TMP 633 E
TMP 633 E
TMP 633 E
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
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 2.5. Analysis of phosphorylated amino acids
GMP was added. In other assays, radioactive ATP
was incubated in the presence of 10 mM AMP, To characterize the phosphoamino acids, protein
GMP, CMP or UMP. In each case, the reaction samples were ¢rst subjected to SDS-PAGE and elec-
was stopped by addition of an equal volume of troblotted onto an Immobilon PVDF membrane
2 Usample bu¡er and the mixture was heated at [12]. Phosphorylated proteins bound to the mem-
100³C for 5 min. brane were detected by autoradiography. The 32 P-
gel band corresponding to the 81-kDa protein was assays 2^4, ATP was replaced by another radioactive nucleotide. In
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
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 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
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-
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This work was supported by the Centre National 680^685.
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