The regulatory subunit of a cGMP-regulated protein kinase A of
Trypanosoma brucei
Tarek Shalaby, Matthias Liniger and Thomas Seebeck{
Institute of Cell Biology, University of Bern, Switzerland
This study reports the identification and characterization of
the regulatory subunit, TbRSU, of protein kinase A of the
parasitic protozoon Trypanosoma brucei. TbRSU is coded
for by a single copy gene. The protein contains an unusually
long N-terminal domain, the pseudosubstrate site involved
in binding and inactivation of the catalytic subunit, and two
C-terminally located, closely spaced cyclic nucleotide
binding domains. Immunoprecipitation of TbRSU copre-
cipitates a protein kinase activity with the characteristics of
protein kinase A: it phosphorylates a protein kinase specific
substrate, and it is strongly inhibited by a synthetic protein
kinase inhibitor peptide. Unexpectedly, this kinase activity
could not be stimulated by cAMP, but by cGMP only.
Binding studies with recombinant cyclic nucleotide binding
domains of TbRSU confirmed that both domains bind
cGMP with K
d
values in the lower micromolar range, and
that up to a 100-fold excess of cAMP does not compete with
cGMP binding.
Keywords: sleeping sickness; protein kinase A; African
trypanosomes; cyclic nucleotide signalling.
The concept of cellular signaling by cyclic AMP (cAMP)
has been maintained throughout evolution, from bacteria to
mammals. However, the only component of this signalling
pathway that has been strictly conserved is the second
messenger molecule itself, cAMP, while the enzymatic

Dictyostelium discoideum [12], or indirectly via extracellu-
lar conversion into adenosine and the subsequent activation
of adenosine receptors in the brain [13].
In mammalian systems, the most extensively studied
downstream effector of cAMP is the cAMP-regulated
protein kinase A (PKA) [14 –18]. According to the current
paradigm, PKA is an R
2
C
2
heterotetramer consisting of two
catalytic and two regulatory subunits. The regulatory
subunits contain a dimerization domain in their N-terminal
regions, followed by an autoinhibitor sequence that
resembles a PKA substrate. This region binds to the active
site of the catalytic subunit, inactivating it while it is in the
R
2
C
2
complex. The C-terminus of the regulatory subunit
contains two adjacent cAMP-binding domains. Domain A is
not accessible for cAMP in the R
2
C
2
complex. cAMP first
binds to domain B, triggering a conformational change that
renders domain A more accessible. The two cAMP-binding
domains are biochemically distinct, both in terms of binding

been identified [3,24], and one of these enzymes, GRESAG
4.4B, has been further characterized [25]. Also, several
cAMP-specific phosphodiesterases have recently been iden-
tified and characterized [26] (S. Kunz, P. Bern, A. Rascon,
S. H. Soderling and J. Beavo, personal communication;
A. Rascon and J. Beavo, personal communication,
University of Seattle, WA, USA). Little is currently known
about the biological role of cAMP signalling in these
organisms. A role for cAMP in the differentiation of long,
slender to short, stumpy forms in the bloodstream of the
mammalian host has been proposed [27]. PKA activity has
also been implicated in a mechanism by which T. brucei
can remove bound host antibody from its cell surface [28].
The enzyme itself has not yet been characterized in any of
the kinetoplastids, although previous work demonstrated the
presence of a PKA-like kinase activity in T. cruzi [29].
The current study describes the identification and
characterization of the regulatory subunit of trypanosomal
PKA (TbRSU). Many of the structural features are well
conserved between TbRSU and its mammalian counterparts.
Despite this overall similarity between mammalian and
trypanosomal regulatory subunits, the trypanosomal homo-
log binds cGMP rather than cAMP, and the trypanosomal
PKA is activated by cGMP, but not by cAMP. TbRSU thus
represents yet another facet in the amazing kaleidoscope of
cyclic nucleotide signalling.
MATERIALS AND METHODS
Materials
Enzymes were obtained from Roche Diagnostics (Rotkreuz,
Switzerland), and culture media were purchased from Difco.

. Cells were cultured in a 22– 24 8C incubator
with no extra CO
2
supplied. Cell viability was checked
using the Trypan Blue exclusion test and was routinely
found to be between 95 and 99%.
Transfection of S2 cells
S2 cells were prepared for transfection by seeding 3 Â 10
6
cells in 3 mL DES
TM
medium into a 35-mm Petri dish. The
culture was incubated at 24 8C until a cell density of
2–4 Â 10
6
mL
21
was reached (6– 16 h). Immediately
before transfection, the following two solutions were
prepared separately (per 35-mm dish). Tube A: 36 mL2
M
CaCl
2
and 19 mg vector DNA, in a final volume of 300 mL
H
2
O. Tube B: 300 mL50mM Hepes, pH 7.1, 1.5 mM
NaH
2
PO

phosphorylation sequence (GFP227-RRRRSII) at its
C-terminus was provided by K. Shokat, Princeton
University, NJ, USA [32]. The plasmid was transfected
into BL21DE, and positive colonies were identified by their
fluorescence under UV light. Liquid cultures were grown to
a D
595
of < 0.4 and were then induced for 4 h with 0.5 mM
isopropyl thio-b-D-galactoside. Cells were suspended in
1–2% of the original culture volume of ice-cold 50 m
M
sodium phosphate, pH 7.0, 300 mM NaCl, and were lysed
by sonication. The lysate was cleared by centrifugation for
20 min at 7000 g, and the recombinant protein was
adsorbed batchwise to Talonw immobilized-cobalt resin
(Invitrogen) and purified according to the manufacturer’s
protocol.
Immunoprecipitation
For the preparation of antibody-coated beads, protein
G–Sepharose beads (Amersham-Pharmacia) were washed
twice in NaCl/P
i
and then suspended as a 50% slurry in
100 m
M phosphate buffer, pH 8.2. Fifty-microliter aliquots
of this slurry were incubated for 1 –3 h at 4 8C in 500 mL
phosphate buffer containing the antibody to be coupled (rat
polyclonal antibody against TbRSU1 or a control polyclonal
rat antibody directed against an irrelevant protein). Beads
were then washed twice in 100 m

times with cold WBI buffer (0.5% NP 40, 0.05%
deoxycholate and 0.05% SDS in NaCl/P
i
, pH 7.5), twice
with cold WBII buffer (125 m
M Tris/HCl, pH 8.2, 500 mM
NaCl, 1 mM EDTA, 0.5% NP40), and finally once with
500 mL kinase buffer (15 m
M NaCl, 5 mM MgCl
2
,10mM
Hepes, pH 7.5).
Protein kinase assay
For assaying protein kinase activity in the immunoprecipi-
tates, the following reaction mix was prepared: 1 mL
[
32
P]gATP ( 5 mCi
:
mL
21
, 150 mM ATP), 4 mLof5Â kinase
buffer (75 m
M NaCl, 25 mM MgCl
2
,50mM Hepes,
pH 7.5), 1 mL kinase substrate (0.5–1 mg), further
additions as required, and H
2
O to a final volume of

pH 7.5, 5 m
M EDTA, 6 M urea. For renaturation, several
procedures were tried, all of which lead to soluble fusion
protein unable to bind to glutathione–Sepharose. Thus, the
fusion protein was purified by gel filtration on a Superdex
200 column, followed by gel electrophoresis. After blotting
the protein to nitrocellulose (Schleicher & Schuell BA 85),
the 50-kDa fusion protein band was excised, dissolved in
dimethylsulfoxide and used for immunization.
Expression of cNMP-binding domains in
Drosophila
S2
cells
For the expression of the cNMP-binding domains of
TbRSU1 in S2 cells, the respective gene fragments were
amplified and cloned into the pMT/V5-His B vector
(Invitrogen). In this vector, expression is regulated by a
metallothioneine promotor, and it allows induction of
expression by the addition of Cu
21
to the growth medium.
The recombinant proteins carry a V5 immunological tag and
a His
6
-tag at their C-termini, which allow for easy detection
and purification. The cNMP-binding domain A (amino acids
231–367) was amplified using primers Adom-F [5
0
-
TAT

cell
s
Cells were collected by centrifugation at 500 g for 5 min at
4 8C. The cell pellet was suspended in ice-cold lysis buffer
(50 mL per mL cell culture; 50 m
M Tris/HCl, pH 7.8,
150 m
M NaCl, 1% Nonidet P-40; Completew protease
inhibitor cocktail was added immediately before use). The
lysate was incubated on ice for 20–30 min, briefly
homogenized in a glass/Teflon homogenizer and finally
centrifuged at 7000 g for 20 min at 2 8C. To the cleared
supernatant, 1/10 volume of a 50% (v/v) suspension of
Talonw beads in NaCl/P
i
was added, and the suspension was
incubated on a rocking platform for 3 h at 4 8C. After
incubation, the suspension was poured into a small column
and was washed extensively with 50 m
M sodium phosphate
buffer, pH 7.0, 300 m
M NaCl. Recombinant protein was
finally eluted with four aliquots of 100 mL elution buffer (50
sodium phosphate, pH 7.0, 300 m
M NaCl, 150 mM
imidazole). The protein containing fractions were pooled,
aliquoted, snap-frozen and stored at 270 8C.
Cyclic nucleotide binding assays
Binding assays were performed in 5 m
M sodium phosphate,

M). Filters were rinsed three times
with 1 mL ice-cold buffer each, thoroughly dried and
counted in a toluene-based scintillator. Dissociation rate
constants were determined by overnight equilibration on ice
of the binding reaction containing 0.4 m
M [
3
H]cGMP. After
the addition of a 100-fold excess of unlabelled cGMP,
aliquots were withdrawn and processed for filtration at time
points between 0 and 30 min. All reactions were done in
triplicate. Binding parameters were determined by curve
fitting using the
PRISM software package of GraphPad Inc.,
San Diego, CA, USA.
RESULTS
Identification of TbRSU1
The DNA database of the T. brucei genome project was
searched for predicted proteins containing putative cAMP-
binding domains. This search resulted in a 600-bp DNA
sequence which was predicted to code for the C-terminal
fragment of a protein with high similarity to the regulatory
subunits of eukaryotic PKAs. From the retrieved sequence,
PCR primers were designed (see Materials and methods)
and were used to amplify the corresponding fragment from
genomic DNA of T. brucei. The resulting PCR fragment of
600 bp was cloned and verified by sequencing. It was then
used to hybridize genomic blots of T. brucei DNA in order
to establish the number of corresponding genes present in
the genome. When genomic DNA was digested with

sequenced. The sequence analysis demonstrated that this
fragment contained the entire open reading frame of the
TbRSU gene (Fig. 1B).
In parallel, a cDNA library of procyclic T. brucei was also
screened with the same PCR fragment, resulting in three
independent phages that all contained a 1500-bp cDNA
fragment. All three were sequenced and were shown to
contain a short 5
0
untranslated region, a complete open
reading frame of 1497 bp, and a 3
0
untranslated region
terminated by a polyA tract. Although all three cDNA
clones were terminated with this sequence, this polyA tract
probably does not represent the polyA tail of the mRNA
because a sequence of 12 adenosine residues following
T3566 was is present in a genomic clone of the T. brucei
genome project (accession number AQ 644384) that extends
beyond this region. The sequences of the open reading
frames of all three cDNAs were identical to that obtained
from the genomic fragment. Upstream of the TbRSU gene,
the 3
0
end of an open reading frame was identified
(nucleotides 1–486 of the genomic fragment), which coded
for an unidentified protein termed TbTAS. The stop codon of
this open reading frame is separated from the start codon of
TbRSU by 1352 bp, including a pyrimidine-rich region.
Predicted amino-acid sequence of TbRSU

asparagine (Asn442) in domains A and B, respectively
(Fig. 3). Sequencing errors or allelic variation at these sites
are unlikely as identical sequences have been obtained by
independent sequencing of TbRSU from different trypano-
some strains (accession nos AQ638897 and AF182823). A
further difference between TbRSU and the PKA regulatory
subunits from other eukaryotes is seen in Val319. All
cAMP-binding domains of the regulatory subunits carry an
alanine residue at this position, while the closely related
cGMP-binding domains of protein kinase G always contain
either threonine or serine residues.
TbRSU
mRNA is more abundant in bloodstream forms
To explore if TbRSU is differentially expressed in the
different life stages of T. brucei, total RNA was extracted
both from bloodstream and from procyclic forms and was
analyzed by Northern blotting and hybridization. RNA
loading was quantitated by ethidium bromide staining to
visualize the ribosomal RNA before blotting the gel, and by
hybridization of the filter with a DNA probe specific for
b-tubulin [35]. The extent of hybridization of both probes
was quantitated using a PhosphorImager. TbRSU mRNA is
clearly detectable in both life cycle stages (Fig. 4A).
Fig. 3. Sequence comparison of cNMP-binding
domains of PKA regulatory subunits and of
protein kinase G. cNMP-binding domains A and
B are indicated by grey boxes. Amino-acid
numbering of the respective proteins is given. A:
Rattus norvegicus type I (accession number
P09456); B: D. melanogaster (P16905); C:

of 56 726.
Similarly to what was observed with TbRSU mRNA, the
TbRSU protein is much more abundant in bloodstream than
in procyclic forms.
Co-immunoprecipitation of PKA with TbRSU
Sequence analysis clearly established TbRSU as a homolog
of the type I regulatory subunits of mammalian PKA. In
order to functionally verify if TbRSU is associated with a
kinase in vivo, TbRSU was immunoprecipitated from whole
cells lysates using the polyclonal rat antibody. Immunopre-
cipitates were first analyzed by immunoblotting with a
polyclonal rabbit antibody against the catalytic subunit of
bovine PKA. In these experiments, the antibody detected a
protein with a M
r
of about 40 000, suggesting that the
catalytic subunit of trypanosomal PKA does in fact
coprecipitate with TbRSU. Inspection of the T. brucei
databases identified several DNA sequences that code for a
homolog of a PKA catalytic subunit. The catalytic activity
of the immunoprecipitates was then analysed by incubation
in kinase reaction buffer in the presence or absence of a
recombinant PKA-specific substrate [32] and 20 m
M cAMP.
Analysis of the reaction products by gel electrophoresis and
autoradiography (Fig. 5) demonstrated that the coimmuno-
precipitates did indeed contain a kinase activity which
phosphorylated the PKA-specific substrate. No phosphoryl-
ation of the substrate was observed when either no antibody,
or an irrelevant antibody, was used for immunoprecipitation,

cation); (D) immunoprecipitation with TbRSU antibody, but without
cell lysate. Beads were incubated for activity assays as follows: lanes 1:
kinase buffer; lanes 2: kinase buffer plus 20 m
M cAMP; lanes 3: kinase
buffer plus PKA-substrate; lanes 4: kinase buffer plus PKA-substrate
plus 20 m
M cAMP
6202 T. Shalaby et al. (Eur. J. Biochem. 268) q FEBS 2001
no effect or inhibited it. While the absence of stimulation by
cAMP was consistent in all of the many independent
experiments carried out (see also below), the inhibitory
effect of cAMP was observed in some, but not in all
experiments.
Phosphorylation of the PKA-specific substrate by the
immunoprecipitates was time-dependent, Mg
21
-dependent
and was quenched by an excess of unlabelled ATP (data
not shown). These results demonstrated that a protein
kinase activity was coimmunoprecipitated with TbRSU
under our conditions. Phosphorylation of the PKA-specific
substrate by this activity suggested that it represented PKA.
This was further corroborated by the observation that
the co-immunoprecipitating kinase activity was inhibited
by the highly PKA-specific peptide inhibitor PKI [36]
(Fig. 6).
PKA activity is stimulated by cGMP, but not by cAMP
When kinase activity of TbRSU immunoprecipitates was
assayed in the presence or absence of 20 m
M cAMP, no

in much less protein (all insoluble). Expression of domain A
alone proved impossible, despite much effort, in agreement
with earlier observations that this domain is highly toxic for
E. coli [37]. Thus, domains A and B were expressed
individually in the Drosophila cell line S2, under the control
of a Cu
21
-inducible metallothionein promoter. Similarly to
what was observed in E. coli, domain B was well expressed,
while domain A again resulted in very poor cell growth and
in low amounts of recombinant protein. The individual
domains A and B were purified by cobalt-affinity
chromatography, and were assayed for cGMP binding
Fig. 6. PKI inhibits the activity of coimmunoprecipitating kinase.
Immunoprecipitates were incubated for 10 min under phosphorylation
conditions with PKA substrate in the presence or absence of PKI
inhibitor peptide (10 mg per 30 mL reaction mix). (A) autoradiogram of
PKA substrate; (B) Coomassie-stained PKA substrate; (C) Phosphor-
Imager analysis of the gel shown in (A).
Fig. 7. The kinase activity which coimmunoprecipitates with
TbRSU is stimulated by cGMP, but not by cAMP. (A) Whole cell
lysates from mammalian COS cells and from T. brucei were
immunoprecipitated with antibody against TbRSU, and the immuno-
precipitates were assayed for PKA activity in the presence or absence of
20 m
M cAMP. (B) Time course of kinase activity of immunoprecipitates
from T. brucei in the presence (grey boxes) or absence (white boxes) of
cGMP. (C and D) Effect of increasing cGMP concentrations on the
kinase activity of immunoprecipitates (autoradiographs). (C
0

subunit of PKA from the parasitic protozoon T. brucei,
TbRSU. Several previous attempts to purify the PKA
holoenzyme from this organism had failed, although an
activity resembling the catalytic subunit could be identified
[29]. Similarly, attempts in several laboratories, including
our own, to demonstrate cAMP-specific protein phosphoryl-
ation in T. brucei were unsuccessful. TbRSU was
originally identified by searching of the T. brucei sequence
databases for putative cAMP-binding proteins. The full
gene was then isolated by screening genomic and
cDNA libraries. Sequence analysis demonstrated that
TbRSU is closely related to the mammalian type I
PKA regulatory subunits, with the only major difference
being the significantly longer N-terminus of the trypanoso-
mal protein.
The two cyclic nucleotide binding domains exhibit
sequence similarities with both the cAMP-binding domains
of the PKA regulatory subunits from yeast to mammals, as
well as with the cGMP-binding domains of protein kinase
G. Unexpectedly, one absolutely conserved arginine residue
in each of the two domains is replaced by Thr318 and
Asn442 in TbRSU. In the bovine regulatory subunit, and by
inference also in all its homologs, these arginine residues
form a hydrogen bond to the phosphate group of the bound
nucleotide [19]. Sequencing errors can be ruled out as a
simple reason for this variation, as this region was
independently sequenced by three different laboratories
using different trypanosome strains. The functional
implication of this amino-acid substitution remains to be
explored. Eight amino acids before Thr318 and Asn442,

stimulation was concentration-dependent, reaching its
maximum at < 20 m
M cGMP. The interaction of TbRSU
with cyclic nucleotides was further investigated using the
recombinant cNMP-binding domains A and B. Both
domains did bind cGMP with K
d
values in the low
micromolar range (7.5 and 11.4 m
M, respectively). This
value is unexpectedly high when compared to the K
d
values
determined for cAMP of mammalian PKA regulatory
subunits (1.2 and 1.7 n
M for domains A and B, respectively
[39]). However, the results are in good agreement with the
PKA activation experiments presented in this study, which
exhibited a maximal activation of the kinase at about 20 m
M
cGMP. This value is almost 200-fold higher than the
apparent activation constant of mammalian PKA (120 n
M;
[17]). Binding of cGMP was not affected by cAMP, up to an
excess of at least 100-fold. Again, the results agree well with
the observations that the kinase activity was not stimulated
by cAMP at concentrations of up to 20 m
M. In marked
contrast to the mammalian regulatory subunit where the two
domains differ considerably in their dissociation rate

We are grateful to Kevin Shokat (Princeton University, Princeton, NJ,
USA) for providing his plasmid for the expression of recombinant PKA
substrate, to Brian Hemmings (Friedrich Miescher Institute, Basel) for
his generous supply of antibody against bovine heart PKA catalytic
subunit, and to Ursula Kurath and Erwin Studer for producing the
trypanosomes. Special thanks go to Min Ku for her careful reading of
the manuscript, and to Michael Boshart (Free University, Berlin) for
communicating unpublished results and for stimulating discussions.
This work was supported by grants 31-046760.96 and 31-058927.99 of
the Swiss National Science Foundation, grant C98.0060 of COST
program B9 of the European Union, and by the UNDP/World Bank/
WHO Special Programme for Research and Training in Tropical
Diseases
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