549 in its C-terminal regulatory domain. SUMOylation can be reverted by the SUMO protease Ulp1. In the presence of glucose, SUMOylation inhibits Snf1 independently of T-loop phosphorylation, counteracting the glucose deprivation response. The authors suggest that upon SUMOylation an intramolecular conformational switch occurs due to the interaction between SUMOylated K549 with a SUMO-interacting motif located near the active site, promptly leading to an inactive conformation of Snf1. On the other hand, the SUMO tag targets Snf1 to ubiquitination via the SUMO-targeted E3 Ubiquitin ligase Slx5–Slx8, leading to degradation and attenuation of Snf1 levels in the cell as a response to glucose sensing.

37The AMPK complex was also recently shown to be post-translationally regulated by SUMOylation, underpinning previous results where AMPK subunits interacted with E2 SUMO conjugating enzyme in a yeast two-hybrid screen. E3 SUMO ligase PIASy specifically modifies AMPKβ2 subunit with the SUMO2 isoform, leading to the formation of poly-SUMO2 chains. The authors used a hypersumoylable mutant (AMPKβ2K262R) to show that SUMOylation of the AMPKβ2 subunit enhances the activity of the AMPK heterotrimeric complex (α2β2γ1), based on increased T-loop phosphorylation of AMPKα2 and on increased phosphorylation of the target acetyl-CoA carboxylase. SUMOylation of AMPKβ2 competes with ubiquitination of the same subunit and antagonizes the ubiquitin-mediated degradation and hence reduction in activity of overall AMPK complex.

38In plants, SnRK1α1 was found to interact with the E2 SUMO conjugating enzyme and with the SUMO protease ESD4 in a yeast two-hybrid screen. Furthermore, in the same study, SnRK1α1 was found to be SUMOylated with both SUMO1 and SUMO3 isoforms in a high-throughput assay in Escherichia coli. The fact that SnRK1α1 has high probability SUMO attachment sites and that SUMOylation has been implicated in the plant abiotic stress response suggests that SUMOylation may be a conserved mechanism for controlling SNF1/AMPK/SnRK1 activity and stability.

39In yeast, deficient protein N-myristoylation results in decreased resistance to nutrient deprivation, and the N-myristoylated protein Sip2 was found to be responsible for this phenotype. Sip2 myristoylation was also implicated in the normal cellular life span. In young cells, myristoylated Sip2 is located at the plasma membrane and sequesters Snf4, the activating γ-subunit of the SNF1 complex. With aging, Sip2 re-localizes from the plasma membrane to the cytoplasm allowing Snf4–Snf1 entry into the nucleus. Nuclear SNF1 subsequently phosphorylates histone H3 and modifies chromatin structure.

40An N-myristoylation consensus site is also present in the Sip1 β-subunit and it was shown to be required for Sip1 relocalization from the cytosol to the vacuolar membrane in response to various types of carbon stress. In glucose-grown cells, Sip1 localizes in the cytosol, and in response to carbon stress it re-localizes with Snf1 to the vacuolar membrane.

41AMPKβ1 and β2 regulatory subunits were shown to be myristoylated in vivo, regulating AMPK activity and its subcellular localization. Myristoylation facilitates AMPK association with cellular membranes, and inhibition of myristoylation prevents the membrane association of AMPK in response to leptin and glucose deprivation. A model has been proposed where the myristoyl group binds to a hydrophobic region within the complex, rendering AMPK inactive when ATP levels are high. The conformational change triggered by AMP binding exposes the myristoyl group thereby allowing the activation of the kinase and membrane association if required. This model can explain the increase in AMPK activity upon removal of the myristoylation site, but also the requirement of this modification for AMP-triggered stimulation of AMPKαT172 phosphorylation and AMPK maximal activation.

42In Arabidopsis, N-myristoyltransferase (NMT) mutants exhibit various degrees of shoot apical meristem abnormalities, with lack of NMT1 causing growth arrest after germination. The developmental arrest of nmt1-1 mutants was caused by deficient myristoylation of a very small set of proteins, including the SnRK1β1 and β2 subunits. GFP fusions of either SnRK1β1 or SnRK1β2 localized to the plasma membrane, whereas G2A substitutions preventing myristoylation relocalized SnRK1β1 to the nucleus and SnRK1β2 to the cytoplasm. This provides a possible explanation for the fivefold increase in SnRK1 kinase activity measured in the nmt1-1 mutant. Myristoylation of β1 or β2-subunits was proposed to sequester the complex to the plasma membrane acting as a negative regulator of the SnRK1 pathway, in accordance with the hypersensitivity to glucose of the nmt1-1 mutant.

43A recent study revealed that AMPK activation is also regulated by the intracellular redox status. Oxidation of two key cysteine residues in the catalytic subunit interferes with the interaction between AMPK and LKB1, blocking AMPK phosphorylation and activity. Thioredoxin1, on the other hand, promotes AMPK activation by reducing these cysteine residues, acting as an essential cofactor during energy starvation.

44To date, regulation by adenylates has been shown for all SNF1/AMPK/SnRK1 kinases (Figure 2;;;; ), and excellent recent reviews are available for AMPK.

45In yeast, AMP does not allosterically activate SNF1 but ADP protects it from dephosphorylation. Although this protection was initially thought to be mediated through ADP binding to Snf4, more recent work indicates that the regulatory subunits are not required for this protection. The current hypothesis is that, after phosphorylating a substrate, ADP would remain in the active site and protect the kinase from dephosphorylation.

46Adenylate regulation of AMPK is known for almost as long as the AMPK itself, providing the basis for its name. Adenylates regulate AMPK at several levels. Firstly, AMPK is allosterically activated by AMP through binding to its γ-subunit (Figure 1B;; ), and this activation has been confirmed to be specific to AMP. Secondly, AMP binding to the γ-subunit of the AMPK complex increases its ability to serve as a substrate for an upstream kinase, recently demonstrated to be LKB1 and not CaMKKβ. A third protein, AXIN, associates with LKB1 and enhances its interaction with AMP-bound AMPK, explaining why only LKB1-dependent phosphorylation of AMPK is stimulated by AMP. Thirdly, binding of the low energy adenylates ADP and AMP to the γ-subunit confers a conformation to the AMPK complex that makes it recalcitrant to dephosphorylation and inactivation by phosphatases. ADP binding was proposed to be the major factor protecting AMPK from dephosphorylation. More recent work questioned this hypothesis by showing that AMP, in its physiological concentration range, has a stronger effect than ADP on AMPK protection against phosphatases.

47The AMPKγ subunit harbors four CBS domains (Figures 1A,B; ), two of which binding adenylates reversibly (1 and 3;; ) and one (4) seemingly binding AMP non-exchangeably. Although initially proposed to have different functions in allosteric activation and protection from phosphatases, two studies suggest that the three adenylate binding sites (1, 3, and 4) are equally important for both functions. It is also important to note that without AMPKβ myristoylation, none of these events can occur.

48In the case of plants, the adenylate sensitivity was assessed only on a purified SnRK1 complex from spinach leaves. It was observed that AMP protects from dephosphorylation when the purified complex is incubated with recombinant mammalian PP2C. Nothing is known about the mechanism underlying this effect but as all the subunits of the trimeric complex are conserved in eukaryotes and the plant subunit complements the corresponding yeast mutant, it is reasonable to assume that the mechanism might be similar. On the other hand, the kinase domain is even more conserved, so a direct protective effect of ADP upon remaining on the active site is also possible.

49In yeast, SNF1 is activated in response to low glucose concentrations and other stresses and quickly inactivated by the addition of glucose (reviewed in ). Interestingly, the glucose analog 2-deoxyglucose, which is phosphorylated but not further metabolized, also inhibits SNF1, whereas 6-deoxyglucose, which cannot be phosphorylated, has no effect. These observations may be explained by a rapid depletion of cellular ATP due to the phosphorylation of 2-deoxyglucose. They may also suggest that glucose phosphorylation is needed for SNF1 repression, in agreement with the interaction of hexokinase2 with the Glc7–Reg1 complex for SNF1 repression. Hexokinase2 activity in turn is repressed by trehalose-6-phosphate (T6P) and T6P synthase 1 (TPS1) to control the influx of glucose into glycolysis and prevent an overconsumption of ATP and metabolic arrest.

50In mammals, high glucose concentrations inhibit AMPK activity. However, high glucose concentrations do not always result in detectable changes in the adenylate charge, suggesting other modes of repression of AMPK activity independent of adenylates. Supporting this, glucose has been reported to have a direct activatory effect on PP1 and PP2A. Amino acids like leucine or glutamine also negatively regulate AMPK. Citrate, produced by the TCA cycle in mitochondria, has been shown to inhibit AMPK activity in the rat hypothalamus during fasting. Furthermore, PP2A was reported to mediate palmitate-induced AMPK inhibition in mice fed with a high fat diet. Glycogen, particularly preparations with a high degree of branching, binds to β-subunits of AMPK leading to an allosteric inhibition of kinase activity. This may suggest that AMPK may sense not only the energy immediately available but also the availability of energy reserves.

51In plants, T6P has arisen as one of the major regulators of SnRK1. T6P is found in trace amounts in most plants, where it is considered to function as a signaling molecule. T6P accumulation is highly correlated with sucrose levels (Figure 2) and hence T6P has been proposed to relay information about carbohydrate availability to other signaling pathways. Inhibition of SnRK1 activity from Arabidopsis seedling extracts by T6P was observed at concentrations in the micromolar range. This inhibition was also observed in extracts from different Arabidopsis tissues and other plants (spinach, broccoli, and cauliflower), with the exception of fully mature leaves, indicating that SnRK1 regulation depends on the developmental stage and probably the type of tissue. Interestingly, no effect of T6P was observed in the activities purified from yeast, nematodes, flies, or human liver, suggesting that this effect is plant specific. Immunoprecipitated or anion-exchange chromatography purified SnRK1 is catalytically active but is no longer inhibited by T6P. However, the inhibition can be restored by supplementing the supernatant from immunoprecipitated seedling extracts indicating that an intermediary factor separable from SnRK1 activity is necessary for inhibition of SnRK1. The need for this intermediary factor is also suggested by the fact that mature leaf supernatant could not restore T6P inhibition. Inhibition of SnRK1 by T6P has also been observed in potato tubers fed with trehalose, in wheat grain and in sugar cane. In agreement with this, plants accumulating elevated T6P levels through overexpression of the E. coli T6P synthase otsA presented an opposite transcriptional profile to that triggered by SnRK1 activation. Similar results were observed in transgenic potato tubers. During wheat grain development, SnRK1-induced and SnRK1-repressed marker gene expression changes in the different tissues of the seed are correlated with changes in T6P levels, further supporting SnRK1 inhibition by T6P. A similar correlation was observed in Arabidopsis seedling extracts. It is also noteworthy that seedling growth arrest on high concentrations of trehalose, due to T6P accumulation, is rescued by overexpression of SnRK1. This suggests that T6P prevents growth on trehalose through the inhibition of SnRK1. Consistent with the inhibition of SnRK1 by T6P, plants silenced for SnRK1α1 and SnRK1α2 senesce early, opposite to plants overexpressing SnRK1α1 or to plants with low T6P levels which have delayed senescence.

52As explained above, T6P is a major regulator of glycolysis in yeast through repression of hexokinase 2. However, plant hexokinase activity seems not to be affected by up to 5 mM concentrations of T6P.

53In addition to T6P, SnRK1 activity is inhibited by other sugars, such as glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), glucose, and sucrose (Figure 2). SnRK1 is repressed by G6P in spinach, sugar cane, and Arabidopsis. G1P inhibits SnRK1 more strongly than G6P and synergistically with T6P. Strikingly, a combination of immunoprecipitation and SnRK1 activity assays suggest that both G1P and G6P, likewise T6P, inhibit SnRK1 via an intermediary factor that is separable from SnRK1. Supply of exogenous non-phosphorylated glucose and sucrose (5–50 mM) to seedlings and mature leaves also inhibits SnRK1 activity, as suggested by gene expression analyses. On the other hand, several studies have reported an induction of SnRK1 activity by sucrose or a SnRK1-dependent activation of gene expression or enzyme activity by sucrose. Such effect may be due to the heterotrophic nature of the material employed, in which SnRK1 may be regulated differently than in autotrophic leaves, or to the high sugar concentrations used, which may trigger stress and defense responses. Finally, inhibition of SnRK1 by ribose-5-phosphate and ribulose 5-phosphate was also observed; however, this inhibition is probably indirect, due to a decrease of ATP availability through ATP-dependent ribulose-1,5-bisphosphate synthesis.

54The crystal structure of the so called Bateman domain (a CBS-domain pair; Figure 1A) of yeast Snf4 and of the γ-subunit of mammalian AMPK revealed the formation of dimers, which in yeast also formed in vivo. Crystallographic dimers were likewise obtained for the kinase domain of Snf1, and co-immunoprecipitation assays of differently tagged catalytic subunits in yeast cells confirmed the existence of these dimers in vivo. The dimerization interface in SNF1 and AMPK is characterized by extensive hydrophobic interactions, involving both conserved and non-conserved residues around the activation loop. However, the exact interface of dimerization remains to be determined, as there are conflicting reports amongst the known structures.

55Importantly, oligomerization of whole heterotrimeric complexes has also been reported. Heterotrimers of truncated SNF1 from Schizosaccharomyces pombe and Saccharomyces cerevisiae expressed in bacteria formed crystallographic dimers, which for Schizosaccharomyces pombe were also found in solution. Even though the crystals of truncated AMPK complex did not show evidence of dimerization, untagged, full-length and enzymatically competent AMPK heterotrimers purified from bacteria did form dimers. Dimers of AMPK heterotrimers and even higher order oligomers were also detected in cellular extracts by Blue Native PAGE. Likewise, gel filtration chromatography data for AMPK revealed a higher molecular weight than expected. This can be merely due to a global non-spherical shape of the heterotrimer, but it has also been proposed to correspond to higher order complexes.

56Dimerization appears to be a reversible concentration-dependent process that can occur both in vitro and in vivo in mammalian cells and it may be particularly important in specific subcellular loci where the kinases are highly concentrated. However, how oligomerization impacts on kinase activity is not understood. The formation of higher order oligomers was associated to an inactive state of the AMPK complex, which upon activation would disassemble into dimeric and monomeric units of the heterotrimeric complex. However, the activation loop of Snf1 becomes inaccessible for phosphorylation by upstream kinases when the catalytic subunits form dimers, suggesting that dimers of heterotrimers would also be inactive.

57When considering the total number of α, β, and γ subunits present in plants, at least 12 heterotrimers can be formed (Figure 1A), but this number increases if alternatively spliced variants are taken into account. The theoretical molecular weight of such heterotrimers ranges from 118 to 165 kDa, but immunoprecipitation of the SnRK1 complex in vivo, coupled to size fractionation, revealed the presence of both catalytic subunits in fractions of higher molecular weight. Supporting the formation of higher order complexes, the maize βγ-subunit was shown to homodimerize in vitro and in vivo. However, it is not yet known whether this impacts on SnRK1 activity or if it is related to specific functions of the βγ-subunit.

58AMPK is known to be affected by an array of chemicals of synthetic (drugs) and natural (xenobiotics) origin, many of which are employed in the treatment of diabetes, obesity and cancer. We will briefly review the effects and mechanisms of action of the best characterized ones, as the regulation of AMPK by drugs and xenobiotics has been specifically reviewed elsewhere recently.

59Most drugs or xenobiotics activate AMPK indirectly by blocking ATP production, either by inhibiting glycolysis (2-deoxyglucose) or oxidative phosphorylation. The latter is the case of mitochondrial poisons like oligomycin and dinitrophenol, phenobarbital, drugs used in the treatment of type 2 diabetes like phenformin, rosiglitazone, and several plant products considered to have health-promoting properties like berberine, resveratrol, or curcumin.

60There are also chemicals that interact with upstream components of the AMPK pathway and indirectly promote its activation, like A23187 and other Ca2+ ionophores, that increase intracelular Ca2+ concentration, activating CaMKKβ.

61Other chemicals activate AMPK through a direct interaction. This is the case for metformin, which interacts with AMPKγ and compound A-769662 and salicylate, which activate AMPK by a similar mechanism involving the β1 and γ subunits. This results in allosteric activation of AMPK and its protection against T172 dephosphorylation. Other small molecule AMPK activators are the related PT1 and C24. PT1 was found in a chemical library screen with inactive truncated human AMPK. It was proposed to bind near the autoinhibitory domain and directly relieve autoinhibition. Finally, a widely used AMPK activator is 5-aminoimidazole-4-carboxamide riboside (AICAR). AICAR is an adenosine analog which is a substrate for adenosine transporters and kinases. It is phosphorylated to the AMP analog, ZMP, and it is capable of activating AMPK, by binding to γ-subunit in a similar manner to AMP. Finally, it is surprising that only one chemical, compound C, has been shown to inhibit AMPK by targeting the kinase domain.

62With the exception of the obligate intracellular parasite Encephalitozoon cuniculi, SNF1/AMPK/SnRK1 are ubiquitously present in eukaryotes, from simple unicellular organisms to complex multicellular ones. These systems have therefore acquired the ability to be regulated by hormones and systemic signals, which in multicellular organisms is essential for the proper coordination of energy balance at the whole-organism level.

63In mammals, AMPK activity is coordinated at the whole-body level through regulation by several hormones and cytokines, including leptin, adiponectin, resistin, ghrelin, insulin, glucagon-like peptide-1, glucocorticoids, inflammatory mediators, and thyroid hormone T3 and T4. Noteworthy, the effect of some hormones on AMPK activity depends on the tissue, and, for example, leptin activates AMPK in adipose tissue and the liver, whilst repressing it in the heart and the hypothalamus. Although in most cases the precise mechanisms underlying hormonal regulation of AMPK are unknown, it is well established that insulin inhibits AMPK in cardiac tissue by activating the Akt/PKB kinase, which can phosphorylate AMPKαS485, thus leading to reduced phosphorylation at AMPKαT172. In the case of thrombin, activation appears to occur through induction of Ca2+ signaling and CaMKKβ activation. Interestingly, chronic TNFα treatment in muscle cells suppresses the AMPK pathway through the induction of the repressor PP2C, suggesting that a connection between hormone signals and energy signaling through the repressive PP2Cs might be conserved in multicellular eukaryotes (see below).

64In the case of plants, an increasing number of studies link SnRK1 to the ABA phytohormone (Figure 2). SnRK1 appears to play a central role in processes well known to be under ABA control, such as seed maturation and germination. Furthermore, Arabidopsis plants overexpressing SnRK1α1 are hypersensitive to ABA during germination and early seedling development, consistent with the phosphorylation by SnRK1α1 of FUSCA3, a central transcription factor regulating seed maturation. Recent work demonstrated that in mature photosynthetic tissues ABA activates SnRK1 through inhibition of its negative regulators, the 2C-type phosphatases ABI1 and PP2CA. This may allow the complementation of the ABA response with a more general one triggered by SnRK1 and directed toward a metabolic and transcriptional reprograming. Additionally, the presence of ABA may potentiate SnRK1 signaling by blocking its inactivation and may allow SnRK1 activation in distant tissues not directly exposed to energy stress. Interestingly, ABA represses SnRK1 signaling via plant-specific SnRK1A-interacting negative regulators during germination and early seedling growth and induces SnRK1 degradation in wheat roots. This suggests the effect of ABA may differ between autotrophic and heterotrophic tissues in a similar manner as animal hormones control AMPK in opposite manner in different tissues.

65The regulation of the SNF1/AMPK/SnRK1 kinases is highly complex, involving, amongst others, multiple post-translational modifications of the catalytic and regulatory subunits, direct and indirect metabolic and hormonal control, and formation of higher order complexes (Figure 2). Regulation is exerted by universal signals like adenylates and sugars as well as by more specific signals like hormones or particular subunits that have evolved to regulate these kinases at the whole organism-level and possibly to serve organism-specific functions. Despite the conservation of some regulatory aspects, such as T-loop phosphorylation by homologous upstream protein kinases, the clear connection established for SNF1 and AMPK between T-loop phosphorylation, adenylate sensing, and kinase activity has not been fully established in plants where additional regulatory mechanisms may be operating.

66On the other hand, the possible interconnection between the different modes of regulation is thus far unknown. Post-translational modifications might have an effect, e.g., on T-loop phosphorylation, either repressing it similarly to what has been described for ubiquitination of AMPK-related kinases or promoting it similarly to what has been described for SUMOylation of the AMPKβ subunit. Furthermore, the functional outcome of a particular mode of regulation might be rather complex and derived from several factors. An example of this is myristoylation of AMPKβ, which is necessary for the adenylate regulation of AMPK, but also for the membrane localization of the complex.

67With regard to regulation by metabolites, hormones, and other compounds, mechanistic knowledge is lacking in most cases. Whether or not the effect of these compounds is direct, indirect involving changes in other metabolites or Ca2+ fluxes, or indirect involving unknown protein factors as reported for T6P in plants, remains to be determined. Moreover, the actual components transducing these signals to SNF1/AMPK/SnRK1 remain to be identified.

68One major challenge lies in the heterogeneity of SNF1/AMPK/SnRK1 complexes. Subunit composition may change in response to specific conditions and may be unique to particular subcellular compartments, tissues or developmental stages. Subunit composition may determine the function of the complex as well as its mode of regulation. Therefore, strategies that allow monitoring specific complexes will ultimately be required for full characterization and understanding of these kinases. Different SNF1/AMPK/SnRK1 complexes are also likely to be recognized and controlled by different upstream regulators, as suggested by the rising number of kinases and phosphatases regulating AMPK and the specificity of some of these to particular tissues.

69Full understanding of plant SnRK1 will also require the identification of further upstream regulatory components as well as a better characterization of their effects. In addition, the passive role of these upstream components, traditionally regarded as being constitutively active should be revisited, as an increasing body of evidence supports metabolic and hormonal regulation at least of the SNF1/AMPK/SnRK1 phosphatases. Identification and characterization of the upstream regulators may also be crucial for understanding the connection of these signaling cascades to other important pathways, as demonstrated for the dual role of ABI1/PP2CA phosphatases in SnRK1 and ABA signaling.

70The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.