From the Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany
Received for publication, June 26, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Phosducin-like protein (PhLP) is a
member of the phosducin family of G-protein Phosducin-like protein
(PhLP)1 was initially cloned
as a product of an ethanol-responsive gene in NG108-15 neuroblastoma
x-glioma cells (1). PhLP has a high sequence homology to the G-protein Regulation of the homologous protein phosducin has been found to occur
by phosphorylation via the serine/threonine kinases PKA, GRK, or
CaM-dependent kinase II (7-11). Phosphorylation of phosducin reduces the affinity for G Materials--
[32P]ATP,
[32P]orthophosphoric acid, and
myo-[2-3H]inositol were purchased from
PerkinElmer Life Sciences. The kinase inhibitors staurosporine,
BAPTA-AM, and
5,6-dichloro-1- Construction of Expression Vectors--
All of the cDNAs
used in these studies were subcloned into pcDNA3. The cDNA for
PhLPL was originally cloned from rat brain (2). The
construction of deletion mutants and Ser/Thr to Ala mutants of
PhLPL were done by a PCR-based strategy and confirmed by
automated sequencing.
Cell Culture, Transient Transfection, and Determination of
Inositol Phosphates--
Human embryonic kidney (HEK) 293 cells were
grown in DMEM, 10% fetal calf serum and were kept in 7%
CO2 humidified atmosphere. Cells were transfected by using
the CaPO4 method (16) on 10-cm dishes at 70%
confluency with a constant total amount of DNA. Transfection efficiency
was usually between 60 and 80% and was equal for all of the cDNAs
used as controlled by transfection of different green fluorescent
protein-tagged constructs. For determination of inositol phosphates,
cells were seeded in six-well plates and labeled with
myo-[2-3H]inositol (2 µCi/ml; specific
activity 21 Ci/mmol) for 16 h in inositol-free RPMI 1640 medium
containing 0.2% fetal calf serum. Unlabeled cells from the same
transfection also seeded in six-well plates were used for Western
blotting to control expression levels. After labeling, cells were
washed once in incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.6 mM CaCl2, 1 g/liter glucose, 20 mM
Na+-HEPES, pH 7.3) and then incubated at 37 °C for 20 min in incubation buffer containing 10 mM LiCl. Reactions
were stopped, and inositol phosphates were extracted as described
previously (17). Inositol determinations were performed in triplicates,
and results were analyzed as the means ± S.E. of at least three
independent experiments. ANOVA and post-test comparison (Bonferroni)
were performed as appropriate. For the control of expression levels,
cells of one well were lysed in 250 µl of hypotonic lysis buffer (1%
Triton X-100, 20 mM Tris base, pH 10.5) for 15 min on ice
and then centrifuged. Supernatants were analyzed by Western blots using
polyvinylidene difluoride membranes (Millipore) and anti-PhLP-CT
antibodies. Detection was performed with goat anti-rabbit-horseradish
peroxidase antibodies and the Uptilight horseradish peroxidase-blotting
kit (Interchim).
Phosphorylation of Phosducins--
Phosducin, PhLPL,
and PhLPS were purified from Escherichia coli as
C-terminally His6-tagged proteins (5). PKA phosphorylation was performed as described (10), and phosphoproteins were visualized by
exposure to Biomax MS films (Eastman Kodak Co.). CK2 kinase assays were
performed with recombinant human CK2 enzyme (Roche Molecular
Biochemicals) or cell extracts. In kinase assays using recombinant
human CK2, a 50-µl volume containing 400 nM recombinant PhLPL, PhLPS or phosducin, CK2 buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl, 5 mM dithiothreitol), 0.04 or 0.3 milliunit of CK2, and 200 µM ATP (1 µCi of
[32P]ATP) was incubated at 30 °C for 30 min or at
37 °C for 120 min. The reaction was terminated by boiling samples in
Laemmli buffer followed by SDS-PAGE, Coomassie Blue staining, and
phosphorimaging. For kinase assays using HEK cell cytosol, a lysate was
prepared by resuspending cells of one 15-cm dish in 5 ml of CK2 buffer (supplemented with 1 mM PMSF), disrupting the cells by
sonication and clearing the lysate by centrifugation (20,000 × g, 10 min). Protein content was determined in the
supernatant by the Bradford method. Phosphorylation was then performed
by adding 150 µg of total protein of cell extract to a 250-µl
reaction with 400 nM His6-tagged protein and 10 µCi of [32P]ATP and incubated at 30 °C for 30 min.
Reactions were then placed on ice, diluted with 5 volumes of ice-cold
pull-down buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl), supplemented with 50 µl of Ni-NTA-agarose
(Qiagen), and rotated at 4 °C for 10 min. The beads were then
washed, boiled, and analyzed by SDS-PAGE and phosphorimaging. Phosphorylation of PhLP in intact HEK 293 cells was done essentially as
described with minor modifications (18). 48 h after transfection and 36 h after seeding in six-well plates, HEK 293 cells were washed in phosphate- and serum-free DMEM supplemented with 20 mM Na+-HEPES, pH 7.4, (labeling buffer) and
then incubated in the same buffer for 30 min. Labeling of the
intracellular ATP pool was then performed with 200 µCi/ml
[32P]orthophosphoric acid (specific activity 10 mCi/ml)
for 2 h at 30 °C. Where indicated, cells were stimulated with 1 mM carbachol for 5 min. Cells were immediately placed on
ice and washed once with ice-cold buffer. The cells then were lysed on
ice with 1 ml of lysis buffer (300 mM NaCl, 50 mM NaF, 5 mM
Na4P2O7, 5 mM EDTA-Na2, 0.1 mM
Na3VO4, 1% Triton X-100, 0.01%
NaN3, 50 mM Tris-HCl, pH 7.2, freshly
supplemented with 10 mM iodoacetamide and 1 mM PMSF). After 20 min, the Triton X-100-insoluble fractions were removed
by centrifugation (20,000 × g for 15 min) and PhLP was immunoprecipitated from the supernatants by the anti-PhLPS
antibody precoupled to protein A-Sepharose for 2 h at 4 °C.
After washing, immunoprecipitates were subjected to SDS-PAGE, and
radiolabeled PhLP was visualized and quantified by phosphorimaging.
Expression of PhLP was controlled by Western blotting of an aliquot
obtained from the centrifuged lysates (with the use of the anti-PhLP-CT antibody).
Tissue Preparation and Dephosphorylation--
Mouse heart,
brain, adrenal gland, and embryo (day 10.5 post-conception) were
homogenized in five volumes of the same lysis buffer as described above
(containing phosphatase inhibitors). After centrifugation and
adjustment of total protein levels in supernatants as indicated,
samples were subjected to SDS-PAGE and Western blotting. In the case of
dephosphorylation experiments, lysis was performed in 40 mM
Tris base, pH 10.5, 1% Triton X-100, and 1 mM PMSF
(phosphatase inhibitors were omitted), samples were cleared by
centrifugation (20,000 × g for 10 min), and protein content was determined by the Bradford method. Dephosporylation was
then performed by adding 50 µg of HEK cell lysate or 100 µg of
brain lysate to a 50-µl reaction with phosphatase buffer (50 mM Tris-HCl, pH 7.5, 2 mM MnCl2,
0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij
35) and with or without 1000 units of Inhibition of Inositol Phosphate Signaling in Intact HEK 293 Cells--
To investigate the functional role of PhLPL and
PhLPS as G
To analyze further the different effects of PhLPL and
PhLPS on G Phosphorylation of PhLPL and PhLPS in HEK
293 Cells--
To analyze the role of phosphorylation in intact cells
as a possible regulatory mechanism, we labeled HEK 293 cells with
[32P]orthophosphate and looked for basal- and
receptor-stimulated phosphorylation. PhLP cDNAs were co-transfected
with M3 muscarinic receptor cDNA. The M3
muscarinic receptor was chosen because of the prominent inositol
phosphate signal that can be achieved by its stimulation (data not
shown). 48 h after transfection, the receptors were stimulated
with carbachol for 5 min (Fig. 3).
PhLPL exhibited a markedly higher degree of basal
phosphorylation compared with PhLPS. However, in both
cases, stimulation of the M3 receptor by carbachol did not
enhance the extent of phosphorylation (Fig. 3A, left
panel). Fig. 3B summarizes a series of similar
experiments demonstrating that the basal phosphorylation of
PhLPL was ~8-fold higher than that of PhLPS,
but that carbachol did not significantly stimulate the phosphorylation
of PhLPL or of PhLPS. Expression was controlled
by Western blotting with the PhLP-CT antibody and showed that both
proteins were equally expressed (Fig. 3A, right panel). Overexpression of PhLPS did not affect the
phosphorylation state of PhLPL (data not shown). These
findings demonstrate that PhLPL was constitutively
phosphorylated in HEK 293 cells, whereas PhLPS only
exhibited very low levels of phosphorylation.
Identification of CK2 as the Kinase Responsible for
PhLPL Phosphorylation in HEK 293 Cells--
To identify
the responsible kinase for PhLPL phosphorylation, a
computer analysis (ScanProsite) of the sequence of the N-terminal 83 amino acids of PhLPL revealed seven putative CK2
phosphorylation sites each within a classical CK2 phosphorylation motif
((S/T)-X-X-(D/E)) (see (Fig.
5A)). To test whether PhLPL could be a substrate
of CK2 phosphorylation in vitro, we performed kinase assays
with human recombinant CK2 and found that recombinant PhLPL
was indeed a substrate, whereas PhLPS and phosducin were
not (Fig. 4A). We used the
potent but unspecific kinase inhibitor staurosporine (22) and the
intracellular Ca2+ chelator BAPTA-AM (23, 24) to test their
effects on recombinant CK2 and found no effect on PhLPL
phosphorylation. On the other hand, we tested two substances that were
known to inhibit CK2: the glycosaminoglycan heparin (EC50
~3 nM) (12, 25) and the nucleoside derivative DRB
(EC50 ~7 µM) (26, 27). In the in
vitro kinase assay, 3 µM heparin completely
abolished PhLPL phosphorylation by CK2 and 100 µM DRB markedly inhibited PhLPL
phosphorylation. To determine whether CK2 is the predominant
PhLPL kinase in HEK 293 cells, we performed kinase assays
using HEK cell lysate and recombinant PhLPL with and
without the addition of kinase inhibitors and recombinant
PhLPS as well as phosducin (Fig. 4B). Previous studies have shown that CK2 is abundantly present in mammalian cell
lysates (12, 28). Here we demonstrate that PhLPL but not
PhLPS or phosducin was predominantly phosphorylated by HEK cell lysate. This phosphorylation was inhibited by heparin (3 µM) and DRB (100 µM), whereas staurosporine
only weakly inhibited and BAPTA-AM weakly stimulated the
phosphorylation of PhLPL. These results provide strong
evidence that CK2 is the predominant kinase in HEK 293 cells that
constitutively phosphorylates PhLPL. We then asked whether
PhLP was also a substrate for PKA phosphorylation in vitro
similar to PKA-dependent phosphorylation of the homologous protein phosducin. PKA kinase assays were done with the same
concentration (400 nM) of purified recombinant phosducin,
PhLPL, and PhLPS (Fig. 4C).
Phosducin was an excellent PKA substrate, whereas both PhLP variants
were not. They roughly showed 10-20-fold less radioactivity incorporated than phosducin. These results demonstrate that the different members of the phosducin family exhibit differential regulation by different kinases.
Identification of a Small Regulatory Region in the
PhLPL N Terminus--
We then asked whether any particular
region of the PhLPL N terminus was responsible for the
weaker functional effects of PhLPL compared with
PhLPS as well as for the constitutive phosphorylation in
HEK 293 cells. As depicted in Fig.
5A, the N terminus of
PhLPL contains several putative phosphorylation sites that
occur in clusters of serines and threonines. Seven of these sites could serve as CK2 phosphorylation sites. We constructed PhLPL
mutants by stepwise shortening of the N terminus. The five
PhLPL constructs were as follows: L5 (amino acids
5-301 with the loss of Thr-2 and Thr-3), L29 (amino acids 29-301 with
the additional loss of Ser-18, Thr-19, Ser-20 and Ser-25), L36 (amino
acids 36-301), L46 (amino acids 46-301 with the additional loss of
Ser-39, Ser-40, Ser-41, and Thr-42), and L58 (amino acids 58-301 with
the additional loss of Ser-54 and Thr-57). The determination of
G Effect of Mutation of Serine 18, Threonine 19, and Serine 20 to Alanines--
We next asked whether the putative CK2
phosphorylation sites of the region compromising amino acids 5-28
might play a functional role in PhLPL regulation, and if
so, which of the four candidates (Ser-18, Thr-19, Ser-20, and Ser-25)
was involved. Two mutants were constructed,
PhLPLA18-20 in which Ser-18, Thr-19, and Ser-20 were
changed to alanines, and PhLPLA25 in which Ser-25 was
changed to an alanine. These constructs were tested for their ability to inhibit G In Vivo Relevance of PhLP Splicing and
Phosphorylation--
Finally, we asked whether the observed phenomenon
of PhLP regulation via N-terminal phosphorylation or splicing might
play a role in different mouse organs, which had been described to contain considerable amounts of PhLPL (heart and brain) (4, 5) and which might also contain PhLPS (adrenal gland) (6). In addition, embryos were chosen because CK2 plays an essential role in
developing cells and cell cycle progression (13). Therefore, we
expected to detect a high level of phospho-PhLPL.
PhLPL was detected by Western blotting in all three organs
as well as in day 10.5 embryo and also in control (empty vector)
transfected HEK 293 cells (Fig.
7A). In contrast,
PhLPS was present in clearly detectable amounts only in the
adrenal gland and in HEK 293 cells after transfection of the
PhLPS cDNA (Fig. 7A). After longer exposure of the film, PhLPS seemed to be present also in heart and
brain but not in HEK 293 cells or embryo (data not shown). In most
instances, PhLPL ran as a doublet, one band corresponding
to the position of recombinant PhLPL purified from E. coli (Fig. 7, A and B, lower arrowhead) and one to the position of cellular PhLPL
(Fig. 7, A and B, upper arrowhead).
Because the presence of phosphate moieties is known to change the gel
mobility, we wondered whether the mutation in Ser-18, Thr-19, and
Ser-20 (PhLPLA18-20) would lead to a similar gel mobility
shift (Fig. 7B). Indeed PhLPLA18-20
corresponded to the lower band in brain and adrenal gland as well as
recombinant PhLPL purified from E. coli
(Fig. 7B). HEK 293 cell lysate and mouse brain lysate were
then treated with G-protein function is regulated by different classes of proteins,
most notably the RGS proteins and the members of the phosducin family. The members of the phosducin family bind to the
G-regulators and
exists in two splice variants. The long isoform PhLPL
and the short isoform PhLPS differ by the presence or
absence of an 83-amino acid N terminus. In isolated biochemical assay
systems, PhLPL is the more potent G
-inhibitor, whereas the functional role of PhLPS is still unclear. We
now report that in intact HEK 293 cells, PhLPS inhibited
G
-induced inositol phosphate generation with ~20-fold greater
potency than PhLPL. Radiolabeling of transfected HEK 293 cells with [32P] revealed that PhLPL is
constitutively phosphorylated, whereas PhLPS is not.
Because PhLPL has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK
cell cytosol in the presence or absence of kinase inhibitors or by
purified CK2. PhLPL was a good CK2 substrate, whereas
PhLPS and phosducin were not. Progressive truncation and
serine/threonine to alanine mutations of the PhLPL N
terminus identified a serine/threonine cluster (Ser-18/Thr-19/Ser-20)
within a small N-terminal region of PhLPL (amino acids
5-28) as the site in which PhLPL function was modified in
HEK 293 cells. In native tissue, PhLPL also seems to be
regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLPL were detected in mouse
brain and adrenal gland. Moreover, the alternatively spliced isoform PhLPS was also found in adrenal tissue. Therefore, the
physiological control of G-protein regulation by PhLP seems to involve
phosphorylation by CK2 and alternative splicing of the regulator.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit (G
) regulator phosducin. Similarly, as phosducin, PhLP has been found to bind to G
with high affinity (2) and act
as a G
-binding protein in intact cells (3). The PhLP mRNA
exists in two major splice variants coding for two proteins, which
differ by the presence (PhLPL) or absence
(PhLPS) of the N-terminal 83 amino acids (1, 4). Subsequent
studies on diverse tissues demonstrated that PhLPL is
expressed in many organs at high protein levels (e.g. brain,
heart, and liver), whereas PhLPS protein levels could not
or only hardly be detected in these organs (4, 5). Direct comparison of
the affinities toward G
showed a 15-fold weaker interaction for
PhLPS compared with phosducin or PhLPL (5).
Therefore, PhLPS had been believed to play only a minor
role in G-protein regulation. Recently, it was reported that
PhLPS could be purified from cultured bovine chromaffin
cells where it might inhibit nicotine-stimulated exocytosis of
catecholamines by a pathway involving a G
·ADP-ribosylation factor 6 complex (6). These findings suggested that alternative splicing of PhLP might indeed occur in at least one organ or
under specific conditions and therefore might play a role in
differential functions of the PhLP isoforms.
, and as a consequence, phosducin looses its ability to regulate G-protein function. In the
case of PhLP, phosphorylation as a regulatory mechanism has been
suggested but has never been demonstrated (10). Here we report that the
G
-regulatory function of PhLPL is inhibited in cells
by N-terminal phosphorylation via casein kinase 2 (CK2), whereas
PhLPS lacks such a regulatory mechanism. CK2 is a
constitutively active and ubiquitously expressed serine-threonine
kinase (12, 13). Our findings further provide strong evidence that the
extended N terminus of PhLPL becomes autoinhibitory
upon phosphorylation by CK2. In contrast, alternative splicing leads to
PhLPS, which is not a substrate for CK2 and thus escapes
this inhibitory mechanism.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranylbenzimidazole (DRB) were
purchased from Calbiochem. Heparin sodium salt (average molecular
weight 6,000) was from Sigma. Primary antibodies used were rabbit
polyclonal anti-PhLPS as described previously (5) and
rabbit polyclonal anti-PhLP-CT. This synthetic peptide was derived from
the very C terminus of PhLPL and PhLPS
(sequence: CHSEDSDLEID). After coupling to keyhole limpet hemocyanin
(14, 15), antibodies were raised in rabbits with two boost injections and collected as serum. Secondary goat anti-rabbit-horseradish peroxidase was from Dianova. Protein A-Sepharose was obtained from
Amersham Biosciences. Phosphorimaging and quantification were done on a
FLA-3000 from Fuji. ScanProsite was accessed at www.expasy.ch.
-protein phosphatase (
-PPase) (New England Biolabs) at 37 °C for 20 min. The
reaction was terminated by boiling in Laemmli buffer followed by
SDS-PAGE and Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-regulators in living cells, we subcloned
the appropriate cDNAs into the pcDNA3 expression vector and
determined the inositol phosphate formation stimulated by the transient
co-transfection of phospholipase C
2
(PLC
2) and G-protein subunits G
1 and
G
2 as described previously (19, 20). Without addition of
any G
-inhibiting proteins, the transfection of
PLC
2 and G
1
2 cDNA (3 µg each)
enhanced basal inositol phosphate accumulation ~5-fold compared with
mock-transfected cells (data not shown). Co-transfection of the
cDNA encoding PhLPL inhibited the G
-stimulated production of inositol phosphates significantly (Fig.
1A). Unexpectedly, the short
splice variant PhLPS turned out to be more effective than
the long splice variant PhLPL, although PhLPS
interacts with purified G
less efficiently than PhLPL
(5). Co-transfection of 8 µg of cDNA for each G
-binding
protein inhibited the total inositol phosphate formation by 32.2 ± 8.4% (n = 13) for PhLPL, 78.3 ± 3.8% (n = 7) for PhLPS, and 75.0 ± 4.0% (n = 3) for GRK2-K220R, which served as positive
control (21). Because inositol phosphate levels of untransfected
control cells were ~20% of the inositol phosphate levels of
PLC
2, G
1
2 co-expressing cells (data not shown), inhibition by ~80%, can be considered full inhibition. Thus, inhibition by PhLPS was almost complete. The
differences between control and PhLPL and between
PhLPL and PhLPS were highly significant
(p < 0.01). The different effects of PhLPL
and PhLPS were not because of different protein expression
as shown by Western blot analysis of the transfected cells (Fig.
1B). The blot with samples from three independent
experiments shows equal expression of PhLPL and
PhLPS within individual experiments and between different experiments.
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Fig. 1.
Inhibition of
G -stimulated inositol phosphate
generation in HEK 293 cells. A, HEK 293 cells were
transiently transfected with 8 µg of cDNA of PhLPL,
PhLPS, GRK2-K220R, or empty vector (control) and
with 3 µg of cDNA of G
1, G
2, and
PLC
2/10-cm dish. Inositol phosphate levels were
determined 42 h later as described under "Experimental
Procedures." (***, p < 0.001; **, p < 0.01 versus control; ##, p < 0.01; and
#, p < 0.05 versus PhLPL).
B, Western blot of three independent experiments comparing
protein expression levels of PhLPL and PhLPS
after transfection. Gels were loaded each with 20% of the cell lysate
from one well of a 6-well plate, and detection was done with the
polyclonal PhLP-CT antibody as described under "Experimental
Procedures."
-function, dose-response experiments were
performed. The cDNA of PhLPL or PhLPS was
diluted with empty vector to maintain the total amount of transfected
cDNA, and inositol phosphates were determined as before. As shown
in Fig. 2A, the
IC50 of PhLPL was 20-fold higher than that of
PhLPS (13.2 and 0.64 µg of cDNA, respectively). Fig.
2, B and C, shows that the transfection of HEK
293 cells with increasing amounts of cDNA did indeed result in the
expression of corresponding amounts of PhLPL or
PhLPS, respectively.
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Fig. 2.
Concentration-dependent effects
of PhLPL and PhLPS on inositol phosphate
generation. A, HEK 293 cells were transiently transfected
with various amounts of cDNA for PhLPL or
PhLPS. The IC50 of PhLPL was
20-fold higher than that of PhLPS (13.2 and 0.64 µg
cDNA/10-cm dish, respectively). B and C,
Western blots demonstrating increasing protein levels of
PhLPL (B) and PhLPS (C)
under the same conditions as in panel A. Note that different
exposure times of the films were used to cover the full range of
protein expression. The polyclonal PhLP-CT antibody was used as
described under "Experimental Procedures."
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Fig. 3.
PhLPL phosphorylation is
constitutive and M3 muscarinic receptor-independent in HEK
293 cells. HEK 293 cells transiently transfected with
M3 muscarinic receptor (2 µg) and PhLPL or
PhLPS cDNA (8 µg) were labeled with
[32P]orthophosphate, stimulated with carbachol (1 mM), and processed as described under "Experimental
Procedures." A, shown are 32P-labeled proteins
as visualized by phosphorimaging (32P) and the
expression control as performed by immunoblot
(IB:PhLP-CT) of one of three independent
experiment with similar results. There is marked basal phosphorylation
in PhLPL, whereas in PhLPS, phosphorylation is
only low in HEK 293 cells. Expression levels of both constructs were
comparable. B, shown is the 32P incorporation in
PhLPL and PhLPS with and without M3
muscarinic receptor stimulation by carbachol (means ± S.E.) of
three to five independent experiments as the fold of basal
PhLPL phosphorylation. Stimulation of PhLPL or
PhLPS phosphorylation by carbachol treatment did not result
in a significant increase. However, there was a 8-fold difference in
the phosphorylation between basal PhLPL and basal
PhLPS (***, p < 0.001). Data were
determined by phosphorimaging as described under "Experimental
Procedures."
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Fig. 4.
Phosphorylation of recombinant phosducin and
PhLP by HEK cell lysate, recombinant casein kinase 2, and protein
kinase A. Equimolar concentrations (400 nM) of
recombinant C-terminally His6-tagged PhLPL
(14.04 µg/ml), PhLPS (10.3 µg/ml), and phosducin (11.62 µg/ml) purified from E. coli were phosphorylated by either
recombinant human casein kinase 2 or by HEK cell lysate or purified
catalytic subunit of PKA. A, recombinant human CK2 (0.04 milliunit/reaction) was used to phosphorylate recombinant
PhLPL, PhLPS, and phosducin at 30 °C for 30 min. After SDS-PAGE and Coomassie Blue staining (Coomassie),
the gel was subjected to phosphorimaging (32P). CK2
phosphorylated PhLPL efficiently, whereas PhLPS
was only phosphorylated weakly (21% PhLPL) and phosducin
was not (2% PhLPL). The concentration of the inhibitors
used was 1 µM for staurosporine (Stauro), 100 µM for BAPTA-AM (BAPTA), 3 µM
for heparin, and 100 µM for DRB. Me2SO
(DMSO) (1% v/v) served as solvent control. B,
HEK 293 cell lysate extracted as detailed under "Experimental
Procedures" was used to phosphorylate recombinant PhLPL
in the absence or presence of kinase inhibitors as well as
PhLPS and phosducin at 30 °C for 30 min. Recombinant
proteins were pulled down by Ni-NTA-agarose and processed as described
under "Experimental Procedures." Control denotes HEK cell lysate
without any recombinant protein and reflects background
phosphorylation. Shown is the gel as visualized by phosphorimaging
(32P) and by Coomassie Blue staining
(Coomassie). PhLPL phosphorylation was inhibited
by heparin and DRB (both known to inhibit CK2) but not by staurosporine
or BAPTA, whereas PhLPS or phosducin was only weakly
phosphorylated. C, recombinant phosducin, PhLPL,
and PhLPS were phosphorylated by the catalytic subunit of
PKA (100 units) at 30 °C for 15 min. Reactions were stopped by
Laemmli buffer, boiled, and separated on 12.5% SDS-PAGE. Shown is the
film (32P) and the corresponding gel
(Coomassie) to demonstrate that PKA phosphorylation of PhLP
isoforms is not significant compared with phosducin.
-dependent inositol phosphate generation showed that
a region within amino acids 5-28 caused the only major step in the
gain of inhibitory function from PhLPL to PhLPS
as demonstrated by the functional difference between L5 and L29 (Fig.
5A, lower panel). The removal of the first 28 amino acids of PhLPL (as in L29, L36, L46, and L58) was
sufficient to gain the full G
-inhibitory activity of PhLPS. Western blots showed that all of the constructs with
the exception of L5 were stable and that the expression levels of the
constructs were comparable (Fig. 5B).
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Fig. 5.
Progressive truncation of the
PhLPL N terminus revealed a short regulatory region between
amino acids 5 and 28. A, the extended N terminus of
PhLPL contains several clusters of putative phosphorylation
sites (S, serine; T, threonine) as depicted in
the upper part of the panel. Arrows indicate the
possible CK2 phosphorylation sites, whereas the numbers over
the arrowheads indicate the amino acids C-terminal of the
cutting point of the truncated constructs of PhLPL. The
removal of the first 28 amino acids of PhLPL
(L29, L29) was sufficient to gain the same
G -inhibitory effect as PhLPS, whereas removing the
first four amino acids (L5) was not. Additional progressive truncation
did not change this effect any further. The inhibition of the inositol
phosphate signal was by 42 ± 11% (n = 5) for
PhLPL, 52 ± 9% (n = 5) for L5,
84 ± 1% (n = 3) for L29, 85 ± 2%
(n = 3) for L36, 84 ± 1% (n = 3)
for L46, 85 ± 2% (n = 5) for L58, and 84 ± 3% (n = 5) for PhLPS (**,
p < 0.01; ***, p < 0.001 versus PhLPL). B, expression control
of the five N-terminally truncated constructs in comparison to that of
PhLPL and PhLPS. Transfections were done as in
inositol phosphate measurements, and 20% of the lysate of one well of
a 6-well plate/lane was used. Shown is one of two Western blots with
similar results. Used was the polyclonal PhLP-CT antibody as described
under "Experimental Procedures."
-dependent inositol phosphate generation.
PhLPLA18-20 showed the same G
-inhibitory capability
as PhLPS and L29 (Fig. 6A), whereas
PhLPLA25 exhibited the same functional capability as
PhLPLA25. The differences between the effects of
PhLPL and PhLPLA25 on the one hand and
PhLPS, L29, and PhLPLA18-20 on the other hand
were highly significant (Fig. 6A). Again, equal expression of the constructs was demonstrated in Western blot experiments (Fig.
6B). These data suggest that the cluster of Ser-18, Thr-19, and Ser-20 contains the phosphorylation sites responsible for the
diminished G
-inhibitory effects of PhLPL, whereas
Ser-25 most probably was not involved. Overexpression of
PhLPL in a 4-fold excess over PhLPS or
PhLPLA18-20 reduced the inhibition of the latter to the
level exerted by PhLPL alone (Fig. 6C),
suggesting that PhLPL competed with PhLPS for
inhibiting G
. A 4-fold overexpression of PhLPL over
PhLPS was controlled by Western blots (data not shown).
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Fig. 6.
Alanine mutation of Ser-18, Thr-19, and
Ser-20 (PhLPL
A18-20) but not of Ser-25
(PhLPL A25)
was sufficient to yield full
G -inhibitory activity.
A, HEK 293 cells were transiently transfected, and the
inositol phosphate generation was determined as before (**,
p < 0.01 versus PhLPL; #,
p < 0.05 versus PhLPLA25 and
p > 0.05 for PhLPL versus
PhLPLA25). B, expression controls exhibit
similar protein levels for all constructs as shown in one of three
Western blots with similar results. Used was the PhLP-CT antibody as
described under "Experimental Procedures." C, effects of
co-expression of various forms of PhLP on inositol phosphate
generation. The amount of PhLP cDNA transfected/10-cm dish is
indicated, whereas total amount of cDNA was held constant with
empty vector (***, p < 0.001 versus
PhLPS; ##, p < 0.01 versus
PhLPLA18-20 and p < 0.001 for all
versus control).
-PPase, which has been described to efficiently
remove phosphates from serine, threonine, and tyrosine residues (29).
This resulted in the loss of the PhLPL high molecular mass
form as detected by Western blotting, whereas the low molecular mass
form of PhLPL became detectable in HEK 293 cells and was
enhanced in mouse brain (Fig. 7C). The contention that the
upper band represents phosphorylated PhLPL was supported by
the observation that phosphorylation of PhLPL by CK2
in vitro caused a reduction of gel mobility (Fig.
7D). Together, these data demonstrate that there is
constitutive phosphorylation of PhLPL in different mouse
tissues and that in brain and adrenal gland non-phosphorylated
PhLPL seems to play a role.
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Fig. 7.
Mouse tissue differentially expresses
PhLPL and PhLPS and exhibits phosphorylated and
non-phosphorylated PhLPL. A, Western blot
analysis of recombinant PhLPL and PhLPS (0.1 pmol), 20 µg of total protein of cell lysates of control transfected,
PhLPL- or PhLPS-transfected HEK 293 cells, and
200 µg of total protein of mouse heart, brain, adrenal gland, and
embryo. Preparation of two different mice was performed in the presence
of phosphatase inhibitors as detailed under "Experimental
Procedures" and showed the same result. Adrenal gland has a prominent
band of the size of PhLPS. Brain and adrenal gland show two
bands: the upper one corresponding to the size of PhLPL in
HEK 293 cells (transfected and endogenous) and the lower one
corresponding to the size of PhLPL purified from E. coli. The PhLPS antibody has considerable
cross-reactivity to the endogenous phosducin, which has an apparent gel
size of 33 kDa. B, close-up view of a Western
blot of 100 µg of total protein of mouse tissue (brain, adrenal
gland, and embryo), 10 µg of total protein of HEK 293 cell lysate
(PhLPL and PhLPL A18-20), and
PhLPL (0.1 pmol) purified from E. coli. In brain
and adrenal gland, doublets are detectable, whereas in embryo, only a
single band is visible. Mutation of the phosphorylation sites Ser-18,
Thr-19, and Ser-20 to Ala caused a shift in mobility of
PhLPL in SDS-PAGE from that of wild-type
PhLPL-transfected HEK 293 cells to that of unphosphorylated
recombinant (rec.) PhLPL and corresponded to the
size of the lower band in mouse brain and adrenal gland. C,
HEK cell lysate (50 µg of total protein) and mouse brain lysate (100 µg of total protein) were prepared as described under "Experimental
Procedures" and subjected to dephosphorylation by 1000 units of
-PPase at 37 °C for 20 min. Controls were incubated without the
phosphatase. In native probes, PhLPL could be
dephosphorylated by
-PPase and changed its gel mobility in a 15%
SDS-PAGE. D, purified recombinant PhLPL (400 nM) was phosphorylated by CK2 (0.3 milliunit) in the
absence or presence of 30 µM heparin at 37 °C for 120 min. SDS-PAGE was performed on 15% gel and subjected to
phosphorimaging. Phosphorylation of recombinant PhLPL by
CK2 caused a gel mobility shift similar to that seen in native
tissue.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits (30, 31) and therefore inhibit G-protein function.
This might result in the disruption of the G-protein cycle, (32, 33) and/or in the inhibition of G
-mediated effects like activation of
PLC
isoforms and G-protein-coupled receptor kinase 2 (9, 34, 35). In
this work, we used the activation of a G
-dependent phospholipase C
2 in HEK 293 cells as a functional
readout of G
-inhibition to demonstrate that PhLP inhibits
G
-functions in living cells. According to present knowledge, PhLP
exists in two isoforms: 1) the long variant PhLPL with an
additional N terminus of 83 amino acids and 2) the short variant
PhLPS, which lacks this N terminus (Figs. 5A and
8) (1). However, the functional differences of these two isotypes in living cells remained to be
elucidated, because (a) in vitro experiments
showed that the short form, which lacks one G
-binding region
according to structural data (Fig. 8) (31), exhibited a markedly
reduced binding affinity toward G
-subunits compared with
PhLPL and phosducin (2, 5) and (b) tissue levels
of the PhLP isoforms appropriate for a physiological role in
G
-inhibition could only be demonstrated for the long form,
PhLPL (4, 5). This finding appeared to suggest that the
physiological G
-regulator should be the long form,
PhLPL. Our present data show that in contrast to this
hypothesis, PhLPS is an effective regulator of
G
-function in HEK 293 cells. The yeast phosducin homolog
resembles PhLPS in size and domain structure (Fig. 8),
suggesting that important G
-regulating properties are contained
in these proteins. Furthermore, our data suggest that the different
forms of PhLP cooperate in inhibiting G
to a variable extent in
that the phosphorylated form of PhLPL appears to have a
partial dominant negative effect on the inhibitory function of
PhLPS.
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Fig. 8.
Topology model of phosducin,
PhLPL and PhLPS and the yeast phosducin homolog
derived from the crystal structure of phosducin (31). Shown are
the phosducin structural domains. The N terminus consists of three
-helices with G
-binding regions and large unresolved areas.
The C terminus is a thioredoxin domain consisting of five
-sheets
and three flanking
-helices (not marked) with G
-binding
regions. PhLPL has all five homologous G
-contact
regions, and PhLPS bears four of the five G
-contact
regions and resembles the yeast phosducin homolog, which also does not
contain the first
-helix and first G
-binding region. As in
phosducin, the PhLPL N terminus is subject to regulation by
phosphorylation (a), most probably by CK2, and this seems to
play an important role in vivo. A further possibility for a
more long term activation of PhLP would be alternative splicing to the
short variant PhLPS (b), which lacks the
possibility of N-terminal regulation by phosphorylation and seems to be
relevant in the adrenal gland. In primitive eukaryotic organisms such
as yeast, the "short phosducin" might be a sufficient principle of
G
-regulation.
PhLPS was found in mouse adrenal gland in amounts comparable with the levels of PhLPL in mouse brain and heart. In the latter organs, tissue levels of PhLPL have been calculated to be sufficient for in vivo regulation of G-proteins (5). The detection of PhLPS in adrenal glands is in line with its purification from bovine chromaffin cells (6). These findings underline the contention that not only PhLPL but also PhLPS is a relevant physiological regulator of G-protein function.
In the case of phosducin, it has been shown that G-protein inhibition is subject to regulation by phosphorylation by several kinases, most notably PKA (7, 8, 10, 11). A PKA consensus motif is also present in PhLP (amino acids 117-121 in PhLPL: GKMT*L)2 but does not appear to serve as a PKA site in vitro or in vivo. However, in HEK 293 cells as well as in mouse organs, PhLPL is apparently constitutively phosphorylated in its N terminus at Ser-18, Thr-19, and Ser-20. CK2 is an ubiquitously expressed and constitutively active serine-threonine kinase (12, 13), which has recently been found to constitutively phosphorylate arrestin-3, another protein of the G-protein-coupled receptor signaling pathway (36). Here, we report that recombinant PhLPL but not PhLPS or phosducin were substrates of CK2 phosphorylation, whereas PKA phosphorylated only phosducin. Therefore, we conclude that the different members of the phosducin family are differentially regulated by different kinases. Further evidence that CK2 is a regulatory kinase for PhLPL comes from the findings that (a) Ser-18, Thr-19, and Ser-20 contain the consensus motif for CK2, (b) mutation of the consensus sites into alanines resulted in a non-phosphorylated form in intact cells, (c) progressive N-terminal truncation or serine/threonine to alanine mutation at these sites both converted PhLPL into a protein with the same potency and efficacy as PhLPS, and (d) phosphorylation of PhLPL by kinase activity from HEK cell lysates was inhibited by two different CK2-specific inhibitors, whereas other kinase inhibitors did not result in inhibition.
Taken together, these findings indicate a regulatory function of the N
terminus of PhLPL and underline the role of N-terminal regulation of the members of the phosducin family. Our data also show
that PhLP isoforms differ in the mode of activity control and that the
G-inhibitory effects of PhLPL are subject to
regulation via the N-terminal domain. Phosphorylation of the N
terminus, in vivo most probably by CK2, diminishes the
ability of PhLPL to inhibit G
-functions. The analysis
of PhLP expression in mouse organs shows that both mechanisms are used
in vivo.
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FOOTNOTES |
---|
* 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.:
49-931-20148400; Fax: 49-931-20148539; E-mail:
lohse@toxi.uni-wuerzburg.de.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M206347200
2 Asterisk in sequence denotes potential phosphorylation site.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PhLP, phosducin-like protein;
PhLPL, long form of PhLP;
PhLPS, short form of PhLP;
G, G-protein
-subunit;
PKA, cAMP-dependent protein kinase;
GRK2, G-protein-coupled receptor kinase 2;
HEK, human embryonic kidney;
BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetra(acetoxymethy)ester;
DRB, 5,6-dichloro-1-
-D-ribofuranylbenzimidazole;
CK2, casein
kinase 2;
CT, C terminus;
ANOVA, analysis of variance;
-PPase,
-protein phosphatase;
PLC, phospholipase.
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