Characterization of a PKA-like kinase from Trypanosoma brucei

Kramer, Susanne (2005): Characterization of a PKA-like kinase from Trypanosoma brucei. Dissertation, LMU München: Fakultät für Biologie

Abstract

The protozoan parasite Trypanosoma brucei causes human sleeping sickness and Nagana in domestic animals and depends on the tsetse fly for dissemination. The complex T. brucei life cycle requires differentiation from the dividing long slender forms via the cell cycle arrested short stumpy forms (both in the mammalian bloodstream) to the procyclic forms of the insect vector. The signaling pathways that regulate differentiation are unknown but there is evidence for an involvement of cAMP. In search of the putative cAMP receptor, three catalytic and one regulatory PKA-like subunits have been previously cloned from T. brucei. The catalytic subunits possess all features of a classical PKA in terms of inhibitor and substrate specificity. It was shown that each of the catalytic PKAlike subunits binds to the regulatory subunit to form a dimeric PKA-like holoenzyme. Most surprisingly, we found that T. brucei PKA-like kinase, despite of its apparent similarities to a PKA, was not activated but instead inhibited by cAMP. Out of several other cyclic nucleotides that were tested on their effects on PKA-like kinase, only cGMP was able to activate the kinase, but in millimolar and thus most likely unphysiological concentrations. Assuming that the activation of PKA-like kinase might depend on its native, subcellular environment, an in vivo kinase assay was established in this work. It is based on the immunological detection of the phosphorylated form of the PKA reporter substrate VASP that was transgenically expressed in T. brucei. Interestingly, results from the in vivo assay did confirm the in vitro data, suggesting that T. brucei PKA-like kinase is in fact inhibited rather than activated by cAMP. Even though these findings challenge the original assumption that T. brucei PKA-like kinase transmits the differentation signal mimicked by cAMP antagonists, data from this work nevertheless provide evidence for an involvement of T. brucei PKA-like kinase in relaying extracellular cues. This is suggested from an increase in in vivo PKA activity in the presence of treatments that have either been shown to induce LS to SS differentiation (etazolate) or to participate in SS to PCF differentiation (cold shock, mild acid stress). In addition, in vivo PKA activity was stimulated with the PDE inhibitor dipyridamole and at hypoosmotic stress. In the context of a putative role for T. brucei PKA-like kinase in the regulation of differentiation, two of the catalytic isoforms (PKAC1 and PKAC2) were of particular interest. We found significant life cycle stage dependent differences in protein expression between the two almost identical isoforms. PKAC1 was nearly exclusively present in bloodstream forms and PKAC2 in procyclic cells. In addition, PKAC1, but not PKAC2 carries a phosphorylation that is restricted to the SS stage. This phosphorylation was mapped to the C-terminal threonine 324 by mass spectrometry. The functions of these life cycle stage dependent differences between PKAC1 and PKAC2 remain unknown. Reverse genetics did not reveal any functional differences between the isoforms, in fact, PKAC1 was even able to complement PKAC2 in procyclic PKAC2 knock-out cells. Results from several reverse genetic experiments indicate that T. brucei PKA-like kinase plays an important role in cell division. Depletion of either PKA-like subunit leads to a cytokinesis block. Depletion of the regulatory PKA-like subunit additionally results in altered basal body segregation. Given that 1) both cytokinesis and basal body movement had been previously suggested to be regulated by the trypanosomal flagellum (Kohl et al., 2003) and 2) the flagellum hosts T. brucei PKA-like kinase (C. Krumbholz, this lab) we propose that trypanosomal flagella act as signaling compartments for coordination of cell division.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Trypanosoma brucei PKA cAMP cGMP
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. April 2005
• 1. Bericht­erstatter:in: Boshart, Michael