Characterization of a PKA-like kinase from Trypanosoma brucei

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.