AJMB  Vol.5 No.3 , July 2015
AMPK Subcellular Localisation in Dictyostelium discoideum
The Dictyostelium discoideum AMP-activated protein kinase (AMPK) snfA subcellular localization was studied in AX2 and stable HPF strains by use of AMPK antipeptide antibody and goat anti-rabbit Alexa-Flour 488-conjugated IgG antibody. The AMPK exhibited cytosolic localization patterns and uniform focalised concentrations in wild type and the strains alike. Constitutive activation and attenuation of the α subunit expression did not affect subcellular distribution of AMPK. However, snfA expression was more intense in strains in which AMPK was constitutively active compared with the AX2 but lesser in attenuation strains. The localisation of the snfA reinforced the putative standing that it had a plethora of cytoplasmic functions. Moreover, the oxidative cellular function would require a ubiquitous system and might coordinately regulate responses to metabolic requirements. Furthermore, the developmental phases of the life cycle would support the cytosolic localization; and since organelles were potentially reorganized or removed entirely during the transition from vegetative living to fruiting body morphology. This study provided insight into the subcellular distribution of AMPK in Dictyostelium discoideum. We demonstrated that AMPK localization was steady in AX2 and derived strains whether constitutively active or anti-sense inhibited depicting extreme genetic states.

Cite this paper
Bokko, P. , Ahmed, A. and Fisher, P. (2015) AMPK Subcellular Localisation in Dictyostelium discoideum. American Journal of Molecular Biology, 5, 105-116. doi: 10.4236/ajmb.2015.53009.

[1]   Hardie, D.G. and Hawley, S.A. (2001) AMP-Activated Protein Kinase: The Energy Charge Hypothesis Revisited. Bio-Essays, 23, 1112-1119.

[2]   Hardie, D.G. (2007) AMP-Activated/SNF1 Protein Kinases: Conserved Guardians of Cellular Energy. Nature Reviews in Molecular Cell Biology, 8, 774-785.

[3]   Rutter, G., Silva, A., Xavier, G. and Leclerc, I. (2003) Roles of 5’-AMP-Activated Protein Kinase (AMPK) in Mammalian Glucose Homeostasis. Biochemical Journal, 375, 1-16.

[4]   Towler, M.C. and Hardie, D.G. (2007) AMP-Activated Protein Kinase in Metabolic Control and Insulin Signaling. Circulation Research, 100, 328-341.

[5]   Viollet, B., Foretz, M., Guigas, B., Horman, S., Dentin, R., Bertrand, L., Hue, L. and Andreelli, F. (2006) Activation of AMP-Activated Protein Kinase in the Liver: A New Strategy for the Management of Metabolic Hepatic Disorders. Journal of Physiology, 574, 41-53.

[6]   Eichinger, L., Pachebat, J.A., Glockner, G., Rajandream, M.A., Sucgang, R., Berriman, M., Song, J., Olsen, R., Szafranski, K., Xu, Q., Tunggal, B., Kummerfeld, S., Madera, M., Konfortov, B.A., Rivero, F., Bankier, A.T., Lehmann, R., HamLin, N., Davies, R., Gaudet, P., Fey, P., Pilcher, K., Chen, G., Saunders, D., Sodergren, E., Davis, P., Kerhornou, A., Nie, X., Hall, N., Anjard, C., Hemphill, L., Bason, N., Farbrother, P., Desany, B., Just, E., Morio, T., Rost, R., Churcher, C., Cooper, J., Haydock, S., van Driessche, N., Cronin, A., Goodhead, I., Muzny, D., Mourier, T., Pain, A., Lu, M., Harper, D., Lindsay, R., Hauser, H., James, K., Quiles, M., Madan Babu, M., Saito, T., Buchrieser, C., Wardroper, A., Felder, M., Thangavelu, M., Johnson, D., Knights, A., Loulseged, H., Mungall, K., Oliver, K., Price, C., Quail, M.A., Urushihara, H., Hernandez, J., Rabbinowitsch, E., Steffen, D., Sanders, M., Ma, J., Kohara, Y., Sharp, S., Simmonds, M., Spiegler, S., Tivey, A., Sugano, S., White, B., Walker, D., Woodward, J., Winckler, T., Tanaka, Y., Shaulsky, G., Schleicher, M., Weinstock, G., Rosenthal, A., Cox, E.C., Chisholm, R.L., Gibbs, R., Loomis, W.F., Platzer, M., Kay, R.R., Williams, J., Dear, P.H., Noegel, A.A., Barrell, B. and Kuspa, A. (2005) The Genome of the Social Amoeba Dictyostelium discoideum. Nature, 435, 43-57.

[7]   Bokko, B.P., Said, F., Bandala, E., Ahmed, A., Annesely, S.J., Huang, X., Khurana, T., Kimmel, A.R. and Fisher, P.R. (2007) Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMPK. Molecular Biology of the Cell, 18, 1874-1886.

[8]   Sung, S., Bisson, S., Koehler, S. and Podgorski, G.J. (1999) The Dictyostelium snf1/AMP-Activated Kinase. Unpublished, EMBL/GenBank ID AF118151.

[9]   Bokko, B.P., Ahmed, A.U. and Fisher, P.R. (2014) Constitutive Activation of AMP-Activated Protein Kinase (AMPK) Propel Mitochondrial Biogenesis. Journal of Cell Biology and Genetics, 4, 15-26.

[10]   Watts, D.J. and Ashworth, J.M. (1970) Growth of Myxameobae of the Cellular Slime Mould Dictyostelium discoideum in Axenic Culture. Biochemical Journal, 119, 171-174.

[11]   Nellen, W., Silan, C. and Firtel, R.A. (1984) DNA-Mediated Transformation in Dictyostelium discoideum: Regulated Expression of an Actin Gene Fusion. Molecular Cell Biology, 4, 2890-2898.

[12]   Fey, P., Compton, K. and Cox, E. (1995) Green Fluorescent Protein Production in the Cellular Slime Molds Polysphondylium pallidum and Dictyostelium. Gene, 165, 127-130.

[13]   Levi, S., Polyakov, M. and Egelhoff, T.T. (2000) Green Fluorescent Protein and Epitope Tag Fusion Vectors for Dictyostelium discoideum. Plasmid, 44, 231-238.

[14]   Damer, C.K., Bayeva, M., Hahn, E.S., Rivera, J. and Socec, C.I. (2005) Copine A, a Calcium-Dependent Membrane-Binding Protein, Transiently Localizes to the Plasma Membrane and Intracellular Vacuoles in Dictyostelium. BMC Cell Biology, 6, 46.

[15]   Kirsten, J.H., Xiong, Y., Davis, C.T. and Singleton, C.K. (2008) Subcellular Localization of Ammonium Transporters in Dictyostelium discoideum. BMC Cell Biology, 9, 71.

[16]   Witke, W., Nellen, W. and Noegel, A. (1987) Homologous Re-combination in the Dictyostelium α-Actin Gene Leads to an Altered mRNA and Lack of the Protein. EMBO Journal, 6, 4143-4148.

[17]   Liu, T., Mirschberger, C., Chooback, L., Arana, Q., Dal Sacco, Z., MacWilliams, H. and Clarke, M. (2002) Altered Expression of the 100 kDa Subunit of the Dictyostelium Vacuolar Proton Pump Impairs Enzyme Assembly, Endocytic Function and Cytosolic pH Regulation. Journal of Cell Science, 115, 1907-1918.

[18]   Winder, W.W. and Hardie, D.G. (1999) AMP-Activated Protein Kinase, a Metabolic Master Switch: Possible Roles in Type 2 Diabetes. American Journal of Physiology, 277, E1-E10.

[19]   Suzuki, A., Okamoto, S., Lee, S., Saito, K., Shiuchi, T. and Minokoshi, Y. (2007) Leptin Stimulates Fatty Acid Oxidation and Peroxisome Proliferator-Activated Receptor Alpha Gene Expression in Mouse C2C12 Myoblasts by Changing the Subcellular Localization of the Alpha2 form of AMP-Activated Protein Kinase. Molecular Cell Biology, 27, 4317-4327.

[20]   Wilson, W.A., Hawley, S.A. and Hardie, D.G. (1996) The Mechanism of Glucose Repression/Derepression in Yeast: SNF1 Protein Kinase Is Activated by Phosphorylation under Derepressing Conditions, and This Correlates with a High AMP:ATP Ratio. Current Biology, 6, 1426-1434.

[21]   Carlson, M. (1999) Glucose Repression in Yeast. Current Opinions in Microbiology, 2, 202-207.

[22]   Pan, D.A. and Hardie, D.G. (2002) A Homologue of AMP-Activated Protein Kinase in Drosophila melanogaster Is Sensitive to AMP and Is Activated by ATP Depletion. Biochemistry Journal, 367, 179-186.

[23]   Carling, D. (2004) The AMP-Activated Protein Kinase Cascade—A Unifying System for Energy Control. Trends in Biochemical Science, 29, 18-24.

[24]   Yang, W., Hong, Y.H., Shen, X., Frankowski, C., Camp, H.S. and Leff, T. (2001) Regulation of Transcription by AMP-Activated Protein Kinase. Journal of Biological Chemistry, 276, 38341-38344.

[25]   Eberhardt, W., Doller, A., Akool, E. and Pfeilschifter, J. (2007) Modulation of mRNA Stability as a Novel Therapeutic Approach. Pharmacology and Therapeutics, 114, 56-73.

[26]   Lee, J.H., Koh, H., Kim, M., Kim, Y., Lee, S.Y., Lee, S., Shong, J., Kim, J., Chung, J. and Karess, R.E. (2007) Energy-Dependent Regulation of Cell Structure by AMP-Activated Protein Kinase. Nature, 447, 1017-1021.

[27]   Kodiha, M., Rassi, J.G., Brown, C.M. and Stochaj, U. (2007) Localization of AMP Kinase Is Regulated by Stress, Cell Density, and Signaling through the MEK→ERK1/2 Pathway. American Journal of Physiology—Cell Physiology, 293, C1427-C1436.

[28]   Salt, I.P., Celler, J.W., Hawley, S.A., Prescott, A., Woods, A., Carling, D. and Hardie, D.G. (1998) AMP-Activated Protein Kinase—Greater AMP Dependence, and Preferential Nuclear Localization, of Complexes Containing the α2 Isoform. Biochemical Journal, 334, 177-187.

[29]   Kodiha, M., Chu, A., Matusiewicz, N. and Stochaj, U. (2004) Multiple Mechanisms Promote the Inhibition of Classical Nuclear Import upon Exposure to Severe Oxidative Stress. Cell Death and Differentiation, 11, 862-874.

[30]   Turnley, A.M., Stapleton, D., Mann, R.J, Witters, L.A., Kemp, B.E. and Bartlett, P.F. (1999) Cellular Distribution and Developmental Expression of AMP-Activated Protein Kinase Isoforms in Mouse Central Nervous System. Journal of Neurochemistry, 72, 1707-1716.

[31]   Pinter, K., Grignani, R.T., Watkins, H. and Redwood, C. (2013) Localisation of AMPK γ Subunits in Cardiac and Skeletal Muscles. Journal of Muscle Research and Cell Motility, 34, 369-378.

[32]   Crute, B.E., Seefeld, K., Gamble, J., Kemp, B.E. and Witters, L.A. (1998) Functional Domains of the α1 Catalytic Subunit of the AMP-Activated Protein Kinase. Journal of Biological Chemistry, 273, 35347-35354.

[33]   Hinas, A. and Söderbom, F. (2007) Treasure Hunt in an Amoeba: Non-Coding RNAs in Dictyostelium discoideum. Current Genetics, 51, 141-159.

[34]   Creighton, J. (2011) Targeting Therapeutic Effects: Subcellular Location Matters. Focus on “Pharmacological AMP-Kinase Activators Have Compartment-Specific Effects on Cell Physiology”. American Journal of Physiology Cell Physiology, 301, C1293-C1295.

[35]   Kim, M. and Tian, R. (2011) Targeting AMPK for Cardiac Protection: Opportunities and Challenges. Journal of Molecular and Cellular Cardiology, 51, 548-553.