From the Institut für Pharmakologie und
Toxikologie, Fakultät für Klinische Medizin Mannheim,
Universität Heidelberg, Maybachstrasse 14-16, D-68169 Mannheim,
the § Institut für Pharmakologie,
Universitätsklinikum Essen, D-45122 Essen, the ¶ Institut
für Physiologische Chemie, Ruhr-Universität Bochum,
D-44780 Bochum, and the
Innere Medizin III-Kardiologie,
Universität Heidelberg, D-69115 Heidelberg, Germany
Received for publication, October 8, 2002, and in revised form, November 29, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
G protein Heterotrimeric G proteins play a pivotal role in many signal
transduction pathways in eukaryotic cells. They consist of a guanine
nucleotide-binding A phosphotransfer reaction that uses G The purpose of this study was to test the hypothesis that NDPK may
represent the unknown co-factor and contributes to the phosphate
transfer via G Preparation of Rod Outer Segment Membranes, Purification of NDPK,
Purification of Gt and its Subunits, and Resolution of the
G
The Gt Purification of Gi/o Proteins, G Phosphorylation and Thiophosphorylation of G Determination of NDPK Activity--
The determination of the
NDPK activity in preparations obtained from solubilized bovine brain
membranes or Gt was performed in a reaction mixture (50 µl total volume) containing 50 mM triethanolamine hydrochloride, pH 7.4, 2 mM MgCl2, 50 µM ATP, and 1 µM [8-3H]GDP
(Amersham Biosciences). The reaction was started by addition of 20 µl
of the protein-containing solutions and conducted for 30 min at room
temperature. Termination of the reaction and analysis of GTP formation
were performed essentially as described (22).
Western Blot Analysis--
Proteins were separated by SDS-PAGE
(10-12% polyacrylamide in the resolving gel or modified gel) (23) and
electrotransferred to nitrocellulose membranes (Schleicher & Schuell).
Blots were washed with TBS (10 mM Tris-HCl, pH 7.4, 154 mM NaCl), incubated overnight at 4 °C with 3% skim milk
in TTBS (TBS + 0.05% Tween 20), washed with TTBS, and incubated with
the respective antisera (anti-G Immunoprecipitation--
The pooled fractions (60 µl) from the
hydroxyapatite column, containing the peak activity of NDPK from
Gt, were phosphorylated in a final volume of 80 µl at
30 °C for 7 min. The reaction was terminated by placing the mixture
on ice and adding an equal volume of 50 mM EDTA, pH 7.4. Precipitation buffer (280 µl), containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 25 mM EDTA, 1 mM dithiothreitol, 1 mM NaF, and 0.2 µM phenylmethylsulfonyl fluoride, was added, followed by
the addition of 10 µl of the same buffer containing 1 mg of protein
A-Sepharose beads. After incubation on ice for 20 min and
centrifugation (26,000 × g, 15 min), the clear
supernatant was taken and supplemented with 0.6 µg of anti-G Treatment of Gt Endoproteinase Glu-C Digest of Thiophosphorylated G Tryptic Digest of Thiophosphorylated Gt G
To identify the two proteins phosphorylated by
[ Enrichment of the G Identification of the NDPK B Isoform in the G Endoproteinase Glu-C and Tryptic Digest of Thiophosphorylated
G
Although G Meanwhile, several laboratories have reported the intermediate
formation of a high energy phosphoamidate bond on a histidine residue
of G The question arises whether NDPK B within the complex is the histidine
kinase required for the phosphorylation of G The NDPK B-enriched fraction obtained from bovine brain reconstituted
the phosphorylation and thiophosphorylation of G dimers can be
phosphorylated in membranes from various tissues by GTP at a histidine
residue in the
subunit. The phosphate is high energetic and can be
transferred onto GDP leading to formation of GTP. Purified G
dimers do not display autophosphorylation, indicating the involvement
of a separate protein kinase. We therefore enriched the
G
-phosphorylating activity present in preparations of the retinal G
protein transducin and in partially purified Gi/o
proteins from bovine brain. Immunoblots, autophosphorylation, and
enzymatic activity measurements demonstrated enriched nucleoside
diphosphate kinase (NDPK) B in both preparations, together with
residual G
dimers. In the retinal NDPK B-enriched fractions, a
G
-specific antiserum co-precipitated phosphorylated NDPK B, and an
antiserum against the human NDPK co-precipitated phosphorylated
G
. In addition, the NDPK-containing fractions from bovine brain
reconstituted the phosphorylation of purified G
. For
identification of the phosphorylated histidine residue, bovine brain
G
and Gt
were thiophosphorylated with guanosine 5'-O-(3-[35S]thio)triphosphate, followed by
digestion with endoproteinase Glu-C and trypsin, separation of the
resulting peptides by gel electrophoresis and high pressure liquid
chromatography, respectively, and sequencing of the radioactive
peptides. The sequence information produced by both methods identified
specific labeled fragments of bovine G
1 that overlapped
in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids
261-267). We conclude that NDPK B forms complexes with G
dimers
and contributes to G protein activation by increasing the high
energetic phosphate transfer onto GDP via intermediately phosphorylated
His-266 in G
1 subunits.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (40-52 kDa), a
subunit (33-43
kDa), and a
subunit (6-10 kDa). The latter two act as a functional
unit and only dissociate upon denaturation. Both G
and G
are
required for receptor-induced G protein activation and can trigger
effector functions (for reviews see Refs. 1-3). Heterotrimeric G
proteins are activated by a GDP/GTP exchange catalyzed by G
protein-coupled receptors. Furthermore, we and other laboratories
provided evidence that phosphotransfer reactions can participate in G
protein activation in vitro by formation of GTP. There is
amble evidence that nucleoside diphosphate kinase (NDPK)1 contributes to G
protein activation by replenishment of GTP from ATP and GDP (for
reviews see Refs. 4-6). Hypotheses suggesting a direct in
situ phosphorylation of GDP bound to G
and monomeric G proteins
(7-9) are most likely based on artifacts. Also complex formation of
NDPK with G proteins and channeling of NDPK-formed GTP into G
(10,
11) have not yet been proven beyond doubt.
subunits as phosphorylated
intermediates has been observed in various tissues (12-16). In this
reaction, the
-(thio)phosphate group of GTP or its analog GTP
S is
transferred onto a histidine residue of G
. Apparently, a
membrane-bound, so far unknown co-factor is required to achieve this
phosphorylation (14, 15). Out of the labile high energy phosphoamidate
bond, the phosphate can be retransferred onto GDP to form GTP, which
then can activates Gi and Gs proteins and thus regulates, for example, adenylyl cyclase activity (17). Nevertheless, the exact significance of this phosphotransfer reaction remains elusive.
. We report here that by the attempts to purify the
co-factor from the retinal G protein transducin (Gt) or
bovine brain membranes, we specifically enriched the NDPK B isoform. We
will further provide evidence for a complex formation of G
with
NDPK B and for a specific phosphorylation of His-266 of
G
1 in this complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phosphorylating Activity from Gt--
Rod outer
segment (ROS) membranes were prepared from illuminated bovine retina
according to Papermaster and Dreyer (18). NDPK was purified to
homogeneity from the cytosol essentially as described (19).
Gt was eluted from the membranes by repeated washing with
the GTP analog, GppNHp (100 µM), as described (12). The
Gt-containing eluate was concentrated by pressure
filtration with a PM 10 membrane (Amicon, Witten, Germany), and unbound
GppNHp was removed by a gel PD-10 gel filtration column (Amersham
Biosciences) (12). About 2 mg of purified Gt was
applied onto a Blue-Sepharose CL6B column (10-ml bed volume) at a flow
rate of 0.5 ml/min and separated into Gt
and
Gt
as described (12).
-containing fractions were pooled and applied onto
a 5-ml hydroxyapatite column (Econo Cartridge CHTII; Bio-Rad) at a flow
rate of 0.3 ml/min. The column was washed with 20 ml of a buffer
containing 10 mM Tris-HCl, pH 6.5, 6 mM
MgCl2, 1 mM dithiothreitol, and 20% (v/v)
glycerol. Bound proteins were eluted with a
KH2PO4 gradient (0-400 mM).
Fractions of 1 ml were collected and analyzed for the content of
Gt subunits, G
-phosphorylating activity, and NDPK
activity. Fractions containing the G
-phosphorylating activity were
pooled and stored at
80 °C.
, and the
G
-Phosphorylating Activity from Bovine Brain
Membranes--
Membranes from bovine brain were prepared as described
(20). Proteins were solubilized from membranes (4 g of protein) by stirring for 1 h at 4 °C in 800 ml of TEM buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA, 20 mM
2-mercaptoethanol) containing 1% (w/v) 1-octyl-1-
-D-thioglucopyranoside (Biomol, Hamburg,
Germany). After centrifugation for 40 min at 110,000 × g, ethylene glycol was added to the supernatants to a final
concentration of 30%. 900 ml of this extract, i.e. ~800
mg of protein, were used for three subsequent steps of liquid
chromatography, carried out at 4 °C, using a fast protein liquid
chromatography device. Elution was monitored by continuous
measurement of absorbance at 280 nm. The extract was first loaded onto
a column (10 × 13 cm) containing 1 liter of DEAE-Sepharose
(Amersham Biosciences) at a flow rate of 0.5 ml/min. After washing with
TEMEC (TEM buffer containing additionally 30% ethylene glycol and
0.9% (w/v) sodium cholate), elution was performed with a linear
gradient of NaCl (0-800 mM, volume 2.2 liters). Fractions
of 12 ml were collected. Gi/o proteins eluted from the
column at about 450 mM NaCl were further purified and
separated into their subunits as described (20, 21). Fractions capable
of phosphorylating G
subunits were identified in the phosphorylation
assay (see below), using 0.3 µg of bovine brain G
as substrate.
Positive fractions eluting at 220-270 mM NaCl were pooled
and concentrated by pressure filtration using a PM 10 membrane. This
pool (about 35 mg of protein) was then loaded onto a column (3 × 14 cm) containing 100 ml of hydroxyapatite (E. Merck, Darmstadt,
Germany) at a flow rate of 0.3 ml/min. The column was washed with
TEMEC. Proteins were eluted with a linear gradient of
K2HPO4/KH2PO4 (0-400
mM, pH 8, volume 300 ml), collecting fractions of 5 ml.
Phosphorylation-positive fractions eluting at phosphate concentrations
of 270-290 mM were pooled and concentrated, and the buffer
was exchanged into HEMEC (20 mM HEPES-NaOH, pH 6.5, 1 mM EDTA, 20 mM 2-mercaptoethanol, 30% ethylene
glycol, 0.9% sodium cholate). This pool (about 6 mg of protein) was
then loaded onto a XK16/20 column (Amersham Biosciences)
containing 25 ml of EMD SO3 650(S) (E. Merck), at
a flow rate of 0.2 ml/min. The column was washed with HEMEC. Elution
from the cation exchange column was performed with a linear gradient of
NaCl (0-1 M, volume 80 ml) in HEMEC. Fractions of 2 ml
were collected. Positive fractions eluting between 350 and 400 mM NaCl were concentrated by centrifugation in a
Centricon-30 device (Amicon). Chromatography media were regenerated according to the respective manufacturer's recommendations.
Subunits and
NDPK--
The indicated amounts of bovine brain or transducin G
dimers and column fractions of the purification procedures were
phosphorylated with 10 nM [
-32P]GTP
(PerkinElmer Life Sciences) for the indicated periods of time at
30 °C in a reaction buffer containing 50 mM
triethanolamine hydrochloride, pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol in a total volume of 20 µl. For
thiophosphorylation of G
, 20 nM
[35S]GTP
S was used, and incubation was for 30 min. The
reaction was terminated by the addition of one volume of sample buffer, followed by 1 h of incubation at room temperature. Proteins were separated by discontinuous SDS-PAGE on gels containing 10-12% (w/v)
acrylamide and autoradiographed.
(T-20; 1:1000) (Santa Cruz
Biotechnology, Inc.), anti-NDPK (C-20; 1:100) (Santa Cruz
Biotechnology, Inc.), anti G
i,o,t,z (D-15;
1:2000) (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-human NDPK
A (1:500) (kind gift of Dr. Ioan Lascu), and rabbit IgG anti human NDPK B (1:500) (kind gift of Dr. Ioan Lascu)) for 3 h at room temperature. Horse radish peroxidase-conjugated anti-rabbit IgG-antibody (1:10,000) was used as secondary antibody. Specific bands
were detected with the enhanced chemiluminescence system (Amersham Biosciences).
(T-20) or 4 µg of anti-NDPK (C-20) antiserum and 5 mg of protein
A-Sepharose beads. The mixture was gently shaken for 2 h at
4 °C. Protein A-Sepharose beads were pelleted and washed three times
with precipitation buffer containing 300 mM NaCl. Bound
proteins were eluted from the protein A-Sepharose beads by adding 50 µl of SDS buffer. The samples were kept at room temperature for at
least 1 h before loading onto a 10% polyacrylamide gel.
with Diethyl
Pyrocarbonate--
Purified Gt
(30 µg) was
incubated for 10 min at room temperature in a buffer (100 µl)
containing 10 mM triethanolamine hydrochloride, pH 7.4, and
10 mM diethyl pyrocarbonate. The reaction was terminated by
the addition of 25 mM EDTA. Free diethyl pyrocarbonate and EDTA were subsequently removed by repeated buffer exchange into 20 mM Tris-HCl, pH 8, 1 mM EDTA, 20 mM
2-mercaptoethanol using a Microcon-10 device (Amicon).
,
Purification, and Sequencing of Peptides--
Bovine brain G
(15 µg of protein) was thiophosphorylated with 100 nM
[35S]GTP
S and 1 µg of NDPK B-enriched co-factor as
described above, in a total volume of 100 µl. The reaction was
terminated by addition of 50 µl of 3-fold concentrated SDS-PAGE
sample buffer and 10 µg of endoproteinase Glu-C (Roche Molecular
Biochemicals). Proteins were digested for 3 h at 37 °C, and the
proteolytic fragments were separated on a Tris-Tricine-based system
(23). After electrotransfer onto a nitrocellulose membrane, the labeled
fragment was identified by autoradiography and excised. The peptide was
eluted, and its amino acid sequence was determined by automated Edman
degradation in an Applied Biosystems, Inc. (Foster City, CA) model 476A
protein sequencer.
,
Resolution, and Sequencing of Peptides--
Unmodified
Gt
(5 µg) was thiophosphorylated with 100 nM [35S]GTP
S and 1 µg of co-factor as
described above for 30 min in a total volume of 40 µl. Thereafter, 10 µg (20 µl) of unlabeled thiophosphorylated Gt
(12), 35 µl of 50 mM NH4CO3, pH
7.7, 5 µl acetonitrile, and 2.5 µg of trypsin were added. After
incubation overnight at 37 °C, the digest was injected directly into
an analytical (2 × 250 mm) Ultrasep Es 100 (E. Merck, Darmstadt,
Germany) C18 reverse phase HPLC column, equilibrated with a
mixture of 90% Solution A (0.2% hexafluoroacetone, NH3,
pH 8.6) and 10% Solution B (0.02% hexafluoroacetone, 84%
methylcyanide). The gradient was run from 10 to 80% Solution B at an
increase rate of 1%/min with a flow rate of 80 µl/min. The
absorbance was monitored at 215 and 295 nm, and peak fractions were
collected. 35S-Labeled peptides were detected by liquid
scintillation counting of 3 µl of each fraction. The sequence of the
peptide in the fraction containing the radioactivity peak was
determined by automated Edman degradation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phosphorylating Activity in Gt and Complex
Formation of Gt
with NDPK--
The
subunits of
the retinal G protein Gt can be transiently
(thio)phosphorylated by GTP or GTP
S and an enzymatic activity present in ROS membranes (12). More recently, it was demonstrated that
soluble preparations of Gt contain NDPK activity and that GTP phosphorylates a 36-kDa protein, most likely G
(22). We therefore attempted to identify the putative co-factor present in the
Gt preparations and investigated whether NDPK is involved in this reaction. For this purpose, Gt was eluted from ROS
membranes with 100 µM of the stable GTP analog, GppNHp,
which does not modify Gt
(12). Gt
and Gt
can be resolved from each other by affinity chromatography on Blue-Sepharose (12, 22). Whereas Gt
has little affinity to the matrix, elution of Gt
requires high salt concentrations (500 mM KCl). Single
protein bands at the apparent molecular weights of Gt
and Gt
were detected by SDS-PAGE and Coomassie Blue
staining (not shown) of the fractions from the first and second peak,
respectively. In search for the Gt
-phosphorylating activity, the fractions were subjected to phosphorylation with [
-32P]GTP. In the Gt
-containing
fraction, two phosphorylated proteins (Mm 18 and 36 kDa;
see Fig. 1) were detected. Measurement of
NDPK activity revealed a formation of 0.2 nmol GTP per mg of protein per min in this fraction. In line with data reported previously (12,
22), no phosphorylated proteins and no NDPK activity was detected in
the Gt
-containing fractions. To separate the two
phosphoproteins from Gt
, ~1 mg of protein from the
second peak was applied onto a hydroxyapatite column, and proteins were eluted with increasing concentrations of potassium phosphate (0-400 mM). Gt
eluted from the column in a single
peak at about 10 mM phosphate (fractions 23-26; see Fig.
1A). No NDPK activity and no phosphorylation by
[
-32P]GTP were detected in the Gt
peak.
The NDPK activity eluted from the column at ~250 mM
phosphate (Fig. 1B). As shown in the inset of
Fig. 1B, this fraction contained both the 18- and 36-kDa proteins phosphorylated by [
-32P]GTP. Immunoblot
analysis showed that this fraction contained NDPK protein,
Gt
, and traces of Gt
.
View larger version (27K):
[in a new window]
Fig. 1.
Resolution of NDPK from
Gt by chromatography on
hydroxyapatite. A, 1 mg of the Gt
pool
was applied onto an Econo Cartridge CHTII column. Bound proteins were
eluted with the indicated potassium phosphate gradient (dotted
line), and the protein content was monitored by absorbance at 280 nm. Gt
(inset) eluted from the column in a
single peak at 10 mM phosphate. B, the NDPK
activity of the eluate fractions was measured as formation of
[3H]GTP as described under "Experimental Procedures."
The fractions containing the NDPK activity peak were pooled and
phosphorylated with [
-32P]GTP. An autoradiograph after
SDS-PAGE is shown (P, inset). The content of NDPK
protein, Gt
, and Gt
was determined by
Western blot (WB, inset).
-32P]GTP, we immunoprecipitated G
and NDPK with
specific antisera after phosphorylation. Identical amounts of IgG were
used as controls. As shown in Fig. 2, the
NDPK-specific antiserum precipitated NDPK as a doublet migrating at
18-20 kDa and an additional phosphoprotein at 36 kDa. The
G
-specific antiserum precipitated phosphorylated G
(36 kDa) and
an additional phosphoprotein at 18-20 kDa. Some minor phosphorylated
bands in the range of 28 to 30 kDa most likely represent degradation
products of phosphorylated G
.
View larger version (64K):
[in a new window]
Fig. 2.
Co-immunoprecipitation of NDPK and
Gt . The
proteins in 60 µl of the NDPK activity pool obtained from the
hydroxyapatite column were phosphorylated with
[
-32P]GTP for 7 min at 30 °C and subjected to
immunoprecipitation with the anti-NDPK antibody (left panel,
right lane) or anti-G
antibody (right panel,
right lane). Identical amounts of IgG were used as control
(left lanes). Autoradiographs after SDS-PAGE are
shown.
-Phosphorylating Activity from Bovine Brain
Membranes and Reconstitution of G
Phosphorylation--
As
Gt differs from other G proteins by its solubility without
detergent, we attempted to purify the G
-phosphorylating activity from another tissue. Because heterotrimeric G proteins are abundant in
bovine brain membranes, and the extent of G
phosphorylation is
rather high compared with membranes of other tissues available in
sufficient amounts (15), we first passed a detergent extract from
bovine brain membranes over a DEAE column. G proteins that elute from
the column at about 450 mM NaCl could no longer be phosphorylated with [
-32P]GTP (15) (not shown).
Apparently, the co-factor that promotes phosphorylation had been
separated from the majority of G proteins. Addition of the fraction
eluting at about 250 mM NaCl to the G protein-containing
fraction or purified bovine brain G
reconstituted the
phosphorylation. The fraction that promoted G
phosphorylation was
further purified by hydroxyapatite chromatography, followed by cation
exchange chromatography using an EMD SO3 650(S) column. After each step, positive fractions were identified by their potential to phosphorylate G
. Equal amounts of protein (1 µg) were used after each step to reconstitute the phosphorylation of purified bovine
brain G
(0.5 µg). Phosphorylated G
subunits were excised from SDS-PAGE gels, and the amount of radioactivity was detected by
liquid scintillation counting, to estimate the specific activity after
each purification step. The data are summarized in Table I. A
small decrease in specific activity after the
EMD SO3 cation exchange
column was noted. However, the chromatogram showed that 97% of total
protein could be removed during this step. Thus, the decrease in
specific activity might have been because of decaying enzymatic
activity in the diluted protein fraction during longer storage at
4 °C. SDS-PAGE and silver staining revealed that the fractions
contained four to five faint protein bands in the molecular range from
20 to 50 kDa (not shown). Similar to the G
-phosphorylating activity
prepared from Gt, NDPK activity (1.8 nmol of GTP formed per
mg protein per min) and NDPK protein (immunoblot,
autophosphorylation) could be detected (Fig.
3A). To test whether this
preparation is able to reconstitute the phosphorylation of bovine
G
, increasing amounts of the EMD SO3 650(S) pool
(0.04-0.4 µg of protein) were phosphorylated with
[
-32P]GTP in the absence and presence of purified
G
(0.5 µg) for 5 min at 30 °C (Fig. 3B).
Conversely, the phosphorylation of increasing amounts of
purified bovine brain G
dimers promoted by a constant quantity of
partially purified factor (200 ng of protein) was studied (Fig.
3C). The phosphorylation of G
increased with the amount
of added co-factor fraction or G
dimer. Maximal phosphorylation was observed with 1-3 µg of G
dimer. Similar data were
obtained when Gt
was used as substrate (Fig.
3D). Previous results showed that pretreatment of membranes
with diethyl pyrocarbonate prevents the phosphorylation of G
because
of the ethoxycarbonylation of histidine residues (13, 24). Similarly,
Gt
pretreated with diethyl pyrocarbonate (10 mM) prior to phosphorylation with the co-factor-containing
fraction exhibited no increase in phosphorylation of the
ethoxycarbonylated Gt
(Fig. 3D). In addition,
phosphorylated G
was sensitive to treatment with hydroxylamine (data
not shown) that cleaves phosphohistidine (25). As shown in Fig.
3E, the co-factor-containing fraction also reconstituted the
thiophosphorylation of G
by [35S]GTP
S. When higher
amounts of the co-factor pool were used, the phosphorylated G
was
detected also in the absence of added G
(Fig. 3, A,
B, and D). Indeed, small amounts of G
could be identified in the co-factor pool by Western blot analysis (Fig. 3A).
Summary of the enrichment of the G-phosphorylating activity from
bovine brain membranes
View larger version (33K):
[in a new window]
Fig. 3.
Reconstitution of the G
(thio)phosphorylation of G
. A, the co-factor
required for G
phosphorylation in solubilized bovine brain membranes
was enriched by subsequent anion exchange, hydroxyapatite, and cation
exchange chromatography as described under "Experimental
Procedures." The fractions obtained from the cation exchange column
at 350-380 mM NaCl were pooled. After phosphorylation with
[
-32P]GTP for 5 min at 30 °C, 800 ng of protein
were subjected to SDS-PAGE. Phosphorylated proteins were visualized by
autoradiography. G
and NDPK were detected by Western blotting with
specific antisera. B, increasing amounts (40-400 ng of
protein) of the co-factor pool (CF) were phosphorylated in
the absence (
) and presence (+) of 0.5 µg of purified bovine brain
G
for 5 min at 30 °C. C, increasing amounts of
G
(10-3000 ng) were phosphorylated in the presence of 200 ng of
the cation exchange fraction. D, Gt
was
treated with 10 mM diethyl pyrocarbonate as described under
"Experimental Procedures." Thereafter, phosphorylation of G
by
[
-32P]GTP was determined with the cation exchange
fraction in the absence (Control) and presence of 2 µg
untreated (Gt
) or diethyl
pyrocarbonate-treated Gt
(Gt
/DEPC). E, 200 ng
of the cation exchange fraction were thiophosphorylated with 20 nM [35S]GTP
S in the absence
(Control) and presence of 1 µg of purified bovine brain
G
for 30 min at 30 °C. Autoradiographs after SDS-PAGE are
shown.
-Phosphorylating
Co-factor Pools--
Two major isoforms of NDPK, NDPK A (nm23-H1) and
NDPK B (nm23-H2), are present in the bovine retina ROS (19) and other
tissues (5, 6). We therefore investigated whether one or both forms had
been enriched during the purification procedures for the
G
-phosphorylating activity by Western blot analysis with
subtype-specific antisera (kindly provided by Dr. Ioan Lascu,
Bordeaux, France) (Fig. 4). Purified recombinant human NDPK A and B (also kindly provided by Dr.
Ioan Lascu) and purified NDPK from the cytosol of bovine ROS served as
positive controls. Although the anti-NDPK A antiserum was more
sensitive than the anti-NDPK B antiserum, only NDPK B was detected in
the G
-phosphorylating fractions obtained from Gt and
bovine brain membranes.
View larger version (38K):
[in a new window]
Fig. 4.
Immunoblot analysis of the NDPK isoforms in
the G -phosphorylating activities from bovine
retina and bovine brain. Purified recombinant human NDPK A
(A, 10 ng; rec) or NDPK B (B, 10 ng;
rec), 500 ng of NDPK purified from the cytosol of bovine
ROS, the G
-phosphorylating activity from bovine retina (30 µl;
Gt
-NDPK), and bovine brain (1 µg; G
-NDPK) were subjected to SDS-PAGE and
immunoblot analysis with the NDPK isoform-specific antisera, rabbit IgG
anti-human NDPK A (A) or rabbit IgG anti-human NDPK B
(B). Duplicate values are shown.
and Identification of the Phosphorylated Histidine in
G
1--
Endoproteinase Glu-C cleaves proteins at the
C-terminal end of glutamate residues and can additionally be used for
in gel digests of proteins in a SDS-containing buffer. Complete
digest of G
1 by endoproteinase Glu-C would produce
several peptides in the range of 1000 to 13,500 kDa. Each contains not
more than three histidine residues. We therefore asked whether we could detect a labeled phosphopeptide after in gel digest with
endoproteinase Glu-C and separation of the peptides by high resolution
SDS-PAGE (19). As shown in Fig.
5A, endoproteinase Glu-C
digest of phosphorylated bovine brain G
produced a single labeled
peptide (Mm ~9 kDa). The same peptide is recognized by
the anti-G
(T-20) antibody (Fig. 5B). To identify the
phosphopeptide, 15 µg of bovine brain G
were thiophosphorylated
with 100 nM [35S]GTP
S and digested with
endoproteinase Glu-C. After SDS-PAGE and electrotransfer onto
nitrocellulose, the labeled peptide was identified by autoradiography
and excised from the blot. The peptide was eluted and sequenced by
Edman degradation (26). The experiment was repeated twice. The first
run produced the sequence
LMXYXXDXII. In the second run,
the 14-amino acid sequence, LMTYSHDNIICGIT, could be identified. This
sequence corresponds to the N terminus of a peptide (amino acid
261-340; Mm 8657.71) that results from endoproteinase
Glu-C digest of G
1 and contains two histidine residues,
His-266 and His-311.
View larger version (51K):
[in a new window]
Fig. 5.
Digest of phosphorylated bovine brain
G with endoproteinase
Glu-C. A, purified bovine brain G
(1 µg) was
phosphorylated with [
-32P]GTP and 200 ng of the NDPK
B-enriched co-factor fraction for 5 min at 30 °C. The reaction was
stopped by addition of sample buffer. In lane 2, proteins
were digested with 0.5 µg of endoproteinase Glu-C for 3 h at
37 °C, and the proteolytic fragments were subjected to SDS-PAGE. An
autoradiograph is shown. B, Western blot analysis of
purified bovine brain G
(1 µg) before (lane 1) and
after (lane 2) digest with 0.5 µg endoproteinase
Glu-C.
1 complexed with different G
subunits is
abundant in bovine brain G
preparations, at least
G
2
x dimers are similarly present
(27). We therefore used the well defined Gt
, i.e. G
1
1, for further
analysis. Unmodified Gt
was thiophosphorylated with
100 nM [35S]GTP
S and the co-factor from
bovine brain (see Fig. 3) and added to 10 µg of unlabeled
thiophosphorylated Gt
. The mixture was digested
overnight with trypsin, the peptides were separated by HPLC on a
C18 reverse phase HPLC column, and peak fractions were collected (Fig. 6A). An
aliquot of each fraction (3 µl) was counted for radioactivity. As
shown in Fig. 6B, the labeled peptide eluted from the column
in a sharp peak at fraction 22. The analysis of this fraction by Edman
degradation revealed the sequence ADQELMXYSHD. It
corresponds to the N terminus of an expected tryptic fragment (amino
acid 257-280; Mm 2659.96) that overlaps with the labeled fragment of the endoproteinase Glu-C digest. Most important, it contains only His-266 of G
1.
View larger version (20K):
[in a new window]
Fig. 6.
Resolution of the tryptic fragments of
Gt by HPLC.
Gt
was thiophosphorylated with
[35S]GTP
S and digested with trypsin overnight as
described under "Experimental Procedures." The proteolytic
fragments were separated by HPLC on a C18 reverse phase
HPLC column. The elution of the peptides was monitored by UV light
absorption at 215 and 295 nm (A), and peak fractions were
collected. An aliquot of 3 µl of each fraction was counted for
35S in a liquid scintillation counter (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits (12-17) that enables the formation of GTP by
transferring this phosphate onto GDP. The phosphorylation of G
requires a co-factor that acts as a histidine kinase (15, 16, 28). In
addition, early studies on an NDPK-like activity leading to formation
of GTP from ATP and GDP in retinal extracts from frogs associated this
so called GDP kinase activity with the Gt
dimer (29).
In this study, we obtained several lines of evidence for a complex
formation of the NDPK B isoform with G
dimers. First, NDPK B and
G
were co-purified from both transducin and solubilized bovine
brain membranes with different purification protocols. Apparently, the
biochemical properties of the NDPK determined the purification of the
complex. In contrast to G
, NDPK binds tightly to Blue-Sepharose,
and this property can be used to purify NDPK (19). Recently, the NDPK
of Drosophila melanogaster was purified by a combination of
anion exchange and hydroxyapatite chromatography (30).
G
1
2 and Gt
exhibit only
a weak interaction with hydroxyapatite and elute from the matrix at
much lower concentrations of phosphate (31, 32). Second, a
G
-specific antiserum co-immunoprecipitated NDPK, and conversely, an
NDPK-specific antiserum co-immunoprecipitated G
from the retinal
preparation. In agreement with our data more indirect evidences for an
interaction of NDPK B with Gt have been described
previously by others (11, 22). The presence of NDPK B and G
or
heterotrimeric G proteins, however, is apparently not sufficient to
obtain the complex formation between G
and NDPK B to reconstitute
the phosphorylation of G
. Most likely, another protein is required
as scaffold for that complex. Recently, it was demonstrated that NDPK
forms a complex with phocein, Eps 15, and dynamin I, a GTPase that
plays a critical role in endocytosis (33). Within this complex, NDPK
interacts with dynamin I through a proline-rich domain, whereas its
interaction with phocein is ill defined. The data, however, indicate
that complexes of NDPK with multiple proteins occur as mechanisms for
local GTP replenishment within cells.
. Protein kinase
activity for NDPK has been described. Serine and threonine phosphorylation by NDPK was found on histone 2b, casein, and ovalbumin (34, 35). Moreover, NDPK phosphorylates the catalytic histidine residue
in ATP citrate lyase (36), and other reports (37-39) indicate that
NDPK is the phosphate donor for the phosphorylation of a histidine in
annexin I. Most recently, Kowluru (28) reported that the
phosphorylation of G
and histone 4 was largely increased by
mastoparan, a known activator of NDPK and G proteins (40, 41). In
accordance with the presumed histidine kinase activity of NDPK B, an
increase in G
phosphorylation was observed in membranes of H10 cells
overexpressing wild-type NDPK B but not its catalytically inactive
mutant H118N (42).
by GTP and GTP
S,
respectively. As observed before in membranes (13, 15, 16),
phosphorylated G
was sensitive to hydroxylamine cleavage (25) and
was prevented by ethoxycarbonylation with diethyl pyrocarbonate (23).
Thus, all our results in the reconstituted systems are in agreement
with earlier results obtained on the (thio)phosphorylation of a
histidine in G
. The data obtained from proteolytic digest of the
thiophosphorylated bovine brain G
and Gt
revealed that His-266 of G
1 is the phosphorylated residue in G
. As shown in Fig. 7, the
imidazolyl side chain of His-266 is freely accessible on the surface of
the heterotrimeric G protein and can therefore be the target of protein
phosphorylation. The seven other histidines are part of the G
propeller structure and thus are unlikely accessible to kinases.
His-266 is conserved in G
2, G
3, and
G
4 but is found not in G
5, which has a
lysine at the analogous position (43). Although we have no proof for this hypothesis at this time, we propose that, based on the high degree
of homology mammalian, G
1-G
4 can be
phosphorylated at His-266. The mainly neuronally expressed
G
5 functionally differs from the other G
subunits. In
accordance with the lack of the respective histidine, we were not able
to detect the phosphorylation of G
5
2 (kindly provided
by Dr. B. Nürnberg, Düsseldorf, Germany) in our
reconstitution assay (data not shown). Two non-mammalian G
subunits
are also lacking a histidine at the analogous position. These are
G
2 of D. melanogaster, which has a
proline at this position, and G
(Ste4p) from Saccharomyces
cerevisiae. Interestingly, Ste4p has a 41-amino acid insertion at
this position. Amino acids within this insertion are target to
phosphorylation, which is necessary for adaptation of the mating
response in yeast (44, 45). In summary, His-266 is apparently not
required for formation of the seven propeller blade G
structure and
is placed on the surface of the heterotrimer at a position where
phosphorylation is likely to occur. Therefore, we propose that NDPK B
phosphorylates the structurally exposed His-266 in G
in a
stoichiometric complex with G
.
View larger version (30K):
[in a new window]
Fig. 7.
Localization of His-266 in the
three-dimensional structure of the
G i1GDP
1
2
heterotrimer. Ribbon diagram (A) and calotte
model (B) of G
i1GDP (magenta),
-
1 (blue), and -
2
(brown). Histidine residues are marked in white.
An arrow points to the position of His-266. The GDP molecule
is given in green.
A phosphotransfer from NTP to His-118 in NDPK B and subsequently onto
His-266 of G and further onto GDP might offer an explanation for
several reports indicating a higher potency of NDPK-formed GTP compared
with exogenously added GTP in G protein activation (10, 46, 47).
However, the three-dimensional structure of the heterotrimeric G
protein (48) (Fig. 7) indicates that His-266 is distant from the GDP
molecule within the G
subunit. Thus, a direct transfer onto
G
-bound GDP is not supported by these structural data. Nevertheless,
the drastic increase in adenylyl cyclase activity as a result of an
overexpression of NDPK B and Gs
(42) might be an
indication for the high efficiency of the phosphotransfer to activate G proteins.
Both purification procedures revealed that the vast majority of G
proteins do not co-purify and thus are not complexed with NDPK B. Therefore, they are most likely not accessible to the phosphate
transfer reaction. Although it is likely that purification, especially
solubilization from the membrane environment, could cause a substantial
dissociation of NDPK B from G proteins, the so far unknown scaffolding
protein mentioned above could be the limiting factor within the complex
of NDPK B and G. Moreover, a low abundance of the NDPK-G protein
complex would fit into a concept where NDPK exclusively regulates the
basal tone of G protein activities and where the vast majority of G
proteins serve the "classical" receptor signal transduction,
as discussed in the accompanying paper (42).
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 49-621-330030; Fax: 49-621-3300333; E-mail: thomas.wieland@urz.uni-heidelberg.de.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210304200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NDPK, nucleoside
diphosphate kinase;
GTPS, guanosine 5'-O-(3-thio)triphosphate;
GppNHp, guanosine-5'-O-(
,
-imino)triphosphate;
ROS, rod outer
segment;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
HPLC, high pressure liquid chromatography;
Mm, molecular
mass.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Offermanns, S., and Simon, M. I. (1996) Cancer Surv. 27, 177-198[Medline] [Order article via Infotrieve] |
2. | Wieland, T., Schulze, R. A., and Jakobs, K. H. (1996) in Molecular Mechanisms of Signalling and Targeting (Wirtz, K. W. A., ed) , pp. 1-24, Springer-Verlag, Berlin, Heidelberg, New York |
3. |
Neves, S. R.,
Ram, P. T.,
and Iyengar, R.
(2001)
Science
296,
1636-1639 |
4. | Piacentini, L., and Niroomand, F. (1996) Mol. Cell. Biochem. 157, 59-63[Medline] [Order article via Infotrieve] |
5. | Kimura, N., Shimada, N., Fukuda, M., Ishijima, Y., Miyazaki, H., Ishii, A., Takagi, Y., and Ishikawa, N. (2000) J. Bioenerg. Biomembr. 32, 309-315[CrossRef][Medline] [Order article via Infotrieve] |
6. | Otero, A. S. (2000) J. Bioenerg. Biomembr. 32, 269-275[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Kikkawa, S.,
Takahashi, K.,
Takahashi, K.,
Shimada, N., Ui, M.,
Kimura, N.,
and Katada, T.
(1990)
J. Biol. Chem.
265,
21536-21540 |
8. | Randazzo, P. A., Northup, J. K., and Kahn, R. A. (1991) Science 254, 850-853[Medline] [Order article via Infotrieve] |
9. |
Zhu, J.,
Tseng, Y. H.,
Kantor, J. D.,
Rhodes, C. J.,
Zetter, B. R.,
Moyers, J. S.,
and Kahn, C. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14911-14918 |
10. | Wieland, T., and Jakobs, K. H. (1992) Mol. Pharmacol. 42, 731-735[Abstract] |
11. | Orlov, N. Y., Orlova, T. G., Nomura, K., Hanai, N., and Kimura, N. (1996) FEBS Lett. 389, 186-190[CrossRef][Medline] [Order article via Infotrieve] |
12. | Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716[Abstract] |
13. |
Wieland, T.,
Nürnberg, B.,
Ulibarri, I.,
Kaldenberg-Stasch, S.,
Schultz, G.,
and Jakobs, K. H.
(1993)
J. Biol. Chem.
268,
18111-18118 |
14. | Hohenegger, M., Mitterauer, T., Voss, T., Nanoff, C., and Freissmuth, M. (1996) Mol. Pharmacol. 49, 73-78[Abstract] |
15. | Nürnberg, B., Harhammer, R., Exner, T., Schulze, R. A., and Wieland, T. (1996) Biochem. J. 318, 717-722[Medline] [Order article via Infotrieve] |
16. | Kowluru, A., Seavey, S. E., Rhodes, C. J., and Metz, S. A. (1996) Biochem. J. 313, 97-107[Medline] [Order article via Infotrieve] |
17. |
Wieland, T.,
Ronzani, M.,
and Jakobs, K. H.
(1992)
J. Biol. Chem.
267,
20791-20797 |
18. | Papermaster, D. S., and Dreyer, W. J. (1974) Biochemistry 13, 2438-2444[Medline] [Order article via Infotrieve] |
19. | Abdulaev, N. G., Karaschuk, G. N., Ladner, J. E., Kakuev, D. L., Yakhyaev, A. V., Tordova, M., Gaidarov, I. O., Popov, V. I., Fujiwara, J. H., Chinchilla, D., Eisenstein, E., Gilliland, G. L., and Ridge, K. D. (1998) Biochemistry 37, 13958-13967[CrossRef][Medline] [Order article via Infotrieve] |
20. | Sternweis, P. C., and Pang, I.-H. (1990) in Receptor-Effector Coupling. A Practical Approach (Hulme, E. J., ed) , pp. 1-30, Oxford University Press, Oxford |
21. | Nürnberg, B., Spicher, K., Harhammer, R., Bosserhoff, A., Frank, R., Hilz, H., and Schultz, G. (1994) Biochem. J. 300, 387-394[Medline] [Order article via Infotrieve] |
22. |
Klinker, J. F.,
and Seifert, R.
(1999)
Eur. J. Biochem.
261,
72-80 |
23. | Schägger, H., and von Jagow, G. (1978) Anal. Biochem. 166, 368-379 |
24. | Miles, E. W. (1977) Methods Enzymol. 47, 431-442[Medline] [Order article via Infotrieve] |
25. | Hokin, L. S., Sastry, P. S., Galsworthy, P. R., and Yoda, A. (1965) Proc. Natl. Acad. Sci. U. S. A. 54, 177-184[Medline] [Order article via Infotrieve] |
26. | Edman, P. (1950) Acta Chem. Scand. 4, 277-282 |
27. |
Asano, T.,
Morishita, R.,
Matsuda, T.,
Fukada, Y.,
Yoshizawa, T.,
and Kato, K.
(1993)
J. Biol. Chem.
268,
20512-20519 |
28. | Kowluru, A. (2002) Biochem. Pharmacol. 63, 2091-2100[CrossRef][Medline] [Order article via Infotrieve] |
29. | Tishchenkov, V. G., and Orlov, N. Y. (1984) Mol. Biol. (Mosc.) 18, 776-785 |
30. | Stenberg, L. M., Stenflo, J., Holmgren, P., and Brown, M. A. (2002) Biochem. Biophys. Res. Commun. 295, 689-694[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Fung, B. K.,
Lieberman, B. S.,
and Lee, R. H.
(1992)
J. Biol. Chem.
267,
24782-24788 |
32. |
Nakamura, F.,
Kato, M.,
Kameyama, K.,
Nukada, T.,
Haga, T.,
Kato, H.,
Takenawa, T.,
and Kikkawa, U.
(1995)
J. Biol. Chem.
270,
6246-6253 |
33. |
Baillat, G.,
Gaillard, S.,
Castets, F.,
and Monneron, A.
(2002)
J. Biol. Chem.
277,
18961-18966 |
34. | Hemmerich, S., and Pecht, I. (1992) Biochemistry 31, 4580-4587[Medline] [Order article via Infotrieve] |
35. | Inoue, H., Takahashi, M., Oomori, A., Sekiguchi, M., and Yoshioka, T. (1996) Biochem. Biophys. Res. Commun. 218, 887-892[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Wagner, P. D.,
and Vu, N. D.
(1995)
J. Biol. Chem.
270,
21758-21764 |
37. |
Muimo, R.,
Banner, S. J.,
Marshall, L. J.,
and Mehta, A.
(1998)
Am. J. Respir. Cell Mol. Biol.
18,
270-278 |
38. |
Muimo, R.,
Hornickova, Z.,
Riemen, C. E.,
Gerke, V.,
Matthews, H.,
and Mehta, A.
(2000)
J. Biol. Chem.
275,
36632-36636 |
39. | Treharne, K. J., Riemen, C. E., Marshall, L. J., Muimo, R., and Mehta, A. (2001) Pflügers Arch. 443 Suppl. 1, S97-S102[Medline] [Order article via Infotrieve] |
40. | Klinker, J. F., Hagelüken, A., Grünbaum, L., Heilmann, I., Nürnberg, B., Harhammer, R., Offermanns, S., Schwaner, I., Ervens, J., Wenzel-Seifert, K., Schultz, G., and Seifert, R. (1994) Biochem. J. 304, 377-383[Medline] [Order article via Infotrieve] |
41. | Odagaki, Y., Nishi, N., and Koyama, T. (1997) Br. J. Pharmacol. 121, 1406-1412[Abstract] |
42. | Hippe, H.-J., Lutz, S., Cuello, F., Knorr, K., Vogt, A., Jakobs, K. H., Wieland, T., and Niroomand, F. (2003) J. Biol. Chem. 278, 7227-7233 |
43. | Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374[CrossRef][Medline] [Order article via Infotrieve] |
44. | Cole, G. M., and Reed, S. I. (1991) Cell 64, 703-716[Medline] [Order article via Infotrieve] |
45. |
Grishin, A. V.,
Weiner, L. J.,
and Blumer, K. J.
(1994)
Genetics
138,
1081-1092 |
46. | Jakobs, K. H., and Wieland, T. (1989) Eur. J. Biochem. 183, 115-121[Abstract] |
47. | Niroomand, F., Mura, R., Jakobs, K. H., Rauch, B., and Kübler, W. (1997) J. Mol. Cell. Cardiol. 29, 1479-1486[CrossRef][Medline] [Order article via Infotrieve] |
48. | Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058[Medline] [Order article via Infotrieve] |