Difference between revisions of "AMPK"

Revision as of 23:09, 4 May 2018

AMPK Protein Kinase AMP-Activated Ver. 0.3.

Structure

The structure with regulation sites is presented on Figure 1.

Figure from (Jeon ‎2016) [1]. Molecular regulation of AMPK and LKB1. (a) Modification of the AMPK α1 (top) and α2 (bottom) subunits by phosphorylation/dephosphorylation, ubiquitination, sumoylation and oxidation/reduction. Pathways marked in red indicate α1- or α2-subunit-specific modifications. Numbers of modified amino acids are based on human proteins, and numbers in parenthesis are those reported in the original research (see text for details). (b) Modification of the AMPK β1 (top) and β2 (bottom) subunits by myristoylation, ubiquitination, sumoylation and glycogen binding. Pathways marked in red indicate β1- or β2-subunit-specific modifications. (c) Modification of the AMPK γ-subunit by AMP, ADP or ATP binding. Binding of AMP to CBS1 induces allosteric activation, and binding of AMP or ADP to CBS3 induces T172 phosphorylation. (d) Modification and regulation of LKB1 by phosphorylation, acetylation, ubiquitination, sumoylation and 4HNE adduction. Arrow indicates activation, and bar-headed line indicates inhibition. α/γ-BD, α/γ-subunit-binding domain; AID, autoinhibitory domain; β-BD, β-subunit-binding domain; CBM, carbohydratebinding module; CBS, cystathionine beta-synthase domain; NLS, nuclear localization signal.

Action

AMPK, a fuel sensor and regulator, promotes ATP-producing and inhibits ATP-consuming pathways in various tissues. AMPK is exists as a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits. The kinase is activated in response to stresses that deplete cellular ATP supplies such as low glucose, hypoxia, ischemia and heat shock. Binding of AMP to the γ subunit allosterically activates the complex, making it a more attractive substrate for its major upstream AMPK kinase, LKB1. As a cellular energy sensor responding to low ATP levels, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies. For example, activation of AMPK enhances both the transcription and translocation of GLUT4, resulting in an increase in insulin-stimulated glucose uptake. In addition, it also stimulates catabolic processes such as fatty acid oxidation and glycolysis via inhibition of ACC and activation of PFK2. AMPK negatively regulates several proteins central to ATP consuming processes such as TORC2, glycogen synthase, SREBP-1 and TSC2, resulting in the downregulation or inhibition of gluconeogenesis, glycogen, lipid and protein synthesis. Due to its role as a central regulator of both lipid and glucose metabolism, AMPK is considered to be a key therapeutic target for the treatment of obesity, type II diabetes mellitus, and cancer. The AMP-activated protein kinase (AMPK) has an important role in the regulation of cellular energy homeostasis. The enzyme is activated under conditions of low ATP, often caused by a variety of stresses and regulates signaling pathways that increase the supplies of ATP available. (http://www.sinobiological.com/ampk-signaling-pathwawy.html)

Kinetics

Pathways

In general, upstream pathways presented at Figure from (Jeon ‎2016) [1].

In general, downstream pathways presented at Figure from (Jeon ‎2016) [1].

AMPK Regulate Multiple Metabolic Processes in Cells.

Figure 3. Substrates of AMPK Regulate Multiple Metabolic Processes in Cells. AMPK is phosphorylated and activated by LKB1 and CAMKK2 in response to stimuli that increase AMP/ADP levels (energy stress) or Ca2+ flux, respectively. Once active, AMPK induces metabolic changes through the phosphorylation of substrates. Some of the best-established metabolic processes regulated by AMPK are shown, together with the relevant substrates (Garcia 2017) [2].

Upstream

Downstream

(Chen 2003) [3]. We have shown that nNOSµ is a target for phosphorylation by AMPK in human skeletal muscle (17). We found a significant increase in phosphorylation of nNOSµ at Ser-1451 immediately after maximal sprint exercise (17) but only modest effects during lower-intensity exercise at 60% VO2 peak (16). In the current study, nNOSµ phosphorylation was also quite variable between subjects and only achieved statistically significant increases at the highest levels of exercise intensity.

Isoforms

AMPK is present in only three heterotrimers in human skeletal muscle: α1/β2/γ1, α2/β2/γ1 and α2/β2/γ3. The approximate distribution of the three AMPK heterotrimers can be estimated to be ∼15% α1/β2/γ1, ∼65% α2/β2/γ1 and∼20% α2/β2/γ3 (Birk 2006) [4].

AMPK α2/β2/γ3 heterotrimer is predominantly activated during exercise in human skeletal muscle (Birk 2006) [4].

Figure from (Jensen 2009) [5]. Skeletal muscle AMPK expression and regulation. (a) Molecular regulation of AMPK activity. (b) AMPK expression in human quadriceps and rodent oxidative and glycolytic muscles and their tentative activation profiles during contraction/exercise.

Location

Within the individual muscle fibres, α2 AMPK has been reported in both the cytosol and nucleus, while α1 seems expressed exclusively in the cytosol (Salt 1998) [6]. a-N-myristoylation of the b subunit may facilitate reversible binding to membranes and activation by upstream kinases (Oakhill et al., 2010; Steinberg and Kemp, 2009; Warden et al., 2001). [7, 8] pmid=25066137

Activity

AMPK phosphorylation (Thr172) and AMPK activity in mammalian muscles are usually strongly positively related. For example, for rat muscle, see figure (Park 2002) [9].

For human muscle see AMPK y3 associated activity after exercise at figure from (Birk 2006) [4]:

There is substantial constitutive AMPK kinase activity at rest. AMPK phosphorylation in resting muscle is possible because significant amount of AMPfree is present in resting muscle, for example see data from (Birk 2006) [4]:

In resting muscle most or all of the phosphorylated AMPK heterotrimers must be α1/β2/γ1 and α2/β2/γ1 (Birk 2006) [4].

Activity for isoforms AMPK-α1 and –α2

The measured values of AMPK activity, possibly depends on substrates and methods. For example in two works with the same exercise (30-s bicycle sprint) different activation patterns was shown:

1. (Chen 2000) [10].30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK- α1 and - α2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser79 (AMPK phosphorylation site) 8.5-fold.

2. (Birk 2006) [4]. In response to high-intensity exercise protocol, there was, surprisingly, no detectable increase in p-AMPK. In line with this, α1-associated activity also decreased significantly, whereas the α2 activity was unchanged. Between the two α2 complexes, only the α2/β2/γ3 activity increased in response to exercise.

Typical values for AMPK α1 and α2 activity are shown in Table:

Even at rest in one article different values (0.5-0.85 pmol/mg/min) of AMPK activity were measured for 3 ocassions (Birk 2006). So data and conclusions about AMPK activity from different articles must be accepted with cautions.

AMPK α1- and α2- associated activity shows different time course during exercise (1 h at 70% peak VO2) and depends on muscle glycogen content (Wojtaszewski 2003) [11].

Data from (Birk 2006) [4] shows that AMPK α2/β2/γ3 heterotrimer is predominantly activated during exercise in human skeletal muscle, only the α2/β2/γ3 subunit is phosphorylated and activated during high-intensity exercise in vivo.

AMPK activity associated with the α2/β2/γ3 heterotrimer was strongly correlated to γ3-associated α-Thr-172 AMPK phosphorylation (r2 =0.84, P <0.001) (Birk 2006) [4].

The phosphorylated γ3 heterotrimers in the exercised state accounted only for 32±5% (n =11) of all the γ3 heterotrimers, still leaving the majority of these unphosphorylated and available for activation (Birk 2006) [4].

Data predict a unique role of the α2/β2/γ 3 heterotrimer in human skeletal muscle, as only this heterotrimer is phosphorylated and activated during the three high-intensity exercise regimes investigated. The α2/β2/γ 3 heterotrimer only constitutes one-fifth of all AMPK heterotrimers, and only one-third of these heterotrimers contain phosphorylated α2 protein, indicating a large pool of spare AMPK signalling within the cell even during high-intensity exercise. Accordingly, only a small fraction (10%) of the total α2 protein pool is phosphorylated during exercise (Birk 2006) [4].

Interestingly, in human skeletal muscle exercise training has been shown to reduce mRNA and γ 3 protein expression (Nielsen et al. 2003; Frosig et al. 2004;Wojtaszewski et al. 2005). As hypothesized, the present finding, that only γ3 heterotrimers are activated in response to exercise, is in accordance with the observation of attenuated AMPK activation during acute exercise after a period of exercise training, even when exercise is performed at the same relative intensity (Nielsen et al. 2003; Yu et al. 2003; Frosig et al. 2004; McConell et al. 2005) from (Birk 2006) [4]. Short term stimulation of rat L6 myotubes with the AMPK activator (AICAR), activates AMPK and promotes translocation of the Na+,K+ -ATPase a1-subunit to the plasma membrane and increases Na+,K+-ATPase activity (Benziane 2012) [12].

Activity regulation

Allosteric activation mechanisms of AMPK by AMP

AMPK works like a delicate instrument. α-Thr172 is just like a start button., once it is phosphorylated, it achieves the initial activity. The γ subunit works like a sensor which senses the allosteric signal of AMP and induces the long range allosteric activation. The allosteric signal will be transduced to the engine, the α subunit, and fully activate the holoenzyme. When ATP and other molecules replace AMP, the allosteric activation is disturbed (Li 2017) [13]. At the same time opposite causal relationship described in review (Gowans 2014) [14] : “Only binding of AMP promotes phosphorylation of Thr172 by LKB1.”

A proposed allosteric activation model

The energy status holds a dynamic equilibrium in eukaryotic cells. In the resting state, AMPK stays in the ATP-bound form and ATP occupies γ-site 1 and γ-site 4. The conformational changes of the γ subunit and the collective upwards movement of the inter-subunit β-sheet make the holoenzyme less compact and also restrict the interaction between α-RIMs and the γ-sites. The α-linker is flexible and the AID stably binds to the kinase domain and maintains it in a relative open, inactive conformation. When the energy level decreases, the concentration of AMP will increase and AMP competitively binds to the AMPK at γ-site 1, γ-site 3 and γ-site 4. This induces substantial conformational changes that enable α-RIM 1 and α-RIM 2 to be recruited to the γ-site 2 and γ-site 3, respectively. Then, the constrained α-linker forces the disassociation of the adjacent AID from the KD, then the AID moves towards and directly binds to the γ-subunit. Thus, the KD is in a more closed, active conformation and the AMPK is allosterically activated (Fig 2D).

Figure 2D from (Li 2017) [13]. In the resting state, the concentration of ATP is high and AMPK is saturated with ATP at γ-site1 and 4, the holoenzyme adopts an relative loose conformation. The AID stably binds to the backside of KD and keeps it in an inactive state, and the α-linker is flexible and disordered in the structure (dotted line). Once the AMP/ATP ratio increases, the AMP will competitively bind to the γ-site1, 3 and 4 and induces lots of conformational changes, then recruits α-RIM1/2 and the induced β-loop. The constrained α-linker forces the disassociation of AID from KD, therefore releases its inhibition effect to KD, and the AID binds to the N-terminus of γ-subunit. The holoenzyme becomes more compact and is allosterically activated.

(b) Exercising human skeletal muscle (exercise regimes – low int.: 40 +- 2% VO2 peak, 20 min; med. int.: 59 +- 1% VO2 peak, 20 min; high int.: 79 +- 1% VO2 peak, 20 min) [59].

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase- α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. (Hawley 2014) [15].

AMPK regulation by phosphorylation

From review (Gowans 2014) [14]. The major kinase responsible for phosphorylation of Thr172 is a complex containing the tumour suppressor LKB1 [18,19]. Although this complex appears to be constitutively active [20], binding of AMP to AMPK not only causes allosteric activation, but also makes AMPK a better substrate for LKB1 [21], and a worse substrate for protein phosphatases [22], thus promoting net phosphorylation and activation. Thr172 can also be phosphorylated by CaMKKs (Ca2+ /calmodulin-activated protein kinase kinases), especially CaMKKβ [23–25], providing an alternative Ca2+ - dependent pathway for AMPK activation. This can occur in the absence of any changes in adenine nucleotides, although changes in cellular AMP can amplify the effect of the Ca2+ - activated pathway due to the protective effect of AMP on Thr172 dephosphorylation [26]. Binding of ADP, as well as AMP, protects AMPK against dephosphorylation by protein phosphatases, but AMP was approximately 10-fold more potent than ADP [38]. Only binding of AMP promotes phosphorylation of Thr172 by LKB1. Our laboratory originally reported that AMP binding to AMPK promoted phosphorylation of Thr172 by LKB1, but not CaMKKβ [23]. However, it was reported more recently that binding of AMP promoted phosphorylation by both LKB1 and CaMKKβ, whereas ADP also promoted phosphorylation by CaMKKβ, as long as the β subunit in the AMPK complex was N-myristoylated [33,39]. We have reinvestigated this and confirmed our original findings that AMP promoted phosphorylation only by LKB1, and not CaMKKβ. We were unable to detect effects of ADP on the rate of phosphorylation by either kinase. The effect of AMP to promote phosphorylation by LKB1 appears to require higher concentrations than its effects on dephosphorylation, suggesting that it may be caused by binding to a different site. Despite the fact that phosphorylation of Thr172 by upstream kinases can cause >100-fold activation when fully dephosphorylated AMPK complexes are incubated with upstream kinases in cell-free assays, we showed that the effects in intact cells are much more modest [38].

From review (Hardie 2016) [16]. Hormones that increase intracellular Ca2+ activate AMPK via phosphorylation of Thr172 by the calmodulin-dependent proteinkinase CaMKKβ [33–35]. This represents an alternative, Ca2+-dependent pathway for AMPK activation that is independent of changes in adenine nucleotides and is therefore considered to be non canonical. However, because AMP binding inhibits Thr172 dephosphorylation, the two pathways can act synergistically [36].

AMPK regulation by glycogen

AMPK β subunits contain a central conserved region that we refer to as the glycogen-binding domain (GBD), which causes AMPK complexes to associate with glycogen in cultured cells and cell-free systems (Hudson et al., 2003; Polekhina et al., 2003). Glycogen availability is known to affect AMPK regulation in vivo. We show that AMPK is inhibited by glycogen, particularly preparations with high branching content. AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen. Even a modest reduction in glycogen content might cause release of significant quantities of AMPK from the polysaccharide so that more of the kinase becomes available to phosphorylate targets (McBride 2009) [17].

Expression

PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAA1)

PRKAA2 Protein Kinase AMP-Activated Catalytic Subunit Alpha 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAA2)

PRKAB1 Protein Kinase AMP-Activated Non-Catalytic Subunit Beta 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAB1)

PRKAB2 Protein Kinase AMP-Activated Non-Catalytic Subunit Beta 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAB2)

PRKAG1 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 1 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG1)

PRKAG2 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 2 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG2)

PRKAG3 Protein Kinase AMP-Activated Non-Catalytic Subunit Gamma 3 (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PRKAG3)

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD006182

Diseases

References

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