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 sp
Trang 1The 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 Kd 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
machinery that generates and transduces the signal exhibits
great variety This is exemplified by the adenylyl cyclases,
which have developed into many different molecular
structures [1 – 3], although their function is invariably to
convert ATP to cAMP A similarly wide range of structure
and sequence diversity of functionally similar enzymes is
found within the cAMP-specific phosphodiesterases
(PDEs) On the basis of sequence comparison as well as
of pharmacological criteria, two distinct classes of
eukaryotic PDEs are currently distinguished, class I and
class II [4,5], with no significant sequence similarities
between them Besides these, many PDEs have been
identified in bacteria that share no significant sequence
homology with either the class I or the class II of the
eukaryotic PDEs [6]
An even greater variety is encountered with the
down-stream effectors of cAMP signalling cAMP can bind
directly to and regulate a number of different ion channels,
such as cyclic nucleotide gated ion channels [7,8] or
hyperpolarization-activated cyclic nucleotide gated chan-nels [9] On the other hand, cAMP can bind to and stimulate drug efflux pumps, e.g in the human erythrocyte [10] Furthermore, recent data have demonstrated that the guanine nucleotide exchange factor Epac is a cAMP-binding protein [11], and that binding of cAMP modulates its activity This interaction potentially allows a crosstalk between cAMP pathways and ras-mediated pathways in cell cycle control
In addition to its many roles as an intracellular messenger, cAMP also can act as an extracellular signalling molecule, either directly, as in the aggregation of the slime mold 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 R2C2heterotetramer 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
R2C2 complex The C-terminus of the regulatory subunit contains two adjacent cAMP-binding domains Domain A is not accessible for cAMP in the R2C2complex 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 kinetics and in their preference for substituted cAMP analogs The three-dimensional structure of the cAMP-binding domain of a bovine type I regulatory subunit has been determined [19] Binding of cAMP to the regulatory subunits releases the active catalytic subunits from the complex These proceed to phosphorylate a plethora of proteins, among them transcription factors such as CREB [20,21] The current view is that most of the downstream effects of cAMP in eukaryotic cells are mediated through
Note: a web site is available at
http://www.izb.unibe.ch/res/seebeck/sehome.html
Correspondence to T Seebeck, Institute of Cell Biology,
University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland.
Fax: 1 41 31 631 46 84, Tel.: 1 41 31 631 46 49,
E-mail: thomas.seebeck@izb.unibe.ch
(Received 27 June 2001, revised 20 September 2001, accepted
1 October 2001)
Abbreviations: PKA, protein kinase A; cGMP, cyclic guanosine
monophosphate; cAMP, cyclic adenosine monophosphate; cNMP,
cyclic nucleoside monophosphate; TbRSU, regulatory subunit of
trypanosomal PKA.
Eur J Biochem 268, 6197–6206 (2001) q FEBS 2001
Trang 2the alteration of transcription via PKA-mediated
phos-phorylation of transcription factors Interestingly, in at least
in some instances, the activity of mammalian PKA appears
to be stimulated by cGMP rather than by cAMP [22]
In the unicellular eukaryote Trypanosoma brucei, the
causative agent of human sleeping sickness in Africa, cAMP
signalling and its role in parasite proliferation and host/
parasite interaction are still poorly understood [23] A large
number of genes coding for different adenylyl cyclases have
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
M A T E R I A L S A N D M E T H O D S
Materials
Enzymes were obtained from Roche Diagnostics (Rotkreuz,
Switzerland), and culture media were purchased from Difco
Radiochemicals were from Dupont-NEN (Regensdorf,
Switzerland), while chemicals were obtained from SIGMA
or Fluka (Buchs, Switzerland) Talonw immobilized-cobalt
resin was from Clontech (Basel, Switzerland) DNA
sequencing was outsourced to Microsynth GmbH, Balgach,
Switzerland where the reactions were run with BigDye
terminators (PE-Biosystems) and were analyzed on an ABI
Prism 377 instrument
Cell culture
T brucei strain 427 (derived form MiTat 15a), was grown as
procyclic forms at 27 8C in SDM medium [30]
Mono-morphic bloodstream forms of strain 221 (MiTat 1.2) were
cultivated as described by Hesse et al [31]
Drosophila Schneider 2 (S2) cells and expression vectors
were obtained from Invitrogen (Carlsbad, CA, USA) Cells
were passaged at cell densities between 6 and
20 106mL21 by splitting at a 1 : 2 to 1 : 5 dilution in
complete DESTM medium (Invitrogen) containing 10%
heat-inactivated fetal bovine serum S2 cells are
density-sensitive and do not proliferate when seeded at less than
5 105 mL21 Cells were cultured in a 22 – 24 8C incubator with no extra CO2 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 3 106 cells in 3 mL DESTMmedium into a 35-mm Petri dish The culture was incubated at 24 8C until a cell density of
2 – 4 106mL21 was reached (6 – 16 h) Immediately before transfection, the following two solutions were prepared separately (per 35-mm dish) Tube A: 36 mL 2M
CaCl2and 19 mg vector DNA, in a final volume of 300 mL
H2O Tube B: 300 mL 50 mM Hepes, pH 7.1, 1.5 mM
NaH2PO4, 280 mM NaCl The contents of tube A were added slowly (over 1 – 2 min) to tube B under continued mixing The final mixture was incubated at room temperature for 30 – 40 min to allow the precipitate to form The suspension was then well resuspended and added dropwise to the medium of the cell culture After incubation for 16 – 24 h, cells were washed twice with medium to remove the calcium-phosphate precipitate, suspended in fresh growth medium, and further incubated Expression of the recombinant protein was induced by the addition of
15 mL 100 mM CuSO4 per 3 mL culture medium (final concentration 500 mM), and protein expression was assayed
12 – 48 h after induction
When stable cell lines were desired, the cells were cotransfected with plasmid pCoHYGRO (Invitrogen) and were selected for growth in 300 mg:mL21hygromycin B Preparation of the PKA-specific substrate
An expression plasmid coding for a 28-kDa His6-tagged green fluorescent protein with a protein kinase A specific 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 D595of < 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 mM
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/Pi and then suspended as a 50% slurry in
100 mMphosphate 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 mM phosphate buffer and once in HB buffer (25 m Tris/HCl, pH 8.0, 50 m NaCl)
Trang 3For immunoprecipitation, 2 108 trypanosomes were
sedimented at 1300 g for 10 min and were washed twice in
ice-cold NaCl/Pi (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM KH2PO4, pH 7.3) The final pellet was
suspended in 240 mL HB buffer and 30 mL Completew
protease inhibitor mix (Roche Molecular Biochemicals)
Thirty microliters lysis buffer (10% deoxycholate, 10%
NP40 in HB buffer) were added, and the mixture was
extensively vortexed After 30 min incubation on ice,
the lysate was centrifuged for 5 min at 10 000 g at 4 8C
200 mL of the supernatant was transferred to a fresh
tube containing 25 mL of the antibody-coated protein
G – Sepharose beads The slurry was gently rocked for 1 h to
overnight at 4 8C Beads were then washed on ice three
times with cold WBI buffer (0.5% NP 40, 0.05%
deoxycholate and 0.05% SDS in NaCl/Pi, pH 7.5), twice
with cold WBII buffer (125 mMTris/HCl, pH 8.2, 500 mM
NaCl, 1 mM EDTA, 0.5% NP40), and finally once with
500 mL kinase buffer (15 mM NaCl, 5 mM MgCl2, 10 mM
Hepes, pH 7.5)
Protein kinase assay
For assaying protein kinase activity in the
immunoprecipi-tates, the following reaction mix was prepared: 1 mL
[32P]gATP (5 mCi:mL21, 150 mMATP), 4 mL of 5 kinase
buffer (75 mM NaCl, 25 mM MgCl2, 50 mM Hepes,
pH 7.5), 1 mL kinase substrate (0.5 – 1 mg), further
additions as required, and H2O to a final volume of
20 mL These 20 mL were added to 10 mL washed
immunoprecipitation beads (corresponding to 2 106
trypanosomes), and the suspension was incubated at 30 8C
for 30 min The reaction was stopped by the addition of
5 mL 5 SDS sample buffer and boiling for 3 min
Expression of a recombinant GST – RSU fusion protein
A sequence fragment of the TbRSU gene recovered from the
T brucei genome project at The Institute for Genetic
Research (TIGR) was used to design two PCR primers
(RSU-1, 50-GAGAGTCGACGCTCAAGGTAGAAGGTA
CGG-30, and RSU-2, 50-AGACTCGAGCTACTTCCTCCC
CTCTGCCC-30; added SalI and XhoI restriction sites
underlined, respectively) The expected 600-bp fragment
was amplified from genomic DNA of T brucei, confirmed
by DNA sequencing and introduced into the multicloning
site of the expression vector pGEX-4T2
(Amersham-Pharmacia) The vector was transformed into Escherichia
coli BL21DE and the recombinant protein was expressed in
high amounts in an insoluble form The protein was
solubilized from the inclusion bodies in 100 mMTris/HCl,
pH 7.5, 5 mM EDTA, 6M 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 inDrosophila 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 Cu21to the growth medium The recombinant proteins carry a V5 immunological tag and
a His6-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 [50 -TATACTAGTATGG(2531)CACTCATCTTGAAGTTGT-30, added Spe I site and start codon, bold underlined] and Adom-R [50-TATCTCGAGA(2938rev)AGGCCACTGAG GAAC-30, added Xho I site underlined] Domain B (amino acids 352 – 499) was amplified using primers Bdom-F [50-TATACTAGTATGC(2921)CGTTCCTCAGTGG-30, added SpeI site and start codon, bold underlined] and Bdom-R [50-TATCTCGAG(3334rev)CTTCCTCCCCT CTG-30, added XhoI site underlined] For amplification of the joint domains (amino acids 231 – 499), primers Adom-F and Bdom-R were used The PCR products were cloned into the pGEM T-Easy vector, verified by DNA sequencing and were finally subcloned into the expression vector pMT/ V5-His B
Purification of recombinant cNMP-binding domains from S2 Drosophilacells
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 mM Tris/HCl, pH 7.8,
150 mM 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/Piwas 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 mMsodium phosphate buffer, pH 7.0, 300 mM NaCl Recombinant protein was finally eluted with four aliquots of 100 mL elution buffer (50 sodium phosphate, pH 7.0, 300 mM 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 mMsodium phosphate,
pH 6.8, 1 mM EDTA, 25 mM 2-mercaptoethanol, 0.2 mM
isobutyl-methyl-xanthine, 1.5 mg purified protein and increasing concentrations of [3H]cGMP (NEN, catalogue
no NET-337) adjusted to a specific activity of
1 mCi:nmol21 cAMP competition and kinetic experiments were carried out in the presence of 0.4 mM [3H]cGMP Initial experiments were carried out in the presence of
500 mg:mL21histone VIII-S, which increased the binding efficiency by about 50% Histone was omitted in later experiments The binding reactions were incubated on ice
qFEBS 2001 PKA regulatory subunit from T brucei (Eur J Biochem 268) 6199
Trang 4overnight Reactions were stopped by the addition of 1 mL
ice cold 10 mMsodium phosphate, pH 6.8, 1 mMEDTA and
were filtered immediately through prewetted Millipore
HAWP filters (0.45 mM) 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 mM[3H]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 thePRISMsoftware package of GraphPad Inc.,
San Diego, CA, USA
R E S U L T S
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 enzymes that did not cut within the DNA sequence of the hybridization probe (Xho I, Stu I, Spe I, Pst I, Nhe I, Kpn I and HindIII), all digests resulted in a single hybridizing band (Fig 1A), strongly indicating that the new gene, TbRSU, is coded for by a single-copy gene The 600-bp PCR fragment was then used to screen a genomic library of
T brucei in a lambda phage vector [33] This screening resulted in several independent phages containing the same
Fig 1 TbRSU is a single-copy gene (A) Digests of genomic DNA of
T brucei were hybridized with a 600-bp PCR fragment representing the
conserved cNMP-binding domain of TbRSU (B) Map of the TbRSU
locus Nucleotides 1 – 483 code for the C-terminus of a protein of
unknown function (TbTAS ) Nucleotides 1838 – 3334 represent the open
reading frame of TbRSU Nucleotides 3335 – 3566 represent a part of the
30untranslated region of TbRSU The grey boxes designated A and B
represent the predicted cyclic nucleotide binding domains of the
TbRSU protein The sequence has been deposited at GenBank under the
accession number AF326975.
Fig 2 Gene and amino-acid sequence of TbRSU The pseudosub-strate sequence is indicated by the grey box The two cyclic-nucleotide binding domains A and B are boxed Shaded boxes in domain A: Glu311 (is Ala in all homologs, see Fig 3); Thr318 (is Arg in all homologs); Val319 (is Ala in cAMP and Thr in cGMP binding domains) Shaded boxes in domain B: Glu435 (is Ala in all homologs); Asn442 (is Arg in all homologs).
Trang 5locus A 3-kb Eco RI fragment was subcloned into
pBlueskriptSK1 and both strands were completely
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 50 untranslated region, a complete open
reading frame of 1497 bp, and a 30 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 30 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
The open reading frame of TbRSU predicts a protein of 499
amino acids, with a calculated Mr of 56 725 (Fig 2)
Overall, the protein shares extensive sequence homology
with mammalian PKA regulatory subunits type I The
N-terminal domain of TbRSU (amino acids 1 – 242) is
longer than the N-termini of its mammalian homologs, and it
bears no identifiable functional domains In analogy to
mammalian type I regulatory subunits, the cysteine residues
Cys15 and Cys67 may be involved in dimer formation,
although such dimers could not be detected in cell lysates
analysed by gel filtration chromatography (data not shown)
In these experiments, TbRSU always migrated as a monomer Residues 202 – 206 (-ArgArgThrThrVal-) rep-resent the pseudo-inhibitor site which is involved in the interaction with the catalytic domain [34] Amino acids
243 – 360 and 363 – 483 form the cyclic nucleotide binding domains A and B, respectively Based on the structural model of the bovine regulatory subunit RIa [17], Glu309 and Glu433 form a hydrogen bond with the 20 hydroxyl of the ribose of the bound cNMP, while Leu310 and Leu434 interact with a nitrogen of the pyrimidine ring of the base Tyr370 and Tyr482 are probably the functional homologs of Trp260 and Tyr371 in bovine RIa, allowing base-stacking with the purine residue Unexpectedly, a strongly conserved arginine residue, which forms a hydrogen bond to the phosphate group, is replaced by threonine (Thr318) and 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:
Caenorhabditis elegans (P30625); D:
D discoideum (P05987); E: S cerevisiae
(P07278); F: Schizosaccharomyces pombe
(P36600); G: TbRSU (AF326975); H:
Homo sapiens protein kinase G (O13237); I:
D melanogaster protein kinase G (O03043).
Filled circles denote amino acids conserved in all
sequences Open squares denote amino acids
which are conserved in all sequences, but differ in
TbRSU.
qFEBS 2001 PKA regulatory subunit from T brucei (Eur J Biochem 268) 6201
Trang 6However, the steady-state level of TbRSU mRNA in
blodstream forms is about five times higher than it is in
procyclic forms
The TbRSU protein is present both in bloodstream and in
procyclic forms
To follow up the results of the Northern blotting experiments
on the protein level, whole cell lysates were analyzed by
immunoblotting, using an affinity-purified polyclonal
antibody raised against recombinant TbRSU (see Materials
and methods) This polyclonal antibody not only recognizes
TbRSU in trypanosomes, but it also detects PKA regulatory
subunits in other organisms such as Saccharomyces
cerevisiae and mammalian cells (Fig 4B) The TbRSU
protein is readily detectable both in bloodstream and in
procyclic forms, and it migrates as a single band of a Mrof
55 000, in agreement with its calculated Mr 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 Mr 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 mMcAMP 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,
or when the TbRSU antibody was used in the absence of cell lysate Unexpectedly, the addition of 20 mM cAMP to the reactions did not stimulate the kinase activity, but had either
Fig 4 TbRSU is more abundant in bloodstream than in procyclic
forms (A) Northern blot analysis Ten-microgram aliquots of total
RNA of procyclic (PC) or bloodstream form (BSF) trypanosomes were
loaded per slot After transfer, the filter was successively hybridized
with a TbRSU probe (a) and a probe for b-tubulin (b) After
electrophoresis, the gel was stained with ethidium bromide to control
for equal loading (c) (B) Hybridization was quantified using a
PhosphorImager (a) Hybridization with TbRSU; (b) hybridization with
a b-tubulin probe Grey bars, procyclics; black bars, bloodstream forms.
(C) Immunoblot analysis (a) The polyclonal antibody raised against
recombinant TbRSU recognizes homologs in a wide spectrum of
species 1, Whole cell lysate from E coli expressing the GST – TbRSU
fusion protein used for raising the antibody; 2, whole cell lysate of
T brucei; 3, whole cell lysate of S cerevisiae; 4, whole cell lysate of
COS (monkey) cells (b) Immunoblot of equivalent amounts of whole
cell lysates of bloodstream (B) and procyclic (P) trypanosomes.
Molecular mass markers are indicated for each panel.
Fig 5 The TbRSU antibody coimmunoprecipitates a protein kinase activity which phosphorylates a PKA-specific substrate Protein kinase activity assays of immunoprecipitates (Top) Coomassie-stained gels, molecular mass markers are IgG heavy chain (50 kDa) and the PKA substrate (30 kDa); (bottom) corresponding autoradiographs, arrowheads indicate the position of the PKA substrate (A) Immunoprecipitation with no antibody; (B) immunoprecipitation with TbRSU antibody; (C) immunoprecipitation with irrelevant antibody (against the phosphodiesterase TbPDE1; S Kunz, personnal communi-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
Trang 7no 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, Mg21-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 mM cAMP, no stimulation of phosphorylation of the PKA-specific substrate could be detected In contrast, control reactions using mammalian COS cell lysates precipitated by the same antibody, exhibited the expected stimulation of kinase activity by cAMP (Fig 7A) This unexpected absence of stimulation of trypanosomal PKA activity by cAMP was consistently observed over many independent experiments (see above) However, when similar experiments were performed with cGMP instead of cAMP, a marked stimulation of kinase activity was consistently observed (Fig 7B – D) The phosphorylation reactions followed a similar time course in the presence and in the absence of cGMP (Fig 7B), but the overall kinase activity was stimulated threefold to fourfold by cGMP The kinase reaction was stimulated to a similar extent in immunoprecipitates from procyclic and bloodstream form trypanosomes (Fig 7C,D), with maxi-mum stimulation reached around 20 mM cGMP These unexpected findings suggested that the trypanosomal TbRSU, in contrast to its homologs in all other eukaryotes analysed so far, is activated by cGMP rather than by cAMP
cGMP binding to the cyclic nucleotide binding domains A and B sites of TbRSU
In order to directly confirm if TbRSU does in fact bind cGMP, expression of the recombinant domains was attempted in E coli Expression of domain B alone produced ample recombinant protein, but all in insoluble form Expression of the combined A and B domains resulted
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 Cu21-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) (C0and D0)
Coomassie stained PKA substrate bl, blank reaction incubated in the
presence of 20 m M cGMP, but without protein substrate (C and C 0 )
procyclics; (D and D0) bloodstream forms.
qFEBS 2001 PKA regulatory subunit from T brucei (Eur J Biochem 268) 6203
Trang 8(Fig 8) Both domains exhibited very similar Kdvalues for
cGMP (domain A: 7.51 ^ 1.97 mM, n ¼ 3; domain B:
11.43 ^ 2.24 mM, n ¼ 3) For both domains, cAMP did
not measurably compete with cGMP binding up to a
100-fold excess of cAMP over cGMP Dissociation rate
constants for cGMP were also very similar between the two
domains (domain A 0.24 min21, n ¼ 3, and domain B
0.36 ^ 0.18 min21, n ¼ 3)
D I S C U S S I O N
The current study reports the identification of the regulatory
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,
another amino-acid substitution peculiar to TbRSU has
occurred: Glu311 and Glu435 replace otherwise invariant
alanine residues Thirdly, Val319 represents another
substitution that sets TbRSU apart from its homologs At
the equivalent position, the other PKA regulatory domains contain an alanine residue while protein kinases G contain serine or threonine The hydroxyl side chain of either one of these residues interacts with the C2 amino group of cGMP and is essential for full activation of cGMP dependent protein kinases [38]
The gene encoding TbRSU is expressed both in the bloodstream and in the procyclic forms of the parasite, but at much higher levels in the bloodstream form The TbRSU protein levels in both life cycle stages closely correspond to the mRNA levels
Immunoprecipitation of TbRSU consistently coprecipi-tated a protein kinase activity exhibiting many character-istics of the catalytic subunit of trypanosomal PKA The coprecipitated kinase is recognized by an antibody against the bovine PKA catalytic subunit, the enzyme phosphorylates a PKA-specific substrate [32], and its activity is strongly inhibited by the rabbit PKI inhibitor peptide [36]
While the protein kinase activity recovered in the immunoprecipitates exhibited all the characteristics of a canonical PKA catalytic subunit, no stimulation by cAMP could be detected On the contrary, cAMP appeared to inhibit the protein kinase activity in some, but not all, experiments Surprisingly, a marked stimulation of protein kinase activity was consistently found with cGMP This stimulation was concentration-dependent, reaching its maximum at < 20 mM 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 Kd values in the low micromolar range (7.5 and 11.4 mM, respectively) This value is unexpectedly high when compared to the Kdvalues determined for cAMP of mammalian PKA regulatory subunits (1.2 and 1.7 nMfor 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 mM
cGMP This value is almost 200-fold higher than the apparent activation constant of mammalian PKA (120 nM; [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 mM In marked contrast to the mammalian regulatory subunit where the two domains differ considerably in their dissociation rate constants (0.15 min21vs 0.04 min21[17]), both domains
of TbRSU behave very similarly (0.24 min21for domain A and 0.36 min21for domain B)
The observation that protein kinase A in T brucei (and probably also in other kinetoplastids) is regulated by cGMP rather than by cAMP implies that cGMP has an important signalling role in this group of organisms Earlier work had demonstrated the presence of cGMP in T cruzi [40], and several members of a family of recently identified cAMP-specific phosphodiesterases of T brucei [26] (A Rascon,
S H Soderling & J Beavo, personal communication) contain one or two GAF-domains [41] that may be involved
in cGMP binding While these phosphodiesterases may represent an interconnection between the cAMP- and the cGMP-signalling pathways in T brucei, the cGMP-regu-lated TbRSU/PKA kinase may well represent the major effector of cGMP signalling in these organisms
Fig 8 The cNMP-binding domains of TbRSU bind cGMP, but not
cAMP Saturation binding of of cGMP to recombinant domains A and B
of TbRSU Data represent one of three very similar experiments.
Trang 9A C K N O W L E D G E M E N T S
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
R E F E R E N C E S
1 Hurley, J.H (1998) The adenylyl and guanylyl cyclase superfamily.
Curr Opin Struct Biol 8, 770 – 777.
2 Chen, Y., Cann, M.J., Litvin, T.N., Iourgenko, V., Sinclair, M.J.,
Livin, L.R & Buck, J (2000) Soluble adenylyl cyclase as an
evolutionarily conserved bicarbonate sensor Science 289,
625 – 628.
3 Naula, C & Seebeck, T (2000) Cyclic AMP signaling in
trypanosomatids Parasitol Today 16, 35 – 38.
4 Nikawa, J., Sass, P & Wigler, M (1987) Cloning and
characterization of the low-affinity cyclic AMP phosphodiesterase
gene of Saccharomyces cerevisiae Mol Cell Biol 7, 3629 – 3636.
5 Sass, P., Field, J., Nikawa, J., Toda, T & Wigler, M (1986) Cloning
and characterization of the high-affinity cAMP phosphodiesterase
of Saccharomyces cerevisiae Proc Natl Acad Sci USA 83,
9303 – 9307.
6 Imamura, R., Yamanaka K., Ogura, T., Hiraga, S., Fujita, N.,
Ishihama, A & Niki, H (1996) Identification of the cpdA gene
encoding cyclic 3050-adenosine monophosphate phosphodiesterase
in Escherichia coli J Biol Chem 271, 25423– 25429.
7 Kraus-Friedman, N (2000) Cyclic nucleotide gated channels in
non-sensory organs Cell Calcium 27, 127 – 138.
8 Broillet, M.C & Firestein, S (1999) Cyclic nucleotide-gated
channels Molecular mechanisms of activation Ann NY Acad Sci.
868, 730 – 740.
9 Clapham, D (1998) Not so funny anymore: pacing channels are
cloned Neuron 21, 5 – 7.
10 Schultz, C., Vaskinn, S., Kidalsen, H & Sager, G (1998) Cyclic
AMP stimulates the cyclic GMP egression pump in human
erythrocytes: effects of probenecid, verapamil, progesterone,
theophylline, IBMX, forskolin and cyclic AMP on cyclic GMP
uptake and association to inside-out vesicles Biochemistry 37,
1161 – 1166.
11 De Rooji, J., Zwartkruis, F.J.T., Verhekgen, M.H.G., Cool, R.H.,
Nijman, S.M.B., Witinghofer, A & Bos, J.L (1998) Epac is a Rap1
guanine nucleotide exchange factor directly activated by cyclic
AMP Nature 396, 474 – 477.
12 Weeks, G (2000) Signalling molecules involved in cellular
differentiation during Dictyostelium morphogenesis Curr Opin.
Microbiol 3, 625 – 639.
13 Rosenberg, P.A & Li, Y (1995) Adenylyl cyclase activation
underlies intracellular cyclic AMP accumulation, cyclic AMP
transport, and extracellular adenosine accumulation evoked by
beta-adrenergic receptor stimulation in mixed cultures of neurons
and astrocytes derived from rat cerebral cortex Brain Res 692,
227 – 232.
14 Taylor, S.S., Radzio-Andzelm, E., Madhusudan, A., Cheng, X., Ten
Eyck, L & Narayana, N (1999) Catalytic subunit of cyclic AMP
dependent protein kinase: structure and dynamics of the active site
cleft Pharmacol Ther 82, 133 – 141.
15 Francis, S.H & Corbin, J.D (1999) Cyclic nucleotide-dependent protein kinase: intracellular receptors for cAMP and cGMP action Crit Rev Lab Sci 36, 275 – 328.
16 Thevelein, J.M & de Winde, J.H (1999) Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway
in the yeast Saccharomyces cerevisiae Mol Microbiol 33,
904 – 918.
17 Francis, S.H & Corbin, J.D (1994) Structure and function of cyclic nucleotide-dependent protein kinases Annu Rev Physiol 56,
237 – 272.
18 Taylor, S.S., Buechler, J.A & Yonemoto, W (1990) cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes Annu Rev Biochem 59, 971 – 1005.
19 Su, Y., Dostmann, W.R.G., Herberg, F.W., Durick, K., Xuong, N.H., Ten Eyck, L., Taylor, S.S & Varughese, K.I (1995) Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains Science 269, 807 – 813.
20 De Cesare, D & Sassone-Corsi, P (2000) Transcriptional regulation by cyclic AMP-responsive factors Prog Nucleic Acid Res Mol Biol 64, 343 – 369.
21 Shaywitz, A.J & Greenberg, M.E (1999) CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals Annu Rev Biochem 68, 821 – 861.
22 Forte, L.R., Thorne, P.K., Eber, S.L., Krause, W.J., Freeman R.H., Francis, S.H & Corbin, J.D (1992) Stimulation of intestinal Cl-transport by heat-stable enterotoxin: activation of cAMP dependent protein kinase by cGMP Am J Physiol 263, C607 – C615.
23 Seebeck, T., Gong, K.W., Kunz, S., Schaub, R., Shalaby, T & Zoraghi, R (2001) cAMP signalling in T brucei Int J Parasitol.
31, 491 – 498.
24 Alexandre, S., Paindovoine, P., Hanocq-Quertier, J., Paturiaux-Hanocq, F., Tebabi, P & Pays, E (1996) Families of adenylate cyclase genes in Trypanosoma brucei Mol Biochem Parasitol 77,
173 – 182.
25 Naula, C., Schaub, R., Leech, V., Melville, S & Seebeck, T (2001) Spontaneous dimerization and leucine-zipper induced activation of the recombinant catalytic domain of a new adenylyl cyclase, GRESAG4.4B Mol Biochem Parasitol 112, 19 – 28.
26 Zoraghi, R., Kunz, S., Gong, K.W & Seebeck, T (2001) Characterization of TbPDE2A, a novel cyclic nucleotide specific phosphodiesterase from the protozoan parasite Trypanosoma brucei J Biol Chem 276, 11559– 11566.
27 Vassella, E., Reuner, B., Yutzy, B & Boshart, M (1997) Differentiation of African trypanosomes is controlled by a density sensing mechanism which signals cell cycle arrest via the cAMP pathway J Cell Sci 110, 2661 – 2671.
28 O’Beirne, C., Lowry, C.M & Voorheis, H.P (1998) Both IgM and IgG anti-VSG antibodies initiate a cycle of aggregation-disaggregation of bloodstream forms of Trypanosoma brucei without damage to the parasite Mol Biochem Parasitol 91,
165 – 193.
29 Ochatt, C.M., Ulloa, R.M., Torres, H.N & Tellez Inon, M.T (1993) Characterization of the catalytic subunit of Trypanosoma cruzi cyclic AMP-dependent protein kinase Mol Biochem Parasitol 57, 73 – 81.
30 Brun, R & Scho¨nenberger, M (1979) Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium Acta Tropica 36, 289 – 292.
31 Hesse, F., Selzer, P.M., Mu¨hlstadt, K & Duszenko, M (1995) A novel cultivation technique for long-term maintenance of blood-stream form trypanosomes in vitro Mol Biochem Parasitol 70,
157 – 166.
32 Yang, F., Liu, Y., Bixby, S.D., Friedman, J.D & Shokat, K (1999) Highly efficient green fluorescent protein-based kinase substrates Anal Biochem 266, 167 – 173.
33 Schla¨ppi, K., Deflorin, J & Seebeck, T (1989) The major
qFEBS 2001 PKA regulatory subunit from T brucei (Eur J Biochem 268) 6205
Trang 10component of the paraflagellar rod of Trypanosoma brucei is a
helical protein that is encoded by two identical, tandemly linked
genes J.Cell Biol 109, 1695 – 1709.
34 Zetterqvist, O & Ragnarsson, U (1982) The structural
require-ments of substrates of cyclic AMP-dependent protein kinase FEBS
Lett 139, 287 – 290.
35 Seebeck, T., Whittaker, P.A., Imboden, M.A., Hardman, N &
Braun, R (1983) Tubulin genes of Trypanosoma brucei: a tightly
clustered family of alternating genes Proc Natl Acad Sci USA 80,
4634 – 4638.
36 Cheng, H.C., Kemp, B.E., Pearson, R.B., Smith, A.J., Misconi, L.,
Van Patten, S.M & Walsh, D.A (1986) A potent synthetic peptide
inhibitor of the cAMP-dependent protein kinase J Biol Chem.
261, 989 – 992.
37 Gosse, M.E., Padmanabhan, A., Fleischmann, R.D & Gottesmann,
M.M (1993) Expression of Chinese hamster cAMP-dependent
protein kinase in Escherichia coli results in growth inhibition of
bacterial cells: a model system for the rapid screening of mutant
type I regulatory subunits Proc Natl Acad Sci USA 90,
8159 – 8163.
38 Taylor, M.K & Uhler, M.D (2000) The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation.
J Biol Chem 275, 28053 – 28062.
39 Døskeland, S.O., Øgreid, D., Ekanger, R.E., Sturm, P.A., Miller, J.P & Suva, R.H (1983) Mapping of the two intrachain cyclic nucleotide binding sites of adenosine cyclic 30,50-dependent protein kinase I Biochemistry 22, 1094 – 1101.
40 Paveto, C., Pereira, C., Espinosa, J., Montagna, A.E., Farber, M., Esteva, M., Flawia, M.M & Torres, H.N (1995) The nitric oxide transduction pathway in Trypanosoma cruzi J Biol Chem 270, 16576– 16579.
41 Ho, Y.S., Burden, L.M & Hurley, J.H (2000) Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor EMBO J 19, 5288 – 5299.