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Glucose deprivation activates a cAMP-independent protein kinase from Trypanosoma equiperdum

Published online by Cambridge University Press:  13 November 2018

Alberto Guevara ,
Cristina Lugo ,
Alejandro J. Montilla ,
Nelson A. Araujo ,
Maritza Calabokis  and
José Bubis Open the ORCID record for José Bubis [Opens in a new window]
Alberto Guevara
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Cristina Lugo
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Alejandro J. Montilla
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela Postgrado en Ciencias Biológicas, Universidad Simón Bolívar, Caracas, Venezuela
Nelson A. Araujo
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
Maritza Calabokis
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
José Bubis*
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
*
Author for correspondence: José Bubis, E-mail: jbubis@usb.ve
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Abstract

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07–51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22–1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm, respectively. The parasite enzyme uses ATP and Mg2+ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β,γ-imidoadenosine 5′-triphosphate and 5′-[p-(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn2+, Mn2+, Co2+, Cu2+, Ca2+ and Fe2+. Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI-α. This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum.

Keywords

Dourineglucose deprivationkemptide kinase activityprotein kinasesprotein phosphorylationsignal transductionTrypanosoma equiperdum
Type
Research Article
Information
Parasitology , Volume 146 , Issue 5 , April 2019 , pp. 643 - 652
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Copyright
Copyright © Cambridge University Press 2018 

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References

Araujo, NA, Guevara, A, Lorenzo, MA, Calabokis, M and Bubis, J (2016) Fluram-kemptide-Lys8 non-radioactive assay for protein kinase A. The Protein Journal 35, 247–255.Google Scholar
Banerjee, C and Sarkar, D (1992) Isolation and characterization of a cyclic nucleotide-independent protein kinase from Leishmania donovani. Molecular and Biochemical Parasitology 52, 195–206.Google Scholar
Bao, Y, Weiss, LM, Braunstein, VL and Huang, H (2008) Role of protein kinase A in Trypanosoma cruzi. Infection and Immunity 76, 4757–4763.Google Scholar
Bloomfield, V, Dalton, WO and van Holde, KE (1967) Frictional coefficients of multisubunit structures. I. Theory. Biopolymers 5, 135–148.Google Scholar
Brun, R, Hecker, H and Lun, ZR (1998) Trypanosoma evansi and T. equiperdum: distribution, biology, treatment and phylogenetic relationship (a review). Veterinary Parasitology 79, 95–107.Google Scholar
Bubis, J and Taylor, SS (1987) Limited proteolysis alters the photoaffinity labeling of adenosine 3′,5′-monophosphate dependent protein kinase II with 8-azidoadenosine 3′,5′-monophosphate. Biochemistry 26, 5997–6004.Google Scholar
Bubis, J, Martínez, JC, Calabokis, M, Ferreira, J, Sanz-Rodríguez, CE, Navas, V, Escalona, JL, Guo, Y and Taylor, SS (2018) The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides. Biochimie 146, 166–180.Google Scholar
Calabokis, M, González, Y, Merchán, A, Escalona, JL, Araujo, NA, Sanz-Rodríguez, CE, Cywiak, C, Spencer, LM, Martínez, JC and Bubis, J (2016) Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate. Journal of Immunoassay and Immunochemistry 37, 485–514.Google Scholar
Darling, PJ, Holt, JM and Ackers, GK (2000) Coupled energetics of λ cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers. Journal of Molecular Biology 302, 625–638.Google Scholar
Dulin, NO, Niu, J, Browning, DD, Ye, RD and Voyno-Yasenetskaya, T (2001) Cyclic AMP-independent activation of protein kinase A by vasoactive peptides. Journal of Biological Chemistry 276, 20827–20830.Google Scholar
Erickson, HP (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biological Procedures Online 11, 32–51.Google Scholar
Ferraris, JD, Persaud, P, Williams, CK, Chen, Y and Burg, MB (2002) cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/osmotic response element-binding protein. Proceedings of the National Academy of Sciences of the USA 99, 16800–16805.Google Scholar
First, EA, Bubis, J and Taylor, SS (1988) Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinases. Identification of a specific interchain disulfide bond. Journal of Biological Chemistry 263, 5177–5182.Google Scholar
Genestra, M, Cysne-Finkelstein, L and Leon, L (2004) Protein kinase A activity is associated with metacyclogenesis in Leishmania amazonensis. Cell Biochemistry and Function 22, 315–320.Google Scholar
Gizaw, Y, Megersa, M and Fayera, T (2017) Dourine: a neglected disease of equids. Tropical Animal Health and Production 49, 887–897.Google Scholar
Hébert, L, Moumen, B, Madeline, A, Steinbiss, S, Lakhdar, L, Van Reet, N, Büscher, P, Laugier, C, Cauchard, J and Petry, S (2017) First draft genome sequence of the dourine causative agent: Trypanosoma equiperdum strain OVI. Journal of Genomics 5, 1–3.Google Scholar
Johansson, E, Majka, J and Burgers, PM (2001) Structure of DNA polymerase δ from Saccharomyces cerevisiae. Journal of Biological Chemistry 276, 43824–43828.Google Scholar
Kemp, BE (1980) Phosphorylation of acyl and dansyl derivatives of the peptide Leu-Arg–Arg-Ala–Ser-Leu–Gly by the cAMP-dependent protein kinase. Journal of Biological Chemistry 255, 2914–2918.Google Scholar
Kemp, BE, Bylund, DB, Huang, TS and Krebs, EG (1975) Substrate specificity of the cyclic AMP-dependent protein kinase. Proceedings of the National Academy of Sciences of the USA 72, 3448–3452.Google Scholar
Kohr, MJ, Traynham, CJ, Roof, SR, Davis, JP and Ziolo, MT (2010) cAMP-independent activation of protein kinase A by the peroxynitrite generator SIN-1 elicits positive inotropic effects in cardiomyocytes. Journal of Molecular and Cellular Cardiology 48, 645–648.Google Scholar
Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.Google Scholar
Lanham, SM and Godfrey, DG (1970) Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology 28, 521–534.Google Scholar
Laurent, TC and Killander, J (1964) A theory of gel filtration and its experimental verification. Journal of Chromatography A 14, 317–330.Google Scholar
Lineweaver, H and Burk, D (1934) The determination of enzyme dissociation constants. Journal of the American Chemical Society 56, 658–666.Google Scholar
Lutz, MP, Pinon, DI and Miller, LJ (1994) A nonradioactive fluorescent gel-shift assay for the analysis of protein phosphatase and kinase activities toward protein-specific peptide substrates. Analytical Biochemistry 220, 268–274.Google Scholar
Luzi, NM, Lyons, CE, Peterson, DL and Ellis, KC (2017) Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate. Analytical Biochemistry 532, 45–52.Google Scholar
MacAla, LJ, Hayslett, JP and Smallwood, JI (1998) Measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide. Kidney International 54, 1746–1750.Google Scholar
Malki-Feldman, L and Jaffe, CL (2009) Leishmania major: effect of protein kinase A and phosphodiesterase activity on infectivity and proliferation of promastigotes. Experimental Parasitology 123, 39–44.Google Scholar
Maller, JL, Kemp, BE and Krebs, EG (1978) In vivo phosphorylation of a synthetic peptide substrate of cyclic AMP-dependent protein kinase. Proceedings of the National Academy of Sciences of the USA 75, 248–251.Google Scholar
Martin, RG and Ames, BN (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. Journal of Biological Chemistry 236, 1372–1379.Google Scholar
Mena-Ulecia, K, Vergara-Jaque, A, Poblete, H, Tiznado, W and Caballero, J (2014) Study of the affinity between the protein kinase PKA and peptide substrates derived from kemptide using molecular dynamics simulations and MM/GBSA. PLoS ONE 9, e109639.Google Scholar
Moore, MJ, Adams, JA and Taylor, SS (2003) Structural basis for peptide binding in protein kinase A. Role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop. Journal of Biological Chemistry 278, 10613–10618.Google Scholar
Naula, C, Parsons, M and Mottram, JC (2005) Protein kinases as drug targets in trypanosomes and Leishmania. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics 1754, 151–159.Google Scholar
Nelson, NC and Taylor, SS (1981) Differential labeling and identification of the cysteine-containing tryptic peptides of catalytic subunit from porcine heart cAMP-dependent protein kinase. Journal of Biological Chemistry 256, 3743–3750.Google Scholar
Nelson, NC and Taylor, SS (1983) Selective protection of sulfydryl groups in cAMP-dependent protein kinase II. Journal of Biological Chemistry 258, 10981–10987.Google Scholar
Ochatt, CM, Ulloa, RM, Torres, HN and Téllez-Iñón, MT (1993) Characterization of the catalytic subunit of Trypanosoma cruzi cyclic AMP-dependent protein kinase. Molecular and Biochemical Parasitology 57, 73–81.Google Scholar
Parsons, M, Worthey, EA, Ward, PN and Mottram, JC (2005) Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. BMC Genomics 6, 127.Google Scholar
Rubin, CS, Rangel-Aldao, R, Sarkar, D, Erlichman, J and Fleischer, N (1979) Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Physicochemical and immunological studies on bovine cerebral cortex protein kinases. Journal of Biological Chemistry 254, 3797–3805.Google Scholar
Sánchez, E, Perrone, T, Recchimuzzi, G, Cardozo, I, Biteau, N, Aso, PM, Mijares, A, Baltz, T, Berthier, D, Balzano-Nogueira, L and Gonzatti, MI (2015) Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes. Parasites & Vectors 8, 536.Google Scholar
Siegel, LM and Monty, KJ (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochimica et Biophysica Acta (BBA) – Biophysics Including Photosynthesis 112, 346–362.Google Scholar
Smith, CM, Radzio-Andzelm, E, Madhusudan, M, Akamine, P and Taylor, SS (1999) The catalytic subunit of cAMP-dependent protein kinase: prototype for an extended network of communication. Progress in Biophysics & Molecular Biology 71, 313–341.Google Scholar
Stevens, JR and Brisse, S (2004) Systematics of trypanosomes of medical and veterinary importance. In Maudlin, I, Holmes, PH and Miles, MA (ed.) The Trypanosomiases, Wallingford, UK: CABI Publishing, CAB International, pp. 1–23.Google Scholar
Sugden, PH, Holladay, LA, Reimann, EM and Corbin, JD (1976) Purification and characterization of the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase from bovine liver. Biochemical Journal 159, 409–422.Google Scholar
Taylor, SS (1989) cAMP-dependent protein kinase. Model for an enzyme family. Journal of Biological Chemistry 264, 8443–8446.Google Scholar
Taylor, SS and Stafford, PH (1978) Characterization of adenosine 3′:5′-monophosphate-dependent protein kinase and its dissociated subunits from porcine skeletal muscle. Journal of Biological Chemistry 253, 2284–2287.Google Scholar
Tielens, AG and van Hellemond, JJ (2009) Surprising variety in energy metabolism within trypanosomatidae. Trends in Parasitology 25, 482–490.Google Scholar
Towbin, H, Staehelin, T and Gordon, J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the USA 76, 4350–4354.Google Scholar
Yang, H, Lee, CJ, Zhang, L, Sans, MD and Simeone, DM (2008) Regulation of transforming growth factor β-induced responses by protein kinase A in pancreatic acinar cells. American Journal of Physiology – Gastrointestinal and Liver Physiology 295, G170–G178.Google Scholar
Zhang, L, Duan, CJ, Binkley, C, Li, G, Uhler, MD, Logsdon, CD and Simeone, DM (2004) A transforming growth factor β-induced Smad3/Smad4 complex directly activates protein kinase A. Molecular and Cellular Biology 24, 2169–2180.Google Scholar
Zhong, H, SuYang, H, Erdjument-Bromage, H, Tempst, P and Ghosh, S (1997) The transcriptional activity of NF-κB is regulated by the IκB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89, 413–424.Google Scholar
Zoller, MJ, Kerlavage, AR and Taylor, SS (1979) Structural comparisons of cAMP-dependent protein kinases I and II from porcine skeletal muscle. Journal of Biological Chemistry 254, 2408–2412.Google Scholar
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