Proceedings of Bioc 462a Honors

Abstract

AMP-activated protein kinase (AMPK) is a metabolic stress sensing protein that regulates cellular energy during times of limited energy. AMPK is made up of three distinctly functioning subunits. The α subunit is composed of a catalytic domain and an autoinhibitory domain that regulates the activity of the protein. The β subunit contains a glycogen-binding domain (GBD) that also regulates the activity. Finally, the γ subunit forms the broad base of the protein. It contains two Bateman domains; each Bateman domain is comprised of two cystathionine β synthase (CBS) segments. The Bateman domains contain the amino acid residues responsible for the binding of AMP or ATP. The three subunits that form this heterotrimer communicate during times of energy depletion or surplus. During times of energy deficiency, an AMP molecule will bind the γ subunit and activate AMPK, hence the name. The conformational chain in the γ subunit is transmitted to the α subunit and activates the protein for phosphorylation. This phosphorylation occurs on Thr-172. Now, the kinase can then deactivate enzymes of a variety of anabolic pathways, such as protein synthesis, and allow the cell to allocate ATP for catabolic mechanisms, such as muscle contractions. Additionally, ATP can also bind to the γ subunit and deactivate the kinase during times of energy excess. Glycogen can inhibit the activity of AMPK by binding to the β subunit and preventing the key Thr-172 residue from being phosphorylated and consequently preventing AMPK from inhibiting anabolic pathways.

Introduction

For a cell to live, tasks such as protein synthesis, transportation of molecules, and metabolism must be performed. These tasks require a source of energy. The main energy source comes from ATP. When a cell undergoes these functions, ATP is consumed. Consequently, energy producing pathways such as glycolysis is concurrently producing ATP to replace those that have been used. The utilization of ATP can yield the byproduct, AMP. AMP normally exists within a cell but in lower levels. During times of energy surplus or when the utilization of ATP is matched with an appropriate production of ATP, the level of AMP is low. However, during times of stress, the cell will consume ATP to the extent that AMP is at a higher concentration. AMP acts like a gauge for cellular energy stress. When this occurs, energy becomes scarce and actions must be taken to rejuvenate the concentration of ATP. Firstly, the cell ceases ATP-consuming pathways that don't contribute to the formation of ATP, such as protein and fatty acid synthesis (Kemp et al., 2003). Secondly, energy producing pathways such as glycolysis (Hue et al., 2002) and food intake (Andersson et al., 2004) are stimulated to increase the amount of ATP to overcome AMP. These important metabolic controls are regulated by AMP-activated protein kinase.
AMP-activated protein kinase (AMPK) is a metabolism conductor that is activated by AMP, which occurs during time of energy exhaustion. An AMP molecule binds to AMPK and allows it to deactivate energy consuming pathways and switch on energy producing pathways (Kemp et al., 2003). Activation and deactivation of these pathways is catalyzed Thr-172 on the α subunit (Hardie, 2007). But other molecules can associate with AMPK to regulate its tendency to perform its energy producing pathways. ATP can also bind competitively to the same site as AMP and deactivate AMPK (Townley et al., 2007).
AMPK is a heterotrimeric kinase composed of a catalytic α subunit, regulatory β subunit and an activating γ subunit. A molecule of glycogen can bind to the β subunit and prevent the essential Thr-172 from being phosphorylated (Polekhina et al., 2005). The binding of AMPK to AMP or ATP and to glycogen are some of the mechanisms that can regulate AMPK. With many different mechanisms where AMPK is involved in up or down regulation of pathways, there are many factors than can effect AMPK's functioning. This property makes this protein appealing for more research into the mechanism of its regulation and function.

Main Section

AMP-activated protein kinase (AMPK) is a heterotrimeric kinase (Hardie, 2003) composed of isoforms of each subunit that are encoded by multiple genes (α1, α2, β1, β2, γ1, γ2, and γ3) (Kahn et al., 2005a). The protein has an elongated triangular structure. The γ subunit forms the base. When the narrow α subunit of the protein is facing up (Fig. 1), the α helices of the γ subunit are oriented horizontally, giving the subunit its width with β sheets are located in the middle. It is within the β sheets where AMP is bound. However, the γ subunit is the not largest subunit in terms of number of residues. Directly above the γ subunit is the β subunit. The β subunit is the smallest of the trimers, composed almost entirely of β sheets. The β subunit is narrower than the γ subunit. AMPK is capped at the top with the α subunit. The α subunit is composed of a combination of α helices and β sheets that are oriented more vertically than the β and γ subunits.

Figure 1. Structure of AMP-activated Protein Kinase. α subunit in red. β subunit in green. γ subunit is in blue. The β flap is shown in magenta. AMP is shown in CPK, ball and stick representation within the middle of the γ subunit. <Larger View>

The α subunit is composed of catalytic, autoinhibitory, and subunit-binding domains (Crute et al., 1998). The catalytic domain is the first 294 residues (Townley et al., 2007). The activity of this protein relies on phosphorylation of the critical residue, Thr-172 (Hawley et al., 1996). Although other residues on the α subunit can be phosphorylated (Kemp et al., 2003), AMPK remains inactive if Thr-172 is not (Hamilton et al., 2002). AMPK kinase is the responsible enzyme for the activation of AMPK through the phosphorylation of Thr-172 (Hawley et al., 1996). But it is a cascade of consecutive reactions by other proteins that will eventually phosphorylate AMPK. The autoinhibitory domain, bound by residues 295-440 (Townley et al., 2007), regulates the activity of AMPK by sites of phosphorylation of serine residues 404 and 485 (B.E. Kemp, D. Stapleton, S. Hamilton, J. Hurley and L.A. Witters, unpublished). However, the specifics of the mechanism of activity of AMPK are not well known.

The C-terminal end is the subunit-binding domain, stretching from residue 441-576 (Townley et al., 2007). Previous works devised a model where the β subunit functions as scaffolding for the α and γ subunits, joining the two subunits. However, according to Kelly A. Wong and Harvey F. Lodish, the β subunit does not connect the α and γ subunit. This conclusion was developed through deletion tests of different expanses of AMPK. In the test, segments of the α subunit is removed and tested for function to determine the region where activation from γ subunit is being transmitted. The α subunit binds to the γ subunit within the catalytic domain (residues 1-312) and the C-terminal end (386-552). The β subunit does not bind to the catalytic domain but actually binds to the C-terminal domain of the α domain, same as the γ subunit (Wong and Lodish, 2006). With this, the C-terminal end of the α subunit can be termed the new scaffold for AMPK.
Shown in Fig.1, the α and β subunit secondary structures intertwine into a compact structure that forms the apex of the triangular AMPK (Townley et al., 2007). The β domain is composed of 298 residues bound to the C-terminal region of the α subunit, residues 386-552 (Wong and Lodish, 2006). As previously stated, this β subunit does not bind to the γ subunit (Wong and Lodish, 2006). New research suggests the β subunit binds only to the α subunit. In the β subunit lies a segment of residues known as the glycogen-binding domain on the N-terminal region. The binding of glycogen is governed by two principles: hydrophobic interactions and hydrogen interactions (Polekhina et al., 2005). Glycogen molecule is “stacked” on the two tryptophan residues by hydrophobic interaction between the tryptophan side chain and the carbon rings of glycogen (Polekhina et al., 2005). In addition, Leu-146 acts as a hook (Fig. 2) for the ringed glycogen molecule to fit into (Polekhina et al., 2005). With the two sets of interactions between glycogen and the hydrophobic residues, glycogen is held in place. To further strengthen the association of glycogen to the subunit, an electrostatic interaction from the α amino group of Asn-150 forms with the oxygen molecule of glycogen. Lys-126 also participates in an electrostatic interaction with the bound glycogen molecule (Polekhina et al., 2005). While a collection of residues play a role in holding of glycogen, the critical residues that are necessary for glycogen to bind are Trp-100 and Lys-126. This idea was tested by site-directed mutagenesis W100G and K126Q. The resulting structure completely lost its ability to bind with glycogen (Polekhina et al., 2003). Trp-100 was one of the hydrophobic residues that buffered glycogen into place by hydrophobic interaction and Lys-120 participated in hydrogen bonding. Further tests revealed N150Q significantly reduced the binding ability of subunit but not completely destroyed. However, when arginine is mutated to a lysine, the glycogen binding domain completely lost its ability to bind with glycogen (Polekhina et al., 2003). It is the amino side chain of either arginine or glutamine that participates in the binding of glycogen by donating hydrogen bonds. Lysine cannot behave in this fashion and thus, disrupts the binding.

Figure 2. Cyclic glycogen molecule is bound to the β subunit by the amino group of 150 and lysine 126. Two tryptophan residues form a base for glycogen to position next and a leucine residue inserts into the ring of glycogen. <Larger View>

High levels of muscle glycogen inhibit the activation of AMPK in rat muscles (Hardie, 2005). When glycogen decreases below a certain level, AMPK is then reactivated and this results in the increase of glycolysis, enhanced insulin sensitivity, and oxidation of fatty acids (Kahn et al., 2005). With glycogen attached, AMPK is directed to a phosphatase that deactivates AMPK (Polekhina et al., 2005) by cleaving the phosphate group on the catalytic serine of the α subunit.
As the name of the protein suggests, AMPK is regulated by AMP. This regulation is centered in the γ subunit. This subunit contains 334 residues that are segregated into different levels of domains (Townley et al., 2007). The γ subunit is split into two domains: Bateman1 and Bateman2. In addition, each Bateman domain is split into two ~60 residue motifs that are comprised of the same group of amino acids (Bateman, 1997). These motifs are known as cystathionine β-synthase (CBS1, CBS2, CBS3, and CBS4). CBS1 and CBS2 are part of the Bateman1 and CBS3 and CBS4 belong to Bateman2. The CBS domains are not catalytic; it was named after the enzyme that isolated the domain (Bateman, 1997). Each Bateman domain binds to one molecule of AMP or ATP. An AMP ligand is the primary activator of the kinase. Similar to glycogen, the binding of ATP to AMPK will hinder the kinase's ability to inhibit energy consuming anabolic pathways, such as protein synthesis, by deactivating the factors involved through phosphorylation (Hardie., 2007). Only one ligand, either AMP or ATP can bind with AMPK, but it is important to note that the binding affinity for ATP is five-fold lower than that of AMP (Scott, 2004). The Bateman domains also exhibits cooperativity - when one Bateman domain is bound to one of the nucleotides, the other domain is more likely to bind with the same ligand. The center of the γ subunit is polar (Townley et al., 2007). Therefore, it is able to accommodate the negatively charge ligands that are bound. Furthermore, the center of the γ subunit (Fig. 1) has a low density of amino acids, providing a pocket (Fig. 3) for ATP or AMP to insert itself. Once AMP or ATP is ligand into the cavity, polar interactions of the Bateman domains stabilize the ligand. In the case of AMP (Fig. 4), it is held within the protein by hydrogen bonds between arginine 139 and 141. The adenine group of AMP is also held by hydrogen bonds with the carbonyl oxygen of alanine 196 and 218. At the bottom of the figure, a Phe-292 keeps AMP in place. A similar set of residues holds ATP in place.

Figure 3. A space-filling rendering of the γ subunit in light blue with AMP bound within the middle. The green region represents surrounding ligand-stabilizing residues. <Larger View>

Figure 4. Stereo view of AMP binding to the γ subunit. The guanidino group of Arginine 139 and 141 donates hydrogen bonds to the phosphate group of AMP. The carbonyl oxygen of alanine 196 and 218 are salt linked with the nitrogens of AMP's adenine. Surrounding residues help to stabilize AMP in the γ subunit. <Larger View>


AMPK is regulated by the identity of the adenine ligand that is bound in the γ subunit. If it is ATP, the kinase will remain inactive. When AMP is bound, AMPK becomes a better substrate to be phosphorylated by an upstream kinase, such as AMPK kinase (Hawley et al., 1995). As a result, AMPK responds to the ratio of AMP and ATP, rather than just the concentration of either ligand alone (Hardie and Carling, 1997). Once activated, AMPK will utilize the phosphorylated Thr-172 to deactivate the enzymes in energy consuming mechanisms (Hardie and Carling, 1997) and initiate energy producing mechanisms through fatty acid oxidation (Chen et al., 2000), glucose transport (Xi et al., 2002), stimulating food intake (Anderson et al., 2004) and other energy producing mechanisms. This results in the deactivation of fatty acid, triglyceride, and cholesterol synthesis (Kemp et al., 1999). The combination of the two counteracting sets of mechanisms is the property of the global influence of AMPK.

Discussion
AMP-activated protein kinase has a very significant role in the proliferation of the cell. AMPK responds to the increase of the ratio of AMP/ATP, which occurs during times of high energy consumption (Hardie and Carling, 1997). AMP binds to the γ subunit of the Bateman regions and activates the protein. The binding of the ligand causes a conformational change that is sensed in the α subunit, causing the catalytic Thr-172 to become phosphorylated (Hamilton et al., 2002). The kinase then transmits the phosphate group directly onto an enzyme of energy consuming anabolic pathways, such as protein synthesis, essentially shutting it down. The phosphorylation of AMPK is facilitated by the binding of AMP and also by other kinases of the same family (Moore et al., 1991).
During unstressed periods in the cell's life, a supply of glycogen can bind to the β subunit. This will position AMPK to a phosphatase that removes the phosphate group off Thr-172, preventing it from shutting down anabolic pathways. It is also during this time that ATP can competitively bind to the same site as AMP and prevent its activation (Townley et al., 2007).
In order for AMPK to stimulate the production of ATP, glucose must be transported into the cell. AMPK signals for the increase in the translocation of the glucose transporter protein GLUT4 to the plasma membrane (Kurth-Kraczek et al., 1999). This function is similar to that of insulin (Sano et al., 2003), which makes AMPK an interest in the study of diabetes. Recent studies have indicated a link between the function of anti-diabetic drugs and AMPK (Hawley et al., 2002). The diabetic drug, metformin, is in current use to treat diabetes functions by the partial activation of AMPK (Fryer et al., 2002). Therefore, AMPK can be a target for new drugs to treat diabetes by inducing the uptake of glucose by cells. One possibility is to create an AMP analogue that has a higher affinity for AMPK than AMP. Since AMP is also involved in other mechanisms, the presence of the AMP analogue signals cellular stress and increase the uptake of glucose.
AMPK deactivates energy consuming pathways by directly phosphorylating the enzymes involved in the synthesis of biomolecules (Hardie and Carling, 1997). In addition, AMPK down regulates genes involved in biosynthetic pathways by phosphorylating transcription factors and co-activators (Hardie, 2007).
One the many proteins that are affect by AMPK is the transcription factor p53. This protein functions as a checkpoint in the cell cycle to control the progression of the cell. When phosphorylated, AMPK in turn phosphorylates p53 on Ser15 and is deactivated (Imamura et al., 2001). This signal conveys the message that the cell does not contain sufficient energy to continue on with the cell cycle and halts progression (Jones et al., 2005). This discovery of cell growth inhibition is of interest in cancer cells. Drugs that promote the function of AMPK may be effective in the treatment of cancer- providing patients and health care providers more time to combat the cancer by slowing down the rate of growth. With a softened growth rate, the formation of malignant tumors can be slowed or may even decline in occurrence.
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