FEMS Microbiology Letters 152 (1997) 333^337

Cyclic AMP stimulates the protein tyrosine kinase activity of

Acinetobacter calcoaceticus
Christophe Grangeasse, Elisabeth Vaganay, Patricia Doublet, Myleéne Riberty,
Alain J. Cozzone *, Bertrand Duclos
Institut de Biologie et Chimie des Proteèines, Centre National de la Recherche Scienti¢que, 7 passage du Vercors, 69367 Lyon, France
Received 14 April 1997; revised 2 May 1997; accepted 5 May 1997

Abstract
The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation
of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP
was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes,
whose functioning is cyclic nucleotide-dependent.
Keywords: Protein tyrosine kinase; Acinetobacter calcoaceticus; Cyclic AMP; Phosphorylation

1. Introduction In prokaryotes, the presence of protein tyrosine

In eukaryotes, a large number of protein tyrosine
kinases have been characterized in both normal and
pathologic situations [1,2]. These enzymes have been
shown to play key roles in the control of various
cellular functions including signal transduction,
growth control, metabolism and malignant transfor-
mation [3,4]. Their activity is stimulated by a number
of regulatory ligands, namely growth factors and
hormones [2,3], as well as certain chemicals such as
hydrogen peroxide [5]. However, unlike the closely
related protein serine/threonine kinases, they display
no dependence on cyclic nucleotides or calcium/
calmodulin [4,6].
* Corresponding author. Tel.: +33 4.72.72.26.72;
Fax: +33 4.72.72.26.01; E-mail: cozzone@ibcp.fr
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 2 1 - 8
kinase activities was suggested by the ¢nding of
phosphotyrosine originally in the proteins of Esche-
richia coli [7] and, since then, in the proteins of a
series of other bacterial species [8]. In particular,
the proteins of Acinetobacter calcoaceticus, a strict
aerobe of the Neisseriaceae family, have been shown
to contain a relatively high proportion of phos-
photyrosine, and the occurrence of a tyrosine kin-
ase activity, which phosphorylates essentially an
81-kDa protein located in the inner membrane
of cells, has been clearly demonstrated in vivo and
in vitro [9]. In this work, further analysis of this
kinase activity was performed by checking the e¡ect
of cyclic nucleotides both quantitatively by measur-
ing the extent of phosphate incorporation into the
81-kDa protein, and qualitatively by determining
the nature of the amino acids phosphorylated in
that protein.
334 C. Grangeasse et al. / FEMS Microbiology Letters 152 (1997) 333^337
2. Materials and methods
2.1. Preparation of cellular extract
Cells of A. calcoaceticus were grown at 30³C in
Luria broth at pH 7.0. After four generations
(OD600 = 1), bacteria were collected by centrifugation
at 5000Ug for 15 min. The bacterial pellet was sus-
pended in a bu¡er containing 25 mM Tris-HCl (pH
7.0), 5 mM MgCl2 , 1 mM dithiothreitol, 1 mM
EDTA and 1 mM phenylmethylsulfonyl £uoride.
Cells were disrupted in a French pressure cell at
16 000 psi. A crude extract was prepared by centri-
fugation for 15 min at 5000Ug after incubation for
30 min at 4³C in the presence of pancreatic deoxy-
ribonuclease I and ribonuclease A (2 Wg/ml each).
2.2. Preparation of inner membranes
The technique used for preparing the inner mem-
brane fraction, which contains the 81-kDa protein,
was adapted from [10]. A crude extract was prepared
as above, in a bu¡er containing 10% (w/v) sucrose. It
was layered onto a two-step gradient consisting of 15
ml of 25% (w/v) sucrose over 5 ml of 70% (w/v)
Fig. 1. Autoradiography of the 81 kDa protein. Inner mem-
branes were incubated for 5 min with radioactive ATP in the ab-
sence (lane 1) or in the presence of cAMP at varying concentra-
tions: 5 mM (lane 2), 10 mM (lane 3) or 20 mM (lane 4). The
81 kDa protein was separated by polyacrylamide gel electropho-
resis and revealed by autoradiography.
Fig. 2. E¡ect of cAMP on the phosphorylation of the 81 kDa
protein. The incorporation of radioactive phosphate into the 81
kDa protein was measured as a function of time of incubation in
the absence (E) or in the presence of cAMP at concentrations of
5 mM (F), 10 mM (a) or 20 mM (b).
sucrose, and centrifuged at 120 000Ug for 2 h. The
material present at the 25/70% sucrose interface was
collected and referred to as total crude membranes.
This fraction was diluted to 10 ml in the bu¡er with-
out sucrose, and layered onto another two-step gra-
dient consisting of 15 ml of 45% (w/v) over 5 ml 70%
(w/v) sucrose. It was centrifuged at 120 000Ug for
3 h. Then, to prepare the inner membranes, the ma-
terial that moved above and at the 45% sucrose in-
terface was collected and diluted to 27 ml with
bu¡er, layered onto a 3 ml pad of 35% sucrose,
and centrifuged at 120 000Ug for 2 h. In these con-
ditions, the material that moved at the 35% sucrose
interface consisted of the inner membrane fraction. It
was collected, diluted with bu¡er, and centrifuged at
200 000Ug for 2 h.
2.3. In vitro protein phosphorylation
Protein phosphorylation was assayed in a total
volume of 20 Wl containing 25 mM Tris-HCl (pH
7.0), 1 mM dithiothreitol, 5 mM MgCl2 , 1 mM
EDTA, 10 WM and 200 WCi/ml of [Q-32 P]ATP (spe-
ci¢c activity 3000 Ci/mmol), and 20^25 Wg of total
protein extracted from inner membranes. In some
assays, the radioactive Q-labelled precursor was 10
WM and 200 WCi/ml of GTP, CTP or UTP. When
 C. Grangeasse et al. / FEMS Microbiology Letters 152 (1997) 333^337 335

Fig. 3. Autoradiography of the phosphoamino acids of the 81 kDa protein. Inner membranes were incubated for 2 min with radioactive
ATP in the absence (a) or in the presence of 10 mM cAMP (b). The 81 kDa protein was separated by gel electrophoresis, extracted from
the gel and subjected to acid hydrolysis. The radioactive phosphoamino acids thus released were separated in two successive dimensions
and revealed by autoradiography. Authentic phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) were analyzed
in parallel and stained with ninhydrin. Their location on the autoradiograms is indicated. The radioactive spots on the right-hand side of
the diagrams correspond to inorganic polyphosphates and unhydrolyzed phosphopeptides [17].

required, 5^20 mM cyclic AMP or 10 mM cyclic
GMP was added. In other assays, radioactive ATP
was incubated in the presence of 10 mM AMP,
GMP, CMP or UMP. In each case, the reaction
was stopped by addition of an equal volume of
2 Usample bu¡er and the mixture was heated at
100³C for 5 min.
2.5. Analysis of phosphorylated amino acids
To characterize the phosphoamino acids, protein
samples were ¢rst subjected to SDS-PAGE and elec-
troblotted onto an Immobilon PVDF membrane
[12]. Phosphorylated proteins bound to the mem-
brane were detected by autoradiography. The 32 P-

2.4. Gel electrophoresis and autoradiography Table 1

E¡ect of various nucleotides on the phosphorylation of the 81
kDa protein
One-dimensional gel electrophoresis was per-
Assay number Nucleotide(s) Radioactivity of protein
formed as described [11]. Migration was carried out
(cpm)
in 10% (w/v) polyacrylamide containing 0.1% (w/v)
1 ATP 1 990
SDS, at 20³C and 10 W per gel. Reference proteins
2 GTP 30
of known molecular mass ranging from 14.4 kDa 3 CTP 25
(lysozyme) to 97.4 kDa (phosphorylase b) were run 4 UTP 25
in parallel. After electrophoresis, gels were soaked in 5 ATP+cAMP 10 590

16% (w/v) trichloroacetic acid for 10 min at 90³C, 6 ATP+cGMP 2 020

7 ATP+AMP 1 970
and incubated overnight in 5% (w/v) trichloroacetic
8 ATP+GMP 2 130
acid and 50 mM Na2 HPO4 . They were stained with 9 ATP+CMP 2 080
0.1% (w/v) Coomassie blue R-250 in 50% (v/v) meth- 10 ATP+UMP 1 940
anol, 7.5% (v/v) acetic acid, and dried under vacuum. Membrane vesicles were incubated with radioactive ATP for 2 min
Radioactive bands were visualized by autoradiogra- either in the absence (assay 1) or in the presence of another nucleo-
phy using direct exposure ¢lms. When required, the tide at a concentration of 10 mM, as indicated (assays 5^10). In

gel band corresponding to the 81-kDa protein was assays 2^4, ATP was replaced by another radioactive nucleotide. In

excised, treated for 4 h at 80³C with 200 Wl of 60%

each case, the radioactivity of the 81 kDa protein was measured
after gel electrophoresis as described in Section 2, and expressed in
(v/v) perchloric acid and 400 Wl of 30% (v/v) hydro-
counts per min. The statistical analysis of three di¡erent measure-
gen peroxide, and its radioactivity was counted in a ments showed that the average deviation was þ 14% of expressed
scintillation £uid. values.
336 C. Grangeasse et al. / FEMS Microbiology Letters 152 (1997) 333^337

labelled protein bands were excised from the Immo- and subjected to acid hydrolysis. The phosphoamino

bilon blot and hydrolyzed in 6 M HCl for 1 h at acids thus liberated were separated in two successive

110³C. The acid-stable phosphoamino acids thus lib- dimensions and revealed by autoradiography. The

erated were separated by electrophoresis in the ¢rst corresponding diagrams (Fig. 3) show that the extent

dimension at pH 1.9 (800 V h) in 7.8% (v/v) acetic of radioactive labelling was greater under treatment

acid/2.5% (v/v) formic acid, followed by ascending with cAMP, as expected. But no labelled phospho-

chromatography in the second dimension in 2-meth- amino acid other than phosphotyrosine was de-

yl-1-propanol/formic acid/water (8 :3 :4, v/v). After tected, as evidenced from comigration with authentic

migration, radioactive molecules were detected by phosphoamino acids. In other words, the cyclic nu-

autoradiography. Authentic phosphoserine, phos- cleotide did not induce any qualitative change in the

phothreonine and phosphotyrosine were run in par- amino acid residues targeted by the protein kinase

allel and visualized by staining with ninhydrin. activity.

In another set of experiments, various nucleotides

were assayed for their capacity either to serve as

3. Results and discussion phosphate donors for phosphorylating 81-kDa pro-

tein or to stimulate this phosphorylation reaction.

In a ¢rst series of experiments, the e¡ect of a vary- The results presented in Table 1 indicate, on the

ing concentration of cAMP on the phosphorylation one hand, that only ATP could be used as nucleotide

of the 81-kDa protein was analyzed. A cellular ex- precursor (assay 1) since no detectable phosphoryla-

tract containing the inner membrane fraction of A. tion occurred at the expense of GTP, CTP, or UTP

calcoaceticus cells was prepared and incubated with (assays 2^4). On the other hand, neither the cyclic
32
[Q- P]ATP in the absence or in the presence of 5^20 nucleotide cGMP (assay 6) nor the nucleoside mono-

mM cyclic nucleotide. After gel electrophoresis, the phosphates AMP, UMP, CMP, GMP (assays 7^10)

amount of radioactivity incorporated into the 81- appeared to be able to stimulate the phosphorylation

kDa protein was estimated by autoradiography. of the 81-kDa protein, in contrast to cAMP (assay

The corresponding pattern (Fig. 1) shows that, in 5).

the presence of cAMP, the phosphorylation of the Together, these data provide evidence that cyclic

81-kDa protein was signi¢cantly increased. AMP has a speci¢c stimulatory e¡ect on the protein

A more precise analysis of the stimulation of phos- tyrosine kinase activity of A. calcoaceticus . To our

phorylation of the 81-kDa protein by cAMP was knowledge, this is the ¢rst example of a kinase of

carried out by measuring the amount of radioactive this type whose functioning is a¡ected by a cyclic

phosphate incorporated in the protein as a function nucleotide, not only in bacteria but in the di¡erent

of time of incubation with di¡erent cAMP concen- eukaryotic systems as well [3,4]. One might envisage

tration. Fig. 2 shows that, in all cases, maximum that the increased labelling of the 81-kDa protein in

incorporation was reached after about 2 min of in- the presence of cAMP could be due, in fact, to the

cubation. At that point, the level of phosphorylation stimulation by this nucleotide of a phosphatase ac-

was increased twofold in the presence of 5 mM tivity, instead of a protein kinase activity. According

cAMP and over ¢vefold in the presence of 10 mM to this hypothesis, the cAMP-stimulated phospha-

cAMP as compared to the control. No further in- tase would extensively dephosphorylate the 81-kDa

crease was observed, however, with 20 mM cAMP. protein before the in vitro phosphorylation assay,

In addition to these quantitative data, it seemed of which would result in an apparently enhanced label-

interest to determine whether the nature of the ami- ling of the 81-kDa protein during incubation with

no acids phosphorylated in the 81-kDa protein was radioactive ATP. To check this possibility, we meas-

the same in the presence and in the absence of ured the phosphatase activity of the inner membrane

cAMP. For this, inner membranes were incubated fraction through its ability to cleave the synthetic

with radioactive ATP in a medium containing or substrate p-nitrophenylphosphate, as previously de-

not 10 mM cAMP, the 81-kDa protein was isolated scribed [13]. No signi¢cant phosphatase activity was

by gel electrophoresis, detected by autoradiography detected with this procedure, which makes the hy-
 C. Grangeasse et al. / FEMS Microbiology Letters 152 (1997) 333^337
pothesis mentioned above unlikely and brings fur-
ther support to the concept that cAMP stimulates
the protein tyrosine kinase activity.
Cyclic AMP has long been known to be involved
in the regulation of several biological processes in
prokaryotes [14]. One of its better characterized roles
is to activate CRP, an allosteric DNA-regulatory
protein, so as to modulate the transcription of a
variety of genes. Other proposed roles concern the
control of cell division, anaerobic growth, sporula-
tion, morphogenesis and virulence of bacteria, to
name only a few. The latter role might be of partic-
ular relevance to the present results, since a number
of reports suggest that protein phosphorylation at
tyrosine plays a crucial role in the bacterial-host
cell interactions and in the signal transduction proc-
esses related to pathogenicity [15,16].
Acknowledgments
This work was supported by the Centre National
de la Recherche Scienti¢que, the Universiteè de Lyon
and the Institut Universitaire de France. Thanks are
due to Emmanuelle Duglas and Alain Bosch for
their help in preparing the manuscript.
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