Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007al., 2006). Based on the literature reviewed, we hypothesize that a cell’s entry into a quiescent statecauses changes in the dynamics of mitochondria and a subsequent change in the morphology of themitochondrial network. In the final part of our project we will investigate whether such changes indeed takeplace and whether they are actually related to the energy-regulating function of AMPK.Besides its essential role in energy homeostasis in the cell, AMPK has been suggested to be involved invarious mitochondrial diseases. Additionally, because of the link between AMPK and cell cycle arrest andapoptosis, results of this research could be valuable for cancer research. Moreover, both mitochondrialmorphology and metabolism are usually altered in cancerous cells, so our insights could help identifyingspecific traits in tumor cells. Lastly, AMPK has also been suggested to play a role in diabetes andischemic reperfusion injury (Bokko et al., 2007).HypothesesOur main hypothesis, summarizing the model described above, is:AMPK and its effects on energy production mechanisms mediate energy-dependent cell cycleprogression in G1 in mammalian cells.We will look at three different aspects of AMPK in our research. First, we will study the effects of activeAMPK on glycolysis, oxidative phosphorylation and fatty acid oxidation. Secondly, we will research therelation between AMPK activation and cell cycle arrest and apoptosis. Lastly, we will look into whethermorphological changes in the mitochondrial network can be induced by AMPK when it causes cell cyclearrest.Hypothesis 1AMPK activation mediates energy production to maintain the cell’s energy balance under acutemetabolic stress. It will do this by influencing the following Energy generating processes: glycolysis,Oxidative phosphorylation (Oxphos) and Fatty Acid Oxidation (FAO).Hypothesis 2AMPK induces cell cycle arrest in response to glucose depletion at multiple points in G1. ProlongedAMPK activation induces apoptosis in early G1 but not in late G1.Hypothesis 3AMPK-induced cell cycle arrest can cause morphological changes in the mitochondrial network.BackgroundAMPK, ATP and MetabolismAdenosine-MonoPhosphate-activated protein Kinase (AMPK) is a protein that plays a regulatory role inthe cell cycle and in the cell’s energy metabolism. Its activation depends on the ATP:AMP ratio in the cell.AMPK is inactive when ATP levels are normal, and active when ATP levels are low (Hardie et al., 2005).When ATP is low the cellular enzyme adenylate kinase converts two molecules of ADP to ATP and AMPas illustrated by the following equation: ADP + ADP ATP + AMP. The action of the enzyme adenylatekinase makes cellular AMP levels dependent on ATP levels (Berg et al., 2006).Since the produced ATP is immediately used by the cell, AMP ratios increase rapidly when a cellsuffers from energy shortage, making AMPK a sensitive sensor of cellular energy levels. AMPK, is aheterotrimeric enzyme which consists of an α1 or α2 catalytic subunit and two regulatory subunits: β1 orβ2 in combination with either γ1, γ2 or γ3 (Tzatsos et al., 2007). The γ-subunits allosterically bind AMP aswell as ATP. AMP, however, has a higher affinity for AMPK than ATP and is needed for AMPK activationvia phosphorylation. This is why AMP and not ATP levels determine AMPK activity in the cell. In addition,binding of AMP prevents dephosphorylation by phosphotases (Long et al., 2006). So far, two upstreamkinases have been shown to activate AMPK by phosphorylating a specific threonine residue (Thr-172): thetumor suppressor LKB-1 and calmodulin-dependent protein kinase kinase β, CaMKKβ, which responds toSCI 332 Advanced Molecular Cell Biology Research Proposal 38
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007changes in calcium levels. In addition, AMPK has been shown to be activated by 5-aminoimidazole-4-carboxamideriboside (AICAR) in LKB-1 deficient HeLa cells. Thus, it is possible to activate AMPK in anLKB-1 independent fashion. AICAR is phosphorylated and mimics AMP, thereby inducing its effect onAMPK without changes in the actual AMP:ATP ratio (Sun et al., 2007). Furthermore, AICAR has beenshown to be a direct activator of AMPK (Kaushik et al., 2001). The use of AICAR as an activator of AMPKexclusively is controversial and previous studies postulate that AICAR influences other pathways besidesAMPK (Woods et al., 2000). Therefore, we will use AICAR only in case constitutively active AMPK is notan option and after the specific effect of energy depletion on the systems under study has beendetermined.Activated AMPK has various effects. Firstly, cell cycle arrest is induced, a phenomenon that hasencouraged researches to name this specific activation of AMPK ‘the energy checkpoint’. Secondly,energy consuming processes are inhibited and energy producing mechanisms are up-regulated (seeFigure 2.3).Figure 2.3: The regulatory role of AMPK in cell metabolism (http://www.innovitaresearch.org).Cellular metabolism can be divided into anabolic and catabolic processes. In anabolic processes, such asprotein synthesis, ATP is used to fuel energy-demanding pathways that build essential molecules fromsmall units. Catabolic metabolism on the other hand, is the process of substrate uptake (oxygen, glucose,fatty acids, etc.) and their conversion into energy through a series of controlled oxidation and reductionreactions. These intracellular biochemical processes result in the production of ATP.Three main catabolic pathways are glycolysis, oxidative phosphorylation and fatty acid oxidation.Glycolysis takes place in the cytosol and is the conversion of glucose to pyruvate, which then can enterthe mitochondrial citric acid cycle. Oxidative phosphorylation is the process of cellular respiration,involving the formation of ATP from ADP and a phosphate group that takes place in the electron transportchain in the inner mitochondrial membrane. Lastly, fatty acid oxidation involves transport of fatty acids intothe mitochondria, and the formation of acyl-CoA with CoA. This is converted to acetyl-CoA, which caneventually enter the citric acid cycle.In general, AMPK is known to inhibit anabolic pathways and activate catabolic pathways uponincreased energy demand in certain differentiated cells. For example, AMPK is involved in glycolysis andfatty acid oxidation, but there is insufficient research indicating a direct relation between AMPK and therates of oxidative phosphorylation in the cell. In some cases, it is even suspected that Oxphos could beinhibited by AMPK activation (Hue et al., 2003), but so far this has only been confirmed via indirectpathways in tumor cells (Wu et al., 2007).Glycolysis, Oxidative Phosphorylation (OXPHOS) and AMPKThe conversion of glucose via pyruvate to lactate is the only way by which the cell can produce energywithout using oxygen. Oxidative phosphorylation further breaks down pyruvate and is a far more efficientATP source. However, certain tumor cells inhibit mitochondrial respiration and mainly glycolysis, despitethe presence of oxygen, an observation called the Warburg effect (Brand et al., 1997).AMPK is thought to function as an emergency signal that is activated upon metabolic stress. Its roleis to conserve ATP levels and in this way restore energy homeostasis (Hue et al., 2003). Glucose uptakeand glycolysis are increased when cells re-enter the cell cycle and start proliferating. This indicates theimportance of glucose as essential energy source for proliferating cells (Brand et al., 1997; Frauwirth andThompson, 2004). Why proliferating cells deal with increased energy requirements by upregulatingglycolysis despite the fact that it is a far less efficient ATP production system than the oxidation of glucose,is still unknown. The most common hypothesis is that this form of energy production protects the cell fromSCI 332 Advanced Molecular Cell Biology Research Proposal 39
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