From the Verna and Marrs McLean Department of
Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030 and the Departments of § Ophthalmology,
Chemistry, and ** Pharmacology, University of Washington,
Seattle, Washington 98195
Received for publication, December 21, 2000, and in revised form, April 2, 2001
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ABSTRACT |
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Inactivation of the visual G protein
transducin, during recovery from photoexcitation, is regulated by
RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS
protein family. Incubation of dark-adapted bovine rod outer segments
with [ Phototransduction in vertebrate rod cells is a prototypical G
protein signal transduction pathway (1, 2). In the activation phase,
the receptor rhodopsin captures a photon and activates the rod's
heterotrimeric G protein transducin
(Gt)1
RGS9-1, the GTPase-accelerating protein (GAP) for Gt Phosphorylation events are often employed to regulate signal
transduction because they can change the activities, subcellular localization, protein-protein interactions, or stability of
transduction components. In ROS, where the reactions of
phototransduction take place, there are many protein kinases and many
phosphoproteins. Kinases including rhodopsin kinase (25), CDK5 (26),
PKC (27), PKA (28), CK II (29), and protein-tyrosine kinases such as Src (30), have all been reported to be present in
photoreceptors. Phosphorylation by endogenous kinases in ROS has been
reported for phototransduction components including rhodopsin (whose
phosphorylation is known to be essential for normal recovery kinetics
(31)), the G ATP has long been known to have profound effects on the kinetics of the
recovery phase of the light response (40, 41), but the mechanisms of
its actions have not been fully determined either. Clearly, one of the
main roles of ATP in recovery is to serve as a substrate for rhodopsin
kinase (3, 42), but it seems likely that other
ATP-dependent reactions, including those catalyzed by other
protein kinases, play an important role as well. We describe here
experiments suggesting that one of these may be phosphorylation of
RGS9-1 and show that this reaction is catalyzed by a protein
kinase not identified previously in ROS.
Reagents--
Buffer reagents, nucleotides and analogues, PKA
catalytic subunit from bovine heart, and PKA substrate
[Val6,Ala7]Leu-Arg-Arg-Ala-Ser-Leu-Gly
([Val6,Ala7]Kemptide) were purchased from
Sigma. Protein kinase inhibitors (A3-HCl, H8-dihydrochloride,
bisindolylmaleimide I-HCl
(2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide), HCl; also known as GF109203X), CaM-binding domain, sangivamycin, and
roscovitine), recombinant CK II, and CK II substrate were purchased
from Calbiochem.
Buffers--
Standard buffers were as follows: buffer A (10 mM HEPES, 100 mM NaCl), buffer B (5 mM HEPES, 0.5 mM MgCl2), buffer C
(10 mM Tris-HCl, 100 mM NaCl, 2 mM
MgCl2, ~20 mg/liter phenylmethylsulfonyl fluoride);
buffer D (10 mM MOPS, 30 mM NaCl, 60 mM KCl, 2 mM MgCl2, 1 mM DTT, ~20 mg/liter phenylmethylsulfonyl fluoride);
buffer E (5 mM Tris-HCl, 2 mM EDTA, 0.2 mM Na3VO4, 15 µM
fenvalerate, 100 nM okadaic acid, 1 mM DTT);
buffer F (50 mM sodium phosphate, 50 mM NaCl,
10 mM KF, 2 mM MgCl2); buffer G
(100 mM NaCl, 5 mM Tris-HCl). For all these
buffers, the pH was adjusted to 7.4-7.5. Other buffer components and
conditions were varied as indicated throughout.
Expression and Purification of Recombinant
Proteins--
His6-tagged RGS9-1 full-length protein in
complex with G Phosphorylation of RGS9-1 in ROS--
Bovine ROS were prepared
in dim red light by sucrose gradient centrifugation (43). After ROS
membranes were collected from the sucrose gradient, they were
homogenized by passage through an 18-gauge needle in buffer C at a
dilution ratio of 1:5, pelleted by centrifugation at 24,000 × g, and then resuspended in buffer C to a rhodopsin
concentration of 100-150 µM and stored at Antibodies and Immunoprecipitation--
Rabbit
anti-RGS9-1c polyclonal antibody and monoclonal anti-RGS9-1 antibody D7
were generated as described previously (4, 5, 20). Mouse
anti-phosphorylated RGS9-1 monoclonal antibodies (A4) were raised
against a peptide from the C terminus of mouse phosphorylated
RGS9-1, KLDRRS(P)QLKKELPPK, where the (P) refers to phosphorylation of the preceding serine residue (Quality Controlled Biochemicals, Inc., Hopkinton, MA), coupled to a carrier protein, KLH
(Sigma) as described previously (45). The mouse sequence, which differs
slightly from the bovine sequence (KLDRRSQLRKEPPPK), was used to assure
reactivity with the phosphoprotein in mice. However, the bovine
phosphoprotein, but not the unphosphorylated form, was also strongly
recognized by the antibody. For use in immunoprecipitation, IgG was
affinity-purified by protein A beads from rabbit anti-RGS9-1c
antisera. Purified IgG was then covalently attached to
CNBr-activated Sepharose 4B-CL from Amersham following the
manufacturer's instructions at a ratio of 10 mg of IgG to 1 ml of
beads. For immunoprecipitation, ROS membranes were washed after ATP
incubation by repeated centrifugation at 84,000 × g for 15 min, homogenized three times with buffer E at 15 µM rhodopsin 0-4 °C, and then solubilized for 30 min
on ice in buffer C with 1% Nonidet P-40 detergent at 60 µM rhodopsin. The insoluble material was removed by
centrifugation for 20 min at 84,000 × g. Typically, 300 µl of solubilized ROS were incubated with 40 µl of IgG-coupled beads for 2.5 h at 4 °C upon mixing on a shaker. The beads were separated from the supernatant by a brief centrifugation and washed three times with the solubilization buffer. Bound proteins were eluted
from the beads by 0.1 M glycine at pH 3.0, concentrated by
trichloroacetic acid precipitation, and redissolved by boiling in the
SDS-PAGE sample buffer. Efficiency of immunoprecipitation was measured
by autoradiography following SDS-PAGE and immunoblot using monoclonal
antibody D7.
Immunoblotting--
Immunoblotting was carried out using a
standard protocol (46) on proteins separated by SDS-PAGE. For
electrophoretic transfer, the buffer used was 25 mM Tris,
192 mM glycine, 0.1% SDS, pH 8.3, and the membranes were
supported nitrocellulose (NitroPure, Osmonics, Inc.). After 60 min at 350 mA (room temperature), membranes were blocked with 5%
nonfat dry milk/TBS solution for 1 h, followed by incubation with
primary antibody for 2 h. Monoclonal antibodies D7 and A4 were
used at a dilution of 1:500, and polyclonal anti-RGS9-1c serum was used
at a dilution of 1:1000. The secondary antibodies used were horseradish
peroxidase-conjugated (Promega) anti-mouse (for monoclonals) or
anti-rabbit (for polyclonals), with detection by
chemiluminescence, using the ECL® system (Amersham Pharmacia Biotech).
For autoradiography, x-ray films were exposed to dried SDS-PAGE gels,
typically for 2-3 days, before being developed.
Detection of in Vivo Phosphorylation--
Mice were maintained
in a dark room (dark-adapted) or in full room light (light-adapted) for
a period of 16 h prior to euthanasia and removal of retinas under
dim red light. The retinas were homogenized by pestle (Kontes) in
1.5-ml microtubes using buffer C with 1% Nonidet P-40 detergent, plus
0.2 mM Na3VO4, 15 µM
fenvalerate, 100 nM okadaic acid to inhibit phosphatase
activities. Immunoprecipitation of RGS9-1 was carried out using rabbit
polyclonal antibodies, as described above, and the immunoprecipitated
protein was analyzed by SDS-PAGE and immunoblotting with the mouse
monoclonal antibody A4 specific for the phosphopeptide.
Identification of the Phosphorylation Site--
ROS were
incubated at 37 °C for 40 min at a concentration of 69 µM rhodopsin with 0.2 mM
[
To verify that the same site is phosphorylated under our standard assay
conditions, phosphorylation reactions were carried out using buffer C
as described under "Phosphorylation of RGS9-1 in ROS", using 5 mM ATP without radiolabel, followed by immunoprecipitation. Immunoprecipitated RGS9-1 was separated by SDS-PAGE and subjected to
in-gel trypsin digestion, and the phosphorylation sites in the tryptic
peptides were identified by mass spectrometry. The method used was a
combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, before and after phosphatase treatment, and on-line capillary liquid chromatography electrospray tandem ion trap mass spectrometry described previously (49). Two-dimensional phosphopeptide mapping was carried out as described (50).
Phosphorylation of Recombinant Proteins--
Purified His-tagged
RGS9-1 in complex with G Tests of Candidate Protein Kinases--
Three different
approaches were used to test the possible involvement of known protein
kinases in RGS9-1 phosphorylation. 1) Activators of PKA (8-Br-cAMP; 50 µM), PKG (8-Br-cGMP; 50 µM), or PKC (PMA;
1, 5, or 10 µM plus 1 or 2 mM
CaCl2, referred to subsequently as PMA/Ca) were added under
our standard assay conditions. The ability of the added cyclic
nucleotide analogues to activate endogenous cyclic
nucleotide-dependent protein kinase activity was verified
by testing their ability to stimulate phosphorylation in ROS
homogenates of [Val6,Ala7]Kemptide using
[ Inhibition by RGS9-1-derived Peptide--
For the peptide
inhibitor, ROS were mixed with ATP in the presence of increasing
concentrations of a synthetic peptide derived from the mouse RGS9-1 C
terminus with the phosphorylation site mutated to Ala: KLDRRAQLKKELPPK
(Quality Controlled Biochemicals, Inc). Reactions were then quenched by
SDS-PAGE sample buffer, and phosphorylation was detected by
Ser475-phosphate-specific monoclonal antibody A4.
Kinase Activity in Fractionated Retinal Membranes--
Retinal
membranes from frozen bovine retinas were prepared in dim red light and
separated by sucrose gradient centrifugation using a standard technique
(43). Samples in the sucrose gradient were then fractionated into 1-ml
aliquots using a gradient puller (Auto-Densi-Flow from Labconco) and
stored at RGS9-1 Is Phosphorylated by an Endogenous Kinase on the ROS
Membranes--
When purified bovine ROS membranes were incubated with
[
To test whether the RGS9-1 phosphorylation level is high
enough to be a potential regulatory mechanism, we studied the
stoichiometry by immunoprecipitating phosphorylated RGS9-1. The highest
phosphorylation level we achieved was 0.45 mol of phosphate/mol of
RGS9-1 after 20 min of ATP incubation (Fig. 1B). However,
the stoichiometry varied among different batches of ROS preparations,
ranging from 0.23 to 0.45 (from three independent experiments; data not
shown), with the differences possibly due to loss of kinase activity
during the ROS preparation. This explanation is supported by the
observation that RGS9-1 kinase is slightly soluble in the isotonic
buffers that were used for ROS preparation (see below). Probably, the phosphorylation stoichiometry is determined by the balance of phosphatase and kinase activity, and phosphatase activity may also vary
somewhat in different preparations. As discussed below, RGS9-1 is not
phosphorylated in our ROS preparations prior to ATP treatment.
RGS9-1 Is Specifically Phosphorylated at Ser475 Near
the Carboxyl Terminus--
When phosphorylated RGS9-1 was digested
with trypsin and the phosphorylated peptide was then extensively
purified by two rounds of reverse-phase HPLC,
Ga3+-immobilized metal affinity chromatography, and two
additional rounds of HPLC with different solvent systems (Fig.
2A) in each separation
procedure, a single major phosphopeptide peak was eluted and collected.
Electrospray ionization MS revealed (Fig. 2B) that this
phosphopeptide has an m/z 583.5 (the precursor
ion, MH1+), corresponding to a
monophosphorylated peptide at Ser475 with a sequence of
S475QLR (calculated average mass for
MH1+ = 583.56). The precursor ion with the loss
of HPO3 was also observed at m/z
503.5 (calculated mass 503.58). The precursor ion underwent elimination
of phosphate
(MH1+-H3PO4, y4) and
elimination of phosphate and water
(MH1+-H3PO4-H2O,
b4). Further decompositions of y4 and b4 yielded ions y1-y3 and b1-b3,
respectively. The observed fragmentation pattern is in agreement with
the sequence SQLR, where the Ser residue is phosphorylated. The same
peptide was identified in two different preparations. These results
demonstrate clearly that the single major phosphorylation site in
RGS9-1 is Ser475.
In a completely different trial to identify the phosphorylation site
under our standard reaction conditions, RGS9-1 was phosphorylated in
buffer C, immunoprecipitated, resolved by SDS-PAGE, and subjected to
in-gel trypsin digestion for MS analysis of the phosphorylation site
using an established procedure (49). Again, Ser475 was
revealed as the only phosphorylation site (data not shown). Under these
conditions, both endogenous RGS9-1 and recombinant phosphorylated
RGS9-1 (see below) each yielded a single major radiolabeled spot upon
two-dimensional phosphopeptide mapping (data not shown).
Recombinant RGS9-1 Can Be Phosphorylated by the Endogenous Kinase,
and Its Phosphorylation Requires Ser475--
To test
further the requirement of Ser475 for RGS9-1
phosphorylation, phosphorylation of both His-tagged RGS9-1 full-length
protein and His-tagged RGS9-1 mutant with Ser475 mutated to
Ala by the endogenous kinase was examined. Only full-length RGS9-1 with
the wild-type sequence was phosphorylated significantly by ROS kinases
(Fig. 3), suggesting that the
Ser475 residue is required for phosphorylation. Only very
weak signals could be detected in the mutant RGS9-1 protein, probably
due to phosphorylation at one or more minor sites. Immunoblotting with the monoclonal Ser475-phosphate-specific antibody A4
confirmed that only the wild-type, and not the mutant protein could be
detected after phosphorylation (data not shown).
Divalent Cation Dependence of RGS9-1 Phosphorylation--
We
tested the requirement of RGS9-1 phosphorylation for metal ions by
performing kinase assays in the presence of different cations. ROS
membranes were first washed with EDTA to remove contaminating metal
ions and then resuspended in buffers containing the desired cations for
phosphorylation. As expected for most phosphotransfer reactions, we
found that Mg2+ was required for RGS9-1 phosphorylation,
because RGS9-1 phosphorylation was completely abolished in the presence
of EDTA or Ca2+ alone and could only be partially restored
by Mn2+ (Fig. 4A).
Chelation of Ca2+ by EGTA in the presence of excess
Mg2+ reduced phosphorylation (Fig. 4C). Free
[Ca2+] calculated to be on the order of
10 Effects of Kinase Activators and Inhibitors--
In order to
determine if the kinase responsible for RGS9-1 phosphorylation is one
of the well characterized protein kinases, we first tried to stimulate
RGS9-1 phosphorylation by adding kinase activators for PKA, PKC, and
PKG, since the Ser475 is located in a sequence similar to
the consensus sequence for PKA and PKG phosphorylation; both PKA and
PKC activities in purified ROS have been reported (27, 28). We found
that RGS9-1 phosphorylation levels did not change significantly in the
presence of 8-Br-cAMP, 8-Br-cGMP, or PMA/Ca2+, although
PMA/Ca2+ did greatly enhance rhodopsin phosphorylation
(Fig. 5, A and B).
Both 8-Br-cAMP and 8-Br-cGMP enhanced the phosphorylation of added
Kemptide substrate in the presence of ROS (Fig. 5G). We then
tested the effects of several kinase inhibitors, including inhibitors
for PKA, PKC, PKG, CK II, CaMK II, rhodopsin kinase, and CDK5/p35. No
significant inhibition of RGS9-1 phosphorylation was observed in any of
these experiments (<10% inhibition at concentrations of 50 times
their reported Ki values as detailed under "Experimental Procedures"; Fig. 5C) except for the PKC
inhibitor bisindolylmaleimide I. Rhodopsin phosphorylation was strongly inhibited by inhibitors of both rhodopsin kinase and PKC (Fig. 5E), verifying their efficacies under our conditions. The
PKA/PKG inhibitor H8 greatly reduced Kemptide phosphorylation in ROS
stimulated by 8-Br-cGMP or 8-Br-cAMP (Fig. 5G), verifying
its potency under our conditions. PDE
We next tested a peptide derived from the mouse RGS9-1 C terminus
containing the phosphorylation site with Ser475 mutated to
Ala (KLDRRAQLKKELPP) for its effect on RGS9-1
phosphorylation. We found that the peptide containing the mutated
phosphorylation site did inhibit RGS9-1 phosphorylation in a
concentration-dependent manner, probably by competing for
the kinase (Fig. 5D). Indeed, the
Ser475-containing peptide was found to be phosphorylated by
ROS membranes (data not shown). Therefore, the RGS9-1 kinase has a
sequence specificity not previously reported for any known protein kinase.
RGS9-1 Kinase Co-purifies with Rhodopsin and RGS9-1 in Fractionated
Retinal Membranes and Is Tightly Membrane-associated--
To determine
whether the RGS9-1 kinase is an endogenous component of ROS or a
contaminant from elsewhere in the retina, we checked the presence of
RGS9-1 kinase activity in fractions from homogenized retina separated
by sucrose gradient centrifugation. In these experiments, we used
recombinant His-tagged RGS9-1 protein as substrate and the monoclonal
Ser475-phosphate-specific antibody A4 to detect the
phosphorylation. There was no detectable phosphorylation of this
recombinant protein on Ser475 prior to incubation with
preparations containing the RGS9-1 kinase, as demonstrated by its lack
of reactivity with the A4 antibody. The major kinase activity peaks
correlated very well with the peaks of rhodopsin and RGS9-1 in those
fractions corresponding to ROS, broken ROS, and unsheared retinal
membranes (Fig. 6A), implying
that within the retina the kinase is predominantly localized to ROS and
probably plays a role in regulation of phototransduction. The lack of
detectable endogenous RGS9-1 phosphorylation in the fractions
containing the minor peak of broken ROS (fractions 25-27) may result
from competition by recombinant protein. The minor kinase activity peak
in the fractions corresponding to soluble proteins (fractions 1-3) can
be attributed to the slight solubility of RGS9-1 kinase in isotonic
buffer (see below). Phosphorylation of endogenous RGS9-1 can be seen to
be more efficient than phosphorylation of recombinant proteins (Fig.
6A, boxed area) when the levels of
phosphorylation are compared with the amounts of substrates that were
present, indicating that the kinase may be localized in close vicinity to or even form a complex with RGS9-1. We were only
able to achieve phosphate incorporation stoichiometries of less than
10% for the recombinant protein.
To characterize the kinase further, we tested whether the kinase
displays similar membrane-binding properties to those of RGS9-1, a
tightly bound peripheral membrane protein, by comparing the kinase
activity remaining on ROS membranes before and after buffer
extractions. All assays were adjusted to the same final ionic strength.
We found that low salt (5 mM ionic strength) and moderate
salt (100 mM ionic strength) extractions removed only about
20% of the kinase activity, but the hypertonic extractions removed at
least 60-70% (Fig. 6B). Assays of kinase activity in the
high salt extracts using recombinant proteins (data not shown) revealed
that, while high salt does inhibit kinase activity somewhat, there is
substantial kinase activity in the extracts, confirming that high ionic
strength extracts the kinase into the supernatant rather than
permanently inactivating it in the membranes. These results indicate
that the kinase itself is tightly membrane-bound and that its membrane
binding has a large electrostatic component. These membrane binding
properties are very similar to those of RGS9-1 (5).
RGS9-1 Phosphorylation in Mouse Retina--
To find out if RGS9-1
phosphorylation actually occurs in vivo, we isolated
endogenous RGS9-1 from freshly dissected mouse retina and used the
Ser475-phosphate-specific monoclonal antibody to detect its
phosphorylation by immunoblotting. The immunoblot (Fig.
7A) demonstrated that RGS9-1
was phosphorylated by endogenous kinase in the retina. A further
comparison of the phosphorylation level between light- and dark-adapted
animals revealed that light reduced signal due to RGS9-1
phosphorylation by about 80% (Fig. 7B). As revealed by
phospho- and site-specific monoclonal antibodies, these results demonstrate clearly that RGS9-1 is phosphorylated on Ser475
in vivo, and that this phosphorylation is regulated by
light, the signal transduced by the pathway whose recovery is regulated by RGS9-1.
The very robust phosphorylation of RGS9-1 can be seen
qualitatively simply by the fact that despite not being a particularly abundant protein (~1 mol of RGS-1/1600 mol of rhodopsin), it
incorporates more phosphate than all but a few other ROS proteins under
a range of different conditions. The significant stoichiometry of
phosphorylation that we observe suggests that under some conditions a
substantial fraction of RGS9-1, and perhaps even all of it, may be
subject to phosphorylation by the endogenous kinase. Immunoblots of ROS purified and washed in the absence of added ATP reveal no detectable Ser475 phosphorylation, suggesting that RGS9-1
phosphorylation is highly dynamic and may be subject to regulation at
the levels of both addition and removal of phosphate.
In contrast to results obtained with purified ROS, immunoblots of
retina from dark-adapted animals reveal Ser475
phosphorylation of RGS9-1 in vivo. The dramatic decrease in
phosphorylation levels observed in animals exposed to light underscores
the physiological regulation of RGS9-1 phosphorylation. Inhibition of
phosphorylation by lowering of calcium is consistent with the direction
of light regulation, because light lowers intracellular
[Ca2+] from the range of hundreds of nanomolar to 10 nM or less (64).
The Ser475 residue phosphorylated by the ROS kinase is
within the RGS9-1 C-terminal domain, which is not conserved in other
RGS proteins. Interestingly, there is another isoform of RGS9 named RGS9-2 in striatum, which results from alternative RNA processing (19,
20). Its amino acid sequence differs from that of RGS9-1 only in the
C-terminal domain beginning at residue 467. Although there is a serine
residue at a similar position (Ser474) in RGS9-2, it is in
a completely different sequence context. Thus, the Ser475
phosphorylation is unique for RGS9-1 and must be highly specific for
photoreceptors, because of the exclusive expression of RGS9-1 in these cells.
Phosphorylation of several other RGS proteins has been reported,
including Sst2, RGS2, RGS3, RGS4, RGS7, and GAIP (34-39). Phosphorylation was found to regulate the function of these RGS proteins by affecting their stability, subcellular localization, GAP
activity, or interactions with other regulatory molecules. For example,
phosphorylation of RGS2 by PKC inhibits its GAP activity, and
phosphorylation of RGS3 and RGS4 translocates them from cytosol to cell
membrane. In the case of RGS9-1, although not fully established, the
function of its unique C-terminal domain is beginning to emerge. Recent
work (14) suggests that it contributes to the tight regulation of GAP
activity of RGS9-1 and there is also evidence suggesting an essential
role in RGS9-1 binding to rod disc membranes (65). Therefore, it will
be interesting to determine whether phosphorylation in the C-terminal
tail has any direct effects on RGS9-1 GAP activity and membrane association.
From the studies we have conducted so far, it is not clear how the
phosphorylation state of RGS9-1 is regulated on a molecular level, or
how its phosphorylation affects its function. Assays of GAP activity
before and after phosphorylation have been difficult to interpret,
because ATP treatment of ROS leads to inactivation of RGS9-1 GAP
activity by a mechanism that seems to be distinct from and to precede
Ser475
phosphorylation.2 Likewise,
assays of recombinant RGS9-1 have so far been rendered ambiguous by the
difficulty in obtaining stoichiometric phosphorylation of the
recombinant protein. As can be seen in Fig. 7, the endogenous membrane-anchored protein is a much better substrate for the
membrane-associated kinase than is the soluble
RGS9-1-G Nevertheless, it would be very surprising if RGS9-1 phosphorylation did
not regulate its function in some way. The high specificity of the
reaction and the localization of the kinase to ROS membranes imply a
role in phototransduction. RGS9-1 interacts with a number of other
proteins in ROS, including G-32P]ATP led to RGS9-1 phosphorylation by
an endogenous kinase in rod outer segment membranes, with an average
stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass
spectrometry revealed a single major site of phosphorylation,
Ser475. The kinase responsible catalyzed robust
phosphorylation of recombinant RGS9-1 and not of an S475A
mutant. A synthetic peptide corresponding to the region surrounding
Ser475 was also phosphorylated, and a similar peptide with
the S475A substitution inhibited RGS9-1 phosphorylation. The RGS9-1
kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of
inhibitors or activators of protein kinase A, protein kinase G,
rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin-dependent kinase 5, at concentrations 50 or more
times higher than their reported IC50 or
Ki values. It was inhibited by the protein kinase C
inhibitor bisindolylmaleimide I and by lowering Ca2+ to
nanomolar levels with EGTA; however, it was not stimulated by the
addition of phorbol ester, under conditions that significantly enhanced
rhodopsin phosphorylation. A monoclonal antibody specific for the
Ser475-phosphorylated form of RGS9-1 recognized RGS9-1 in
immunoblots of dark-adapted mouse retina. Retinas from light-adapted
mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is
phosphorylated on Ser475 in vivo, and the
phosphorylation level is regulated by light and by
[Ca2+], suggesting the importance of the modification in
light adaptation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit. The activated Gt
, in its GTP-bound form,
then activates its downstream effector cGMP phosphodiesterase (PDE), which in turn hydrolyzes cGMP and lowers the cellular cGMP level to
close the cGMP-gated cation channels. In the recovery phase, rhodopsin
is deactivated by mechanisms involving phosphorylation and arrestin
binding (3), and Gt
is deactivated by hydrolysis of its
bound GTP. The intensity and duration of the G protein-coupled signaling is determined by the balance between reactions that amplify
or sustain the amount of activated Gt
-GTP and those that
dampen or terminate it. Therefore, rhodopsin deactivation by
phosphorylation and GTPase acceleration on Gt
are two major mechanisms for regulation in the recovery stage of normal vision.
, is
an important regulator of phototransduction and a key mediator of the
recovery to a dark state (4-6). It belongs to the ubiquitous RGS
(regulators of G protein signaling)
family of GAPs (for reviews, see Refs. 7-9) and shares with them a
conserved catalytic core or RGS domain that is responsible for the GAP
activity (10). Like most other RGS proteins (11-13), RGS9-1 also
contains multiple additional functional domains, which have been
speculated to be involved in GTPase regulation (14). These include a G
protein
-like domain, which tethers RGS9-1 to its partner subunit,
G
5L (15, 16), a
dishevelled/EGL-10/pleckstrin domain (17, 18), and a C-terminal domain
unique to RGS9-1 (4, 19, 20). Deletion of the RGS9 gene in mice results
in profound slowing of photoresponse recovery and transducin GTP
hydrolysis (21), and adding exogenous RGS9 catalytic core to excised
patches from ROS dramatically attenuates the phototransduction cascade
(22). It has been proposed that RGS9-1-regulated GTP hydrolysis is the
rate-limiting step in phototransduction (23, 24), but this hypothesis
has yet to be tested in a definitive way. Thus, modulation of GTPase
accelerating activity provides a plausible mechanism for attenuating,
sensitizing, speeding up, or slowing down light responses in order to
accommodate changes in background light or other conditions.
-binding protein phosducin
(32), Gt
(30), and the inhibitory
subunit of
cGMP phosphodiesterase (26, 33), but the roles of most of these
phosphorylation reactions in regulation of phototransduction are not
currently well understood. Recently, phosphorylation of other RGS
proteins, such as Sst2, RGS2, RGS3, RGS4, RGS7, and GAIP, has been
reported to modulate their functions (34-39). Therefore, it is likely
that the function of RGS9-1 may also be subject to regulation by
mechanisms involving phosphorylation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5S was expressed in Sf9
cells using a baculovirus vector and affinity-purified as described
(14). Polymerase chain reaction mutagenesis was used to generate a
construct that was identical to the His-tagged RGS9-1 except for
mutation of Ser475 to Ala (S475A). The expression and
purification of the mutant protein was essentially the same as that of
the full-length His-RGS9-1. Full-length recombinant RGS9-1 had to be
prepared and used as a complex with G
5S
because the protein is not expressed in a stable form in the absence of
G
5S or the natural partner subunit (16) of
RGS9-1, G
5L (14). Truncated recombinant
constructs containing only the RGS and C-terminal domains of RGS9-1
were not effective substrates for endogenous ROS kinases (data not shown).
80 °C.
Phosphorylation experiments were carried out in the dark. Kinase assays
were performed by incubating ROS with ATP at 30 °C in buffer C in
the presence of 1 mM DTT and 10 mM
NH2OH (to minimize photoisomerized rhodopsin
phosphorylation), unless otherwise stated. ATP was added to final
concentrations of 2-5 mM, with [
-32P]ATP
specific activity ranging from 40 to 100 Ci/mol. Phosphorylation was detected by autoradiography of immunoprecipitated RGS9-1 (see below) following SDS-PAGE. The quantity of phosphate incorporation into
RGS9-1 was determined by scintillation counting of the RGS9-1 bands
excised from SDS-PAGE gels following immunoaffinity isolation of RGS9-1
as described below. The amount of RGS9-1 in the sample was determined
by densitometry of the Coomassie Blue-stained gel using known amounts
of bovine serum albumin as standards. Differences in dye binding by the
standards and RGS9-1 were accounted for by UV absorbance
spectrophotometry of recombinant His-tagged RGS9-1, which was purified
from Escherichia coli in guanidine HCl by affinity chromatography. The extinction coefficient for RGS9-1 of 93,910 M
1-cm
1
calculated from its amino acid sequence (44) was used to determine the
amount of protein present, which was found to be 0.98 ± 0.01 of
that determined by dye binding using standards.
-32P]ATP (0.56 Ci/mmol) in buffer F. After the
phosphorylation reaction, all separation procedures prior to HPLC were
carried out at 0-4 °C. The phosphorylated membranes were collected
by centrifugation (86,000 × g for 10 min) and washed
several times with buffer A and then twice with buffer B before being
dissolved in 125 mM Tris-HCl, pH 6.5, containing 4% SDS
(w/v), 20% glycerol (v/v), 2.5 mM reducing agent
tris-(2-carboxyethyl) phosphine hydrochloride, 2.5 mM
MgCl2. Phosphorylated proteins were separated by 12% (w/v) preparative SDS-PAGE gels. The gels were stained with Coomassie Blue
R-250 and destained, and the RGS9-1 band was identified by its mobility
as calibrated by immunoblotting with monoclonal antibody D7 on an
identical gel run in parallel. After the gels were washed with water
and then pH 7.8 sodium bicarbonate solution, the RGS9-1 band was
excised and pulverized, and the protein was extracted by shaking with
10 mM Tris-HCl, pH 7.8, containing 1%
-mercaptoethanol (v/v), 0.2% SDS (w/v) for 5 h. The gel was extracted again with extraction buffer and water, and the combined extracts were
vacuum-dried. The dried extract was redissolved in water and subjected
to centrifugation to remove insoluble material. The resulting
supernatant was mixed with 100% trichloroacetic acid (w/v) to a final
concentration of 10% trichloroacetic acid to precipitate proteins. The
supernatant was subjected to another round of precipitation by 15%
trichloroacetic acid. The pellets were pooled and washed sequentially
with acetone, acetone/methanol (1:1), and water. The pellet was
digested with trypsin (10-20 µg) in 400 µl of 12.5 mM
1,3-bis[tris(hydroxymethyl)-methylamino]propane, a pH buffer, pH 7.9, containing 2 M urea, 0.125% mercaptoethanol (v/v), 1 mM CaCl2. The suspension was incubated at room
temperature for 7 h with occasional vortexing. Insoluble material
was separated by centrifugation and treated again with trypsin until
32P was undetectable in the pellet. Phosphopeptides were
isolated by chromatography with detection by scintillation counting.
After each elution, a single major 32P-containing peak was
collected, vacuum-dried, and used for the next step. Reverse-phase HPLC
was performed using a C18 HPLC column (Vydac 201HS52; 2.1 × 250 mm) with binary solvent systems (solvent A: H2O/0.1%
trifluoroacetic acid; solvent B: CH3CN/0.1%
trifluoroacetic acid; solvent C: H2O/0.2% HFBA; solvent D:
CH3CN/0.2% HFBA) and linear gradients. The first gradient
was 100% A/0% B to 10% A/90% B in 30 min at 0.3 ml/min, and the
second was from 100% A/0% B to 75% A/25% B in 50 min at 0.2 ml/min.
The peptide was further purified by Ga3+-immobilized metal
affinity chromatography (47) using 0.2 ml of Chelex-Sepharose (Amersham
Pharmacia Biotech). Finally, two additional rounds of HPLC were carried
out; one was identical to the second gradient described above, and the
last was also identical to the second except that solvents C and D were
used. The major peak was dried down and subjected to analysis by
electrospray mass spectrometry and tandem mass spectrometry (MS/MS)
using a Sciex API III triple quadrupole mass spectrometer fitted with a
nebulization-assisted electrospray ionization source (PE/Sciex, Thornhill, Ontario). For tandem MS/MS, precursor ions were selected with the first of three quadrupoles (Q1) for collision-induced dissociation with argon in the second quadrupole (Q2), and product ions
were scanned by the third quadrupole (Q3) (48).
5S was mixed with
ROS and [
-32P]ATP at 30 °C for the time indicated.
Final concentrations of proteins and reagents were 60 µM
rhodopsin, 0.2 µM His-RGS9-1 or 0.2 µM
His-RGS9-1-S475A, and 2.5 mM [
-32P]ATP
(100 Ci/mol). Reactions were then quenched by the addition of equal
volumes of SDS-PAGE sample buffer. Phosphorylation of His-RGS9-1 was
determined by autoradiography following SDS-PAGE, and the amounts of
His-RGS9-1 in the reactions were determined by immunoblotting using
rabbit anti-RGS9-1c polyclonal antibody.
-32P]ATP, with detection by binding to
phosphocellulose paper, followed by scintillation counting (51). The
concentrations used are 1000 times the reported Ka
value for PKA activation (52) and 100 times the reported
Ka value for PKG activation (53). Efficacy of PMA/Ca
for PKC stimulation under our conditions was verified by the previously
characterized phosphorylation of rhodopsin (54, 55), using
[
-32P]ATP and autoradiography. 2) Known substrates for
candidate kinases were added and tested for phosphorylation using
[
-32P]ATP and either autoradiography of SDS-PAGE gels
or phosphocellulose paper binding and scintillation counting. Those
that were phosphorylated (PKA/PKG and CDK5 substrates) were then used
to test the efficacy of activators (see above) and inhibitors (see
below). Lack of phosphorylation (CK II substrate) was taken as evidence
for absence of the kinase after verification of the ability of added
kinase to phosphorylate the substrate. The substrate used for PKA and PKG was [Val6,Ala7]Kemptide (Sigma) at 100 µM, the substrate for CK II was a substrate peptide
(Calbiochem) at 100 µM, and the substrate used for CDK5 was the
subunit of ROS cGMP phosphodiesterase (PDE6), PDE
(26, 33) at 400 nM. Peptide phosphorylation was assayed using
[
-32P]ATP under the same conditions as for RGS9-1
phosphorylation, with detection by binding to phosphocellulose paper,
followed by scintillation counting (51). Phosphorylation of proteins was also detected using [
-32P]ATP and autoradiography
following SDS-PAGE. 3) Known inhibitors were added, and their effects
on RGS9-1 phosphorylation were determined by measuring RGS9-1
phosphorylation in immunoblotting using
Ser475-phosphate-specific monoclonal antibody A4.
Inhibitors used were as follows: for PKA and PKG, H8-dihydrochloride
(60 µM) (Ki for PKA = 1.2 µM; Ki for PKG = 0.48 µM (56)); for rhodopsin kinase, sangivamycin (10 µM) (Ki = 180 nM (57));
for CDK5/p35, roscovitine (20 µM) (IC50 = 0.2 µM (58)); for CK II, A3-HCl (250 µM)
(Ki = 5.1 µM (59)); for PKC,
bisindolylmaleimide I-HCl (GF 109203X,
3-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide, HCl; Calbiochem) (500 nM) (Ki = 10 nM (60, 61)); for CaM-dependent kinase II (CaMK
II), CaM-binding domain (2.5 µM)
(Ki = 52 nM (62)). Inhibitors were
preincubated with ROS at room temperature for 15 min before the
addition of [
-32P]ATP to start phosphorylation. The
efficacy of the inhibitors under our conditions was verified by
determining their effects on phosphorylation of endogenous (PKC and
rhodopsin kinase) or added (PKA/PKG, CK II, CDK5) substrates by
endogenous (PKA/PKG, CDK5) or added (CK II) enzyme.
80 °C. To check the kinase activity in these fractions,
20 µl of each fraction was mixed with purified His-RGS9-1 (in complex
with G
5s) and ATP at 25 °C for 10 min.
Final concentrations of proteins and reagents were as follows: 60 µM rhodopsin (in the peak fraction), 200 nM
His-RGS9-1, 5 mM ATP. Reactions were quenched by adding equal volumes of standard SDS-PAGE sample buffer. Phosphorylation of
His-RGS9-1 was detected by immunoblotting using
Ser475-phosphate-specific monoclonal antibody A4, and the
amount of endogenous RGS9-1 and recombinant proteins in each sample was determined by the anti-RGS9-1c polyclonal antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, radioactivity was detected in a protein
migrating to the same position as that of RGS9-1. To determine if this
phosphoprotein is indeed RGS9-1, we incubated ROS with
[
-32P]ATP, washed away free ATP, and
immunoprecipitated RGS9-1 from detergent-solubilized ROS. Two strongly
labeled radioactive bands were detected in the total ROS proteins,
migrating to the positions corresponding to rhodopsin and RGS9-1 (Fig.
1A), respectively. At the
position of RGS9-1, the radioactivity came from a detergent-soluble and
a detergent-insoluble species, implying the presence of two co-migrating phosphoproteins. Both the autoradiogram and the immunoblot showed that the detergent-soluble phosphoprotein was quantitatively precipitated by anti-RGS9-1 antibody, identifying it as phosphorylated RGS9-1. The detergent-insoluble phosphoprotein was later identified by
mass spectrometry analysis to be tubulin (29, 63) (data not shown).
Comparison of the radioactive signals from RGS9-1 and other ROS
proteins after [
-32P]ATP labeling clearly reveals
RGS9-1 as one of the major targets for phosphorylation in ROS under our
phosphorylation conditions.
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Fig. 1.
Phosphorylation of RGS9-1 by endogenous
kinase. A, ROS membranes (60 µM
rhodopsin) were incubated with 5 mM
[ -32P]ATP as described under "Experimental
Procedures" for 15 min in the dark. After washing to remove ATP,
membranes (ROS) were extracted by detergent as described,
yielding pellet and supernatant (Sup't) fractions. RGS9-1
was immunoprecipitated, again yielding pellet (IP) and
supernatant (After IP) fractions. Identical SDS-PAGE gels
loaded with equal proportions of each fraction were visualized by dye
staining (Coomassie), autoradiography (Auto-Rad),
or immunoblotting with monoclonal RGS9-1 antibody D7
(Western). Upper arrows, RGS9-1; lower
arrows, rhodopsin. B, ROS were incubated with
[
-32P]ATP for the times indicated. The stoichiometry
of RGS9-1 phosphorylation was determined as described under
"Experimental Procedures." The curve drawn is a nonlinear least
squares fit of the data to a single exponential function with a rate
constant of 0.36 min
1.
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Fig. 2.
Isolation of tryptic phosphopeptide by
reverse-phase HPLC and identification of the phosphorylation site by
mass spectrometry. A, HPLC elution profiles of the
RGS9-1 phosphopeptide(s) in the presence of 0.1% trifluoroacetic acid
or 0.2% HFBA. The proteolysis and preliminary separations of peptides
were as described under "Experimental Procedures." The RGS9-1
phosphopeptide was purified using a C18 HPLC column in the presence of
0.1% trifluoroacetic acid (solid line with open
circles). The 32P radioactivity-containing fractions
were pooled, dried down, and further purified on the same column in the
presence of 0.2% HFBA (dashed line with solid
circles) as described under "Experimental Procedures."
B, tandem MS/MS of the RGS9-1 phosphopeptide purified by
HPLC in the presence of 0.2% HFBA (the major
32P-radioactivity peak in Fig. 2A). MS/MS
spectrum of MH1+ precursor ion
(m/z 583.5) of the phosphorylated
S475QLR yielded ions of y1-y4 (all dephosphorylated) and
b1-b4 (all dephosphorylated and dehydrated). The same site was
identified from two independent phosphorylation experiments.
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Fig. 3.
Phosphorylation of recombinant RGS9-1.
ROS membranes were incubated with purified His-tagged
RGS9-1-G 5s complex (rRGS9)
and [
-32P]ATP. Phosphorylation of His-RGS9-1 or
His-RGS9-1-S475A was detected by autoradiography following SDS-PAGE
(32P). The amount of RGS9-1 in each sample was
verified by immunoblot using a polyclonal anti-RGS9-1c antibody
(RGS9 antibody). Upper bands are recombinant proteins
(rRGS9), and lower bands are endogenous RGS9-1
(RGS9).
7 M was sufficient to restore
full activity (Fig. 4C). For these immunoblots, we used
monoclonal antibody A4 that specifically recognizes
Ser475-phosphate. It was raised against a phosphopeptide
derived from murine RGS9-1 (KLDRRS(P)QLKKELPP) and was found
to react with RGS9-1 only in ATP-treated ROS (Fig. 4B).
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Fig. 4.
Cation requirements for RGS9-1
phosphorylation. A, purified bovine ROS membranes were
homogenized at 15 µM rhodopsin in buffer G plus 1 mM EDTA and centrifuged twice to remove contaminating metal
ions. ROS were then homogenized in the following buffers once and
resuspended in corresponding buffers to a final concentration of 60 µM rhodopsin: EDTA, buffer G with 1 mM EDTA; Mg2+, buffer G with 2 mM MgCl2 and 0.1 mM EDTA;
Mn2+, buffer G with 2 mM
MnCl2 and 0.1 mM EDTA; Ca2+,
buffer G with 2 mM CaCl2 and 0.1 mM
EDTA. RGS9-1 was phosphorylated in these buffers as described for those
in buffer C, and phosphate incorporation was detected by
autoradiography in immunoprecipitated RGS9-1 after SDS-PAGE
(32P). Equivalent loading of immunoprecipitated
RGS9-1 was verified by immunoblot using monoclonal antibody D7
(RGS9 antibody). Control, phosphorylation of
RGS9-1 in ROS membranes without any washes. B, specificity
of monoclonal antibody A4. Proteins in ROS membranes were analyzed by
SDS-PAGE and immunoblotting with mAb A4 after incubation with (+ ATP) or without ( ATP) ATP. Similar results
were obtained with recombinant RGS9-1 isolated from insect cells (data
not shown). C, inhibition by Ca2+ chelation.
ROS, ROS without ATP incubation; ROS + ATP, ROS plus 5 mM ATP plus 500 nM
CaCl2, [Ca2+]
500 nM;
EGTA, ROS plus 5 mM ATP plus 4.0 mM
EGTA, [Ca2+]
1 nM; Ca (150 nM), ROS plus 5 mM ATP, 4.0 mM
EGTA, 2.9 mM CaCl2, [Ca2+] = 142 nM; Ca (300 nM), ROS plus 5 mM ATP, 4.0 mM EGTA, 3.3 mM
CaCl2, [Ca2+] = 285 nM.
Mg2+ was present at 6 mM total concentration in
all samples. The program WinMAXC written by Chris Patton (Stanford
University) was used to calculate free Ca2+
concentrations.
was phosphorylated by an
endogenous kinase, most likely CDK5/p35 (33), and the phosphorylation
was inhibited 67% by added roscovitine, verifying its efficacy as well
(Fig. 5F). No phosphorylation of the CK II substrate in ROS
membranes was observed, and there were no effects on phosphorylation of any protein detectable upon the addition of the CK II inhibitor A3-HCl.
However, this inhibitor did reduce phosphorylation of the CK II
substrate peptide by added CK II in the presence of ROS. Since no
activity of CK II or CaMK II was detected using either substrate (CK
II) or inhibitors (CK II and CaMK II), it seems likely that they are
not responsible for RGS9-1 phosphorylation and also are absent in our
membrane preparation. Thus, we can rule out, to varying degrees of
certainty, the involvement in RGS9-1 phosphorylation of all protein
kinases known to be present in ROS, as well as of additional kinases
whose presence is less certain. Although the inhibition by the PKC
inhibitor bisindolylmaleimide I raises the possibility of a previously
uncharacterized PKC isozyme or PKC-like enzyme in ROS, the inhibitor we
used can also inhibit kinases other than PKC, such as phosphorylase
kinase, (IC50 = 0.7 µM at 250 µM ATP (60)). The kinase responsible for RGS9-1 phosphorylation is clearly distinct from the PKC isozyme responsible for PMA-induced phosphorylation of rhodopsin (54, 55).
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Fig. 5.
Effects of kinase activators and inhibitors
on RGS9-1 phosphorylation. A, ROS membranes were
incubated with 5 mM [ -32P]ATP (40 Ci/mol)
at 60 µM rhodopsin in buffer C in the presence of
8-Br-cAMP (cA) or 8-Br-cGMP (cG). The reactions
were allowed to proceed at 30 °C for 15 min and then quenched by
SDS-PAGE sample buffer. RGS9-1 phosphorylation was detected by
autoradiography following SDS-PAGE. The positions of RGS9-1 in
A and B are indicated by arrows.
B, phosphorylation of RGS9-1 in the presence of PMA (0, 1, 5, or 10 µM) and CaCl2 (0 or 2 mM, upper panel; 0 or 1 mM, lower panel) was performed as in
A, with detection by 32P in the upper
panel and immunoblotting using
Ser475-phosphate-specific antibody A4 (S475-P
antibody) in the lower panel. C,
ROS were preincubated at room temperature for 15 min in buffer C with
one of the following kinase inhibitors at the concentrations listed
under "Experimental Procedures": CK II, A3-HCl;
PKC, bisindolylmaleimide I-HCl; CaMK II, CaM-binding domain;
RK, sangivamycin; CDK5, roscovitine,
PKA/PKG, H8 dihydrochloride. ATP was then added to a final
concentration of 0.2 mM, and the reactions were allowed to
proceed for 15 min. before adding SDS-PAGE sample buffer to quench.
RGS9-1 phosphorylation was determined as in C. D,
RGS9-1 was incubated with 5 mM ATP in the presence of
the peptide inhibitor KLDRRAQLKKELPPK. Increasing concentrations of
the peptide (peptide) were used: 0, 0.5, 2.0, and 8.0 mM. RGS9-1 phosphorylation was determined as in
C. E, ROS were incubated with rhodopsin kinase
inhibitor (San; 2 µM) or PKC inhibitor
(Bis; 100 nM) and 5 mM
[
-32P]ATP at 60 µM rhodopsin in buffer C
for 15 min. Rhodopsin phosphorylation was monitored by autoradiograms
(32P) and Coomassie (Coom.) staining
following SDS-PAGE. F, recombinant PDE-
(400 nM) was phosphorylated with or without CDK5/p35 inhibitor
(Rosc.; 20 µM) in the presence of ROS (60 µM rhodopsin) and 5 mM
[
-32P]ATP in buffer C. PDE-
(P-
)
phosphorylation was measured by autoradiograms (32P)
following SDS-PAGE. G,
[Val6,Ala7]Kemptide (Kemp.; 100 µM) was phosphorylated by ROS kinases (ROS; 60 µM rhodopsin) in the presence of 0.2 mM
[
-32P]-ATP, 8-Br-cAMP (camp, 50 µM), or 8-Br-cGMP (cGMP, 50 µM),
with or without PKA/PKG inhibitor (H8; 60 µM).
Kemptide phosphorylation was measured by phosphocellulose binding and
scintillation counting. H, ROS (60 µM rhodopsin) were incubated with 0.2 mM
[
-32P]ATP in the presence of CK II peptide substrate
(Sub.; 100 µM), CK II inhibitor
(A3, 250 µM), or recombinant CK II (5 units/µl). Phosphorylation of CK II substrate was determined as in
G.
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Fig. 6.
Co-purification of RGS9-1 kinase activity
with rhodopsin and RGS9-1 in fractionated retinal membranes and tight
membrane binding of RGS9-1 kinase. A, ROS were purified
by a standard discontinuous sucrose density gradient technique (43).
The sucrose gradient after ultracentrifugation was fractionated into
1-ml fractions. Rhodopsin concentrations in each of the fractions were
determined by absorbance at 500 nm and were normalized to the highest
concentration value. RGS9-1 kinase activity in each of the fractions
was detected using His-tagged RGS9-1 as substrate, as described under
"Experimental Procedures." The boxed area
shows an enlarged view of the fractions corresponding to the rhodopsin
peak. ROS, fraction containing peak of ROS; Broken
ROS, fraction containing peak of broken ROS membranes;
Pellet, fraction containing unsheared retinal membranes;
RGS9 antibody, immunoblot using anti-RGS9-1c antibody;
S475-P antibody, immunoblot using monoclonal
anti-Ser475-phosphate antibody A4. B,
purified ROS membranes were homogenized by repeated passage through a
23-gauge needle at 15 µM rhodopsin in one of the
following buffers: isotonic buffer (Iso.) (100 mM NaCl, 5 mM Tris, pH 7.4, 2 mM
MgCl2, 1 mM DTT); hypertonic buffer
(Hyper.) (1 M NH4Cl, 5 mM Tris, pH 7.4, 1 mM DTT); hypotonic buffer
(Hypo.) (5 mM Tris, pH 7.4, 0.5 mM
MgCl2, 1 mM DTT). Membranes were then collected
by centrifugation at 84,000 × g for 20 min. The
washing step was repeated three times for each sample, and the washed
ROS were finally resuspended in buffer C to a rhodopsin concentration
of 60 µM for ATP incubations. Endogenous RGS9-1
phosphorylation was detected by autoradiography
(32P) following immunoprecipitation from detergent
extracts and SDS-PAGE, and the amount of immunoprecipitated RGS9-1 was
verified by immunoblot using the monoclonal antibody D7 (RGS9
antibody). ROS, RGS9-1 phosphorylation on unwashed
membranes.
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Fig. 7.
RGS9-1 phosphorylation in mouse retina.
A, RGS9-1 was immunoprecipitated from dark-adapted
(Dark) or light-adapted (light) mouse retinas and
immunoblotted with Ser475-phosphate-specific antibody
(S475-P antibody). The amount of RGS9-1 in the samples was
measured on the same blot using monoclonal anti-RGS9-1 antibody D7
(RGS9 antibody). B, RGS9-1 phosphorylation was
compared between dark- and light-adapted animals by densitometry of
films. Three data points from immunoblots similar to A were averaged,
and RGS9-1 phosphorylation = (average densities of RGS9-1 bands in
Ser475-phosphate-specific antibody Western blots)/(average
densities of RGS9-1 bands in monoclonal anti-RGS9-1 antibody D7 Western
blots).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5S complex, and no
Ser475 phosphorylation of the latter can be detected on the
complex as purified from insect cells.
5L, the PDE
inhibitory subunit of cGMP phosphodiesterase, and Gt
in
addition to its interactions with the kinase and with ROS membranes. Moreover, sensitivity and kinetics of photoresponses vary with the
intensity of excitation light and background light. Because of its
pivotal position in the inactivation phase of the light response,
modulation of RGS9-1 GAP activity through phosphorylation is a
plausible mechanism for bringing about some of these changes. Even if
GAP activity turns out not to be regulated by phosphorylation, RGS9-1
phosphorylation has revealed the presence of a membrane-associated protein kinase that appears to be distinct from those previously identified in ROS. This kinase is clearly active in ROS in
vivo, and its activity is regulated by light. Identification and
characterization of this kinase at the molecular level should provide
insight into its functional role and will be facilitated by the
procedures we describe here for extracting it in soluble form and for
assaying it in the absence of ROS membranes.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Jing Huang for helping in the preparation of the monoclonal anti-phosphorylated peptide antibody A4.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Research Grants EY08061 and EY11900 and Training Grant EY07001, the Welch Foundation, a grant from Research to Prevent Blindness (RPB) (to the University of Washington, Department of Ophthalmology), and a grant from the E. K. Bishop Foundation.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.: 713-798-6994; Fax: 713-796-9438; E-mail: twensel@bcm.tmc.edu.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.M011539200
2 C. Cowan and G. Hu, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: Gt, G protein transducin; CaM, calmodulin; CaMK II, CaM-dependent kinase II; CK II, casein kinase II; GAP, GTPase-accelerating protein(s); HFBA, heptafluorobutyric acid; MS, mass spectrometry; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; ROS, rod outer segment(s); PDE, phosphodiesterase; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; 8-Br-cAMP, 8-bromo-cyclic AMP; 8-Br-cGMP, 8-bromo-cyclic GMP.
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