From the Departments of Ophthalmology,
¶ Pharmacology, and
Chemistry, University of
Washington, Seattle, Washington 98195 and the § Verna
and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, Texas 77030
Received for publication, November 19, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Inactivation of the visual G-protein transducin
by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP)
RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light- and
Ca2+-sensitive phosphorylation on Ser475.
Because there are much higher RGS9-1 levels in cones than in rods, we
investigated whether Ser475 is phosphorylated in rods using
Coneless mice and found that both the phosphorylation and
its regulation by light occur in rods. Therefore, we used rod outer
segments as the starting material for the purification of RGS9-1
kinase activity. Two major peaks of activity corresponded to protein
kinase C (PKC) isozymes, PKC Transducin (Gt),1 the
visual G-protein, is inactivated when its bound GTP is hydrolyzed
to GDP, a free phosphate dissociates from the catalytic site, and the
RGS9-1 contains a G-protein- We previously reported that RGS9-1 is phosphorylated by an endogenous
kinase in ROS membranes with an average stoichiometry of 0.2-0.45 mol
of phosphates/mol of RGS9-1 (18). A single major site of
phosphorylation was identified by a combination of mass spectrometric
analysis and mutagenesis as Ser475. The kinase responsible
has been only partially characterized. Initial studies revealed that
the RGS9-1 kinase is a peripheral membrane protein that differs from
protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II,
casein kinase II, and cyclin-dependent kinase 5. In
addition, it is sensitive to the PKC inhibitor bisindolylmalemide I. The kinase activity was also inhibited by EGTA and restored by
submicromolar [Ca2+]. The kinase co-purifies with
rhodopsin in sucrose gradients and can be extracted in buffers of high
ionic strength (18). We also developed a monoclonal antibody specific
for the Ser475-phosphorylated form of RGS9-1 and found that
it recognized RGS9-1 in immunoblots of dark-adapted mouse retina (18).
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. Phosphorylated RGS9-1 is present
exclusively in the cholesterol-rich detergent-insoluble membrane
domains (Rafts), whereas the unphosphorylated form can be readily
extracted with mild detergent (19). These results demonstrate
differences in the subcellular localization between phosphorylated
RGS9-1 and its unphosphorylated form.
In another study, cAMP-dependent protein kinase (PKA)
inhibitor H89 reduced RGS9-1 phospholabeling by more than 90%, whereas dibutyryl-cAMP stimulated it 3-fold (20). These studies suggested PKA
as the major kinase responsible for RGS9-1 phosphorylation in ROS. The
PKA catalytic subunit was found to phosphorylate recombinant RGS9-1,
and mutational analysis of a fragment of RGS9 (residues 276-431)
identified the phosphorylation sites as Ser427 and
Ser428. However, in ROS, RGS9-1 phosphorylation required
the presence of free Ca2+ ions and was inhibited by light
(20).
The current work focused on the identification of the protein kinase(s)
and the protein phosphatase responsible for regulating phosphorylation
of RGS9-1. We used bovine ROS as a starting material and carefully
fractionated protein kinase activities, quantitatively accounting for
the RGS9-1 kinase activity. We report here that two PKC isozymes from
ROS, PKC Proteins--
Recombinant kinases, phosphatases, and antibodies
were purchased as follows: PKA and PKC Anti-RGS Antibodies--
Monoclonal antibody D7 was raised
against bacterially expressed RGS9-1 as described previously (16).
Mouse anti-phosphorylated RGS9-1 monoclonal antibody (A4) was raised
against a C-terminal peptide derived from mouse phosphorylated RGS9-1,
(KLDRRpSQLKKELPPK, where pS represents phosphorylated serine); Quality
Controlled Biochemicals, Inc., Hopkinton, MA), and coupled to a carrier
protein, keyhole limpet hemocyanin (Sigma) (18). Rabbit
anti-RGS9-1c polyclonal serum was generated as described previously
(1).
Expression and Purification of Recombinant RGS9-1 and R9AP--
RGS9-1 proteins, with either a glutathione S-transferase or
His6 tag on the N terminus, bound to G Phosphorylation of RGS9-1 in Vivo--
All animal experiments
employed procedures approved by the University of Washington Animal
Care Committee and conformed with recommendations of the American
Veterinary Medical Association Panel on Euthanasia. All animals were
maintained in complete darkness, and all manipulations were done under
dim red light employing Kodak number 1 safelight filters
(transmittance >560 nm). Coneless mice were obtained from
J. Nathans (Johns Hopkins University) and GCAP1/2 knockout mice from
J. Chen (Southern California University). The analysis of RGS9-1
phosphorylation of mouse retinas was carried out essentially as
described previously (18). Briefly, 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 with 10 mM Tris-HCl,
100 mM NaCl, 2 mM MgCl2, ~20
mg/liter phenylmethylsulfonyl fluoride with 1% Nonidet P-40 detergent,
plus 0.2 mM Na3VO4, 15 µM fenvalerate, and 100 nM okadaic acid to
inhibit phosphatase activities. Immunoprecipitation of RGS9-1 was
carried out using rabbit polyclonal antibodies (18), and the
immunoprecipitated protein was analyzed by SDS-PAGE and immunoblotting
with the mouse monoclonal antibody A4 specific for the phosphopeptide
and phosphorylation-independent antibody D7 at a dilution of 1:500. The
secondary antibody used was horseradish peroxidase-conjugated
(Promega), and detection was done by chemiluminescence using the ECL®
system (Amersham Biosciences). RGS9-1 phosphorylation was compared
between dark- and light-adapted animals by densitometry of films. Three
data points from immunoblots were averaged, and relative
phosphorylation levels were calculated as follows: relative RGS9-1
phosphorylation = (average densities of RGS9-1 bands in
Ser475-phosphate-specific antibody immunoblots)/(average
densities of RGS9-1 bands in monoclonal anti-RGS9-1 antibody D7
immunoblots). Phosphorylation levels were normalized to the relative
phosphorylation value obtained in wild type mice.
Kinase Assays for Kinase Purification and
Characterization--
The RGS9-1 kinase activity was routinely assayed
using the KLDRRSQLKKGLPPK peptide derived from the
C-terminal region of RGS9-1 that is phosphorylated in vitro
in ROS suspensions (18). This site is also phosphorylated in RGS9-1
in vivo (18). In the standard conditions, phosphorylation
was carried out in 10 mM BTP, pH 7.5, 5 mM
MgCl2, containing 13 mg/ml peptide with and without 0.1 mM cGMP, 0.1 mM cAMP, 0.01 mM DAG,
1 mM PMA, 50 mM Ca2+, 50 mM EGTA, 3 µM CaM, and 230 mg/ml PS. The
phosphorylation reaction was initiated by an addition of
[
Alternatively, qualitative assays of RGS9-1 kinase activity were
carried out using similar conditions as described for the peptide
assays, using the recombinant His6-RGS9-1/G Phosphorylation of RGS9-1 by Recombinant PKC for Functional
Assays--
His6- or glutathione
S-transferase-tagged RGS9-1 full-length protein with G Purification of RGS9 Kinase--
ROS were purified in dim red
light from fresh or frozen bovine retinas as described (22). All
procedures were performed on ice or at 4 °C. Bovine ROS (obtained
from 200 retinas) were homogenized with 50 ml of water, and the
membranes were collected by centrifugation at 100,000 × g for 20 min. The water extraction was repeated a second
time, and two additional extractions were performed in 10 mM BTP, pH 7.5, containing 100 mM NaCl. RGS
kinase(s) was extracted using 10 mM BTP, pH 7.5, containing
1 M NH4Cl. The high salt extraction was
repeated twice, and the obtained supernatants were combined (~150 ml)
and loaded onto hydroxyapatite (1.5 ml) at a flow rate of 15 ml/min.
The column was washed with 300 mM NaCl in 10 mM
BTP, pH 7.5, and proteins containing the RGS9-1 kinase activity were
eluted with 300 mM K2HPO4 in 10 mM BTP, pH 7.5. Salt was removed by dialysis against 10 mM BTP, pH 7.5, and proteins were chromatographed on a
DEAE-Sepharose (5 ml; Amersham Biosciences) column pre-equilibrated
with 10 mM BTP, pH 7.5, at a flow rate of 0.8 ml/min.
Unbound proteins were removed by 10 mM BTP buffer, pH 7.5, whereas remaining proteins were eluted with a linear gradient of 0-1
M NaCl in 10 mM BTP buffer, pH 7.5. Fractions
with kinase activity were pooled together, dialyzed, and applied to a
heparin-Sepharose column previously equilibrated with 10 mM
BTP, pH 7.5. Proteins were eluted with a linear gradient of 0-0.4
M NaCl in 10 mM BTP, pH 7.5. RGS9-1 kinase
activity was monitored using the peptide or recombinant RGS9-1
immunoblot assays as described above.
PKGII Expression--
HEK cells at 40% confluence in 10-cm
plates were transfected by calcium phosphate precipitation with 20 µg/plate of pCMV.CGKII (a gift from M. Uhler) (23). After 26 h,
the cells were washed with PBS buffer, scraped, and homogenized.
Expression of the enzyme was confirmed by immunoblot using anti-PKGII
(M. Uhler).
P-RGS Dephosphorylation--
Dephosphorylation was carried out
for 20 min at 30 °C in 15 µl of 10 mM BTP pH 7.5, with
the addition of 0.4 units of phosphatase, 5 mM
MgCl2, and 5 µg of bovine ROS. The reaction was quenched by the addition of SDS-PAGE sample buffer. After separation in SDS-PAGE
gels, dephosphorylation/phosphorylation was visualized by immunoblot
and A4 antibody.
SDS-PAGE and Immunoblotting--
SDS-PAGE was performed
according to Laemmli (24) with 12% polyacrylamide gels. For
immunoblotting, membranes were blocked with 2% bovine serum albumin in
20 mM Tris, pH 8.0, containing 150 mM NaCl and
0.05% Tween 20 and incubated for 1 h with primary antibodies (D7
and A4), 1:1000 or 1:500. When secondary antibody conjugated with
alkaline phosphatase (Promega) was used, the dilution was 1:7000, and
antibody binding was detected using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as a substrate. When secondary antibody conjugated with horseradish peroxidase (Promega) was used, the
dilution was 1:5000, with detection by chemiluminescence using the
ECL® system (Amersham Biosciences).
Immunolocalization--
For flat mount localization, bovine eyes
obtained from a local slaughterhouse were held briefly on ice prior to
dissection. For localization in mouse and bovine retinas, the anterior
segment and vitreous humor were removed from each eye, and the eye cups were immersed for 6 h in chilled 4% formaldehyde in 0.086 M sodium phosphate buffer, pH 7.3. Pieces of retina (5 mm2) were excised, washed in phosphate buffer, and embedded
in 5% agarose in phosphate-buffered saline. Sections of retina (100 µm thick) were cut with a VT1000E vibrating microtome (Leica). Antibody labeling was assessed by indirect immunofluorescence. Sections
were first incubated in normal goat serum diluted 1:50 in ICC buffer
(phosphate-buffered saline containing 0.5% bovine serum albumin, 0.2%
Triton X-100, and 0.05% sodium azide, pH 7.3) for 1 h to reduce
nonspecific labeling. Mouse monoclonal RGS9 antibody
(A280 = 0.69) diluted 1:25 in ICC buffer was
added to retinal sections for incubation overnight in a humidified
chamber. Negative controls for antibody labeling were produced by
omitting primary antibody from the incubation buffer or by preadsorbing RGS9 antibody with purified full-length RGS9 coupled to CNBr-activated Sepharose (0.5 ml, 1 mg/ml RGS9). After repeated washing in buffer, sections were incubated for 4 h in indocarbocyanine
(Cy3)-conjugated goat anti-mouse antibody (Jackson ImmunoResearch,
Inc.) diluted 1:200 in ICC buffer. Sections were washed repeatedly with
ICC buffer, mounted in 5% n-propyl gallate in glycerol,
coverslipped, and examined on a Bio-Rad MRC 600 laser-scanning confocal
microscope. Scan head parameters influencing image intensity and
resolution (pinhole aperture, PMT gain and black level, and attenuation
of laser by neutral density filters) were standardized for all samples. Single plane and Z series images were collected and stored as unprocessed files. Images for publication were processed with Adobe
Photoshop 3.0.
Single-turnover GTPase Assays--
Single-turnover GTPase assays
were performed as described previously (1, 25). PKC-phosphorylated or
-unphosphorylated RGS9-1 were mixed with urea-washed ROS, purified Gt,
PDE RGS9-1 Binding to R9AP Vesicles--
The binding assays were
carried out as described recently.2 E. coli
expressed His6-R9AP was purified in detergent and
reconstituted into lipid vesicles containing
L- RGS9-1 Phosphorylation in Transgenic Mice--
RGS9-1 localizes in
rod and cone photoreceptors in the bovine retina (16). Previously, we
showed that RGS9-1 is phosphorylated in ROS preparations (18). We
reinvestigated localization of RGS9-1 using the D7 antibody raised
against bacterially expressed RGS9-1 and the flat mount section of
bovine retina. The flat mount immunolocalization revealed a
significantly higher level of RGS9-1 in cones rather than in rods as
shown at lower (Fig. 1Aa) and higher magnification (Fig. 1Ab). Immunolabeling of RGS9-1
using the cross-section confirmed higher cone expression (Fig.
1Ac). Preincubation with purified protein abolished RGS9-1
immunoreactivity (data not shown).
Because of the higher expression levels of RGS9-1 in cone
photoreceptors, we sought additional proof that RGS9-1 is
phosphorylated in ROS. One possible explanation for our previous
results on RGS9-1 phosphorylation in ROS was that isolated ROS were
contaminated by RGS9-1 from cone outer segments, where it is highly
enriched, and consequently, phosphorylation was a cone-specific
phenomenon. Therefore, to evaluate RGS9-1 phosphorylation in rods, we
employed mice lacking cone photoreceptors (Coneless mice)
(26). RGS9-1 immunoprecipitated from retinal homogenates obtained from
dark- and light-adapted wild type and Coneless mice was
subjected to electrophoresis, transferred to Immobilon membrane, and
probed for RGS9-1 phosphorylation with the Ser475
phosphate-specific antibody A4 (18). Immunoblotting with the D7
anti-RGS9-1 antibody revealed that the total amount of
immunoprecipitated RGS9-1 was similar in wild type and
Coneless, as well as in light- and dark-adapted mice (Fig.
1B). Comparison of the phosphorylation level between dark-
and light-adapted mice revealed that light reduced phosphorylation by
~50% in both wild type and Coneless mouse retina (Fig.
1B). These results demonstrate that RGS9-1 phosphorylation
occurs in rod cells in vivo and is regulated by light.
Because RGS9-1 phosphorylation is regulated in vitro by
Ca2+, we also measured RGS9-1 phosphorylation in mice
lacking GCAP1/GCAP2, two Ca2+-binding proteins in
photoreceptors (27, 28). We found similar results in those mice as
compared with wild type mice, suggesting that GCAP1 and GCAP2 are not
involved in the regulation of RGS9-1 phosphorylation (data not shown).
Extraction of RGS9-1 Kinase--
To identify protein kinases
responsible for phosphorylation of RGS9-1 at Ser475, bovine
ROS was fractionated, and the kinase activity was quantitatively assessed. ROS were used as starting material, because they are readily
accessible for biochemical procedures. Two specific assays were
utilized to determine RGS9-1 kinase activity. The first assay involves
phosphorylation of full-length recombinant RGS9-1 obtained from
Sf9 cells, in a complex with G
Fractionation of RGS9-1 kinase was accomplished by washing bovine ROS
membranes with H2O, 0.1 M NaCl, and 1 M NH4Cl (Fig. 2A). RGS9-1 peptide was
phosphorylated by all three extracts, with the highest phosphorylation
level by the H2O extract (Fig. 2C). However, the
specific full-length RGS9-1 kinase activity was found only in the 1 M NH4Cl extract using the A4-site-specific antibody. The two phosphorylated bands detected in the A4 immunoblot corresponded to the tagged recombinant (rRGS9-1) and the endogenous RGS9-1 (Fig. 2C, inset). These data indicated
that ~38% of the total kinase activity toward the peptide was RGS9-1
specific, tightly membrane associated, and could be extracted with 1 M NH4Cl. We also observed that another 32% of
the total activity, which remained in the H2O and 0.1 M NaCl fractions, was not RGS9-1 specific and could be
easily removed during the extraction process. The remaining ROS pellet
contained ~30% RGS9-1 kinase activity, which was refractory to all
of the above extraction procedures (data not shown).
Purification of RGS9-1 Kinase from ROS--
RGS9-1 kinase
extracted from the ROS membranes with 1 M NH4Cl
was routinely chromatographed on hydroxyapatite (data not shown) followed by DEAE-Sepharose. From the DEAE-Sepharose column, the bulk of
the activity was eluted by a salt gradient as a single peak (Fig.
2D). Fractions 7-11 contained a majority of the RGS9-1 kinase activity (Fig. 2D). This chromatography also
separated the RGS9-1 kinase(s) from the endogenously phosphorylated
RGS9-1, which did not bind to DEAE-Sepharose (data not shown).
Subsequent heparin-Sepharose chromatography of the kinase
fractions eluted from DEAE-Sepharose resulted in the important
separation of three RGS9-1 kinase activity peaks (Fig.
3A), with peak 1 being more variable between preparations in terms of maximal activity. The small
amounts of these kinases and the partial nature of the purification precluded us from direct sequence identification of these kinases; however, full separations of these activities allowed further biochemical characterization. In summary, using conventional column chromatography, we were able to identify three unique fractions of
RGS9-1 kinase activity, suggesting the involvement of multiple kinases
in the phosphorylation of Ser475 within RGS9-1.
PS-dependent Stimulation of RGS9-1
Kinase(s)--
Next, we tested if the kinase activities obtained from
the Heparin-Sepharose column were stimulated by common kinase
modulators like cGMP; cAMP; DAG, PS, and Ca2+; PS, PMA, and
Ca2+; CaM and Ca2+; and EGTA. Using the peptide
as a substrate, we found that the activities of RGS9-1 kinases in peaks
2 and 3 were elevated in the presence of DAG, PS and Ca2+,
with kinase peak 3 having the most dramatic increase (Fig.
3A). In fact, the total contribution of peak 3 to the RGS9-1
kinase activity increased from 40 to 77% upon stimulation,
constituting the largest fraction of activity from these three peaks.
On the other hand, when the full-length recombinant RGS9-1/G Identification and Localization of PKC Isozymes--
Using
immunoblot analysis with antibodies raised against recombinant PKC
isozymes, we found that PKC Phosphorylation of RGS9-1 by PKC Isozymes--
Because of the
presence of PKC
The specificity of PKC phosphorylation of RGS9-1 may be partially
determined by the membrane environment in which they normally interact.
Although the predominant site in RGS9-1 phosphorylated by the
endogenous kinases in ROS membranes is Ser475, and an
RGS9-1 mutant lacking that site is not efficiently phosphorylated, both
recombinant highly active PKC
We also tested other kinases phosphorylated sequence corresponding to
the RGS9-1 Ser475 phosphorylation site (38). We compared
PKG, PKA, and Ca2+/CaM II kinase for their abilities to
phosphorylate recombinant RGS9-1 or its peptide (Fig.
6, A and B). The
phosphorylation level by PKA and CaM kinase II was much lower than that
by PKC isoforms. Although PKGI did show strong phosphorylation of
RGS9-1, immunoblotting of different fractions with specific antibodies
produced against PKGI and PKGII, together with immunocytochemistry
(data not shown), did not provide any evidence for the presence of PKG
in photoreceptor cells (Fig. 6C). With all three kinases,
PKGI, PKA, and Ca2+/CaM II kinase, positive results were
obtained with their specific peptides (Fig. 6B), indicating
that all enzymes were reactive in our assays.
Dephosphorylation of RGS9-1 by PP2A--
Since
phosphorylation of endogenous RGS9-1 is light-dependent
(18), both phosphorylation and dephosphorylation should be regulated
processes. To identify the phosphatase responsible for dephosphorylation, we tested PP1, PP2A, and PP2B (Fig.
7). Using the Ser475
phosphorylation-specific antibody A4, we found that PP2A was capable of
removing the phosphate from Ser475 (Fig. 7, lane
5), whereas neither PP1 nor PP2B could catalyze the
dephosphorylation reaction efficiently. Importantly, PP2A was
previously isolated from ROS (39-43). These findings suggest that PP2A
is possibly the RGS9-1 phosphatase that can specifically dephosphorylate RGS9-1 at Ser475.
Functional Consequences of RGS9-1 Phosphorylation--
RGS9-1 is
the GTPase-accelerating protein for Gt Phosphorylation Occurs in Rods--
RGS9-1 is highly expressed in
cone but is also present in rod photoreceptors cells (Fig.
1A) (16). To obtain additional evidence that the observed
phosphorylation of RGS9-1 (18) occurs in rods, which are a readily
accessible source for biochemical isolation, we measured RGS9-1
phosphorylation in transgenic mice lacking cones. We demonstrated that
phosphorylation of RGS9-1 takes place in dark-adapted rods and then
dephosphorylation occurs when mice are exposed to light. There were no
significant differences between Coneless mice and wild type
mice (Fig. 1B), strengthening the argument that RGS9-1
phosphorylation is taking place in rods.
Multiple Forms of PKC Involved in the RGS9-1
Phosphorylation--
By purification of RGS9-1 kinase(s) from ROS
extracts, we identified that multiple forms of PKC are involved in
RGS9-1 phosphorylation at Ser475. PKCs constitute a protein
family that displays lipid- and cofactor-stimulated kinase activity
(44, 45). Three groups of PKC isozymes were identified that display
unique requirements for Ca2+. The conventional PKCs encoded
by
PKC
Among all of the kinase candidates we tested, we also found that PKG
robustly phosphorylated RGS9-1 at Ser475 (Fig. 6), but PKA
and Ca2+/CaM-dependent kinase II did
not. However, we, as well as other groups, were not able to find any
evidence of the presence of PKGI or PKGII isoforms in either purified
ROS or the outer segment of mouse photoreceptors.
Localization of PKC Isozymes--
Although immunocytochemical
methods were generally not successful in localizing PKC
Based on biochemical studies, PKC was suggested to be involved in
several processes of phototransduction, including rhodopsin phosphorylation (33, 59). Phosphorylation of rhodopsin is involved in
lowering the catalytic efficiency of photoactivated rhodopsin in the
activation of Gt (60, 61). These authors suggest that most of the PKC
in photoreceptor outer segments is of the conventional type and that
most, if not all, of this conventional PKC activity comes from a novel
isozyme(s) (34). Subsequent studies (62, 63) have not supported a
physiologically significant role for PKC in rhodopsin phosphorylation.
Further studies to resolve the role of PKCs in phototransduction,
including phosphorylation of RGS9-1 will require a combination of
biochemical experiments in conjunction with in vivo studies
that take advantage of mouse genetics.
Phosphatases and Dephosphorylation of RGS9-1--
Of the three
most common enzymes, protein phosphatase 2A, dephosphorylates
phosphorylated RGS9-1 at Ser475. Protein phosphatase 2A has
been identified as one of the many enzymes dephosphorylating
phosphorylated rhodopsin in ROS (39-43). This enzyme is not regulated
by Ca2+ or phospholipids. Therefore, the regulation of
RGS9-1 phosphorylation is on the level of protein kinases.
Unique RGS9-1 C-terminal Sequence and Its Kinase--
RGS9 belongs
to the R7 RGS subfamily and is expressed in retina and brain as the
spliced forms RGS9-1 and RGS9-2, respectively (7). The retina-specific
human RGS9-1 contains 484 amino acid residues, whereas the larger form
in the brain contains ~675 residues (14). Except for a unique
C-terminal portion (difference begins at residue 467 in RGS9-2), both
variants are identical. The 18-amino acid long tail characteristic for
RGS9-1 was not found even in very closely related RGS sequences. The
function of this unique sequence is still unknown. However, some data
suggested its importance in the interactions of RGS9-1 with targets
(10, 17, 65). As highlighted in the sequence alignment (Fig.
2B), the C-terminal part of RGS9-1 contains
Ser475 that is phosphorylated by PKC.
Functional Consequences of RGS9-1 Phosphorylation--
PKC
phosphorylation of RGS9-1 in vitro resulted in dramatic
reduction in its affinity for its membrane anchor, R9AP (Fig. 8), which
would be expected in vivo to reduce its catalytic activity toward Gt and PKC
. A synthetic peptide
corresponding to the Ser475 RGS9-1 sequence and RGS9-1 were
substrates for recombinant PKC
and PKC
. This phosphorylation was
removed efficiently by protein phosphatase 2A, an endogenous
phosphatase in rod outer segments, but not by PP1 or PP2B.
Phosphorylation of RGS9-1 by PKC had little effect on its activity in
solution but significantly decreased its affinity for its membrane
anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKC
immunostaining was at higher levels in cone outer segments than in rod
outer segments, as was found for the components of the RGS9-1 GAP
complex. Thus, PKC-mediated phosphorylation of RGS9-1 represents a
potential mechanism for feedback control of the kinetics of
photoresponse recovery in both rods and cones, with this mechanism
probably especially important in cones.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit reassociates with its partner
subunits. The rate of
hydrolysis is regulated by the protein's association with a
GTPase-accelerating protein (GAP). This GAP has been identified to be
RGS9-1 (1), a member of a family of regulatory proteins for G
GAPs
termed the regulators of G-protein signaling (RGS) family (reviewed in
Refs. 2 and 3). The GAP activity of RGS9-1 is further enhanced by the
-subunit of the phototransduction effector cGMP phosphodiesterase
(PDE
) (1, 4). The acceleration of GTPase activity is essential for
timely recovery of dark conditions of photoreceptor cells (5) (for
recent reviews, see Refs. 6 and 7).
-subunit-like domain (8) that is
employed for functional association with the G
5L subunit (9-12).
This subunit regulates GAP activity by the effector subunit, PDE
,
and induces and stabilizes RGS9-1 (5, 10, 13). ROS membranes from
knockout mice lacking RGS9 hydrolyze GTP more slowly than ROS membranes
from control mice. Moreover, in electrophysiological measurements, the
flash responses of RGS9
/
rods recovered much more slowly
than normal after illumination (5). Alternative splicing of the RGS9
gene yields two molecular forms of RGS9, RGS9-1 and RGS9-2, that vary
only in the amino acid sequence of the C terminus. RGS9-1 is a
retina-specific transcript, whereas RGS9-2 is expressed in the brain
(1, 14, 15). Immunolocalization using monoclonal antibodies and
confocal microscopy revealed that RGS9-1 is present in cones at
significantly higher levels than in rods, implying that the RGS9-1
level is a potentially important determinant of the faster response
kinetics and lower sensitivity of mammalian cones compared with rods
(16). RGS9-1 also forms a complex with a 25-kDa photoreceptor-specific
phosphoprotein, RGS9-1 anchor protein (R9AP). R9AP binds to the
N-terminal domain of RGS9-1 and anchors RGS9-1 to the disk membrane
(17).
and PKC
, co-purify with RGS9-1 kinase activity, that
both phosphorylate RGS9-1 at Ser475, and that protein
phosphatase 2A is responsible for the RGS9-1 dephosphorylation at this site.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
from Sigma; PKGI, anti-PKGI
antibody, and Ca2+/CaM II kinase from Calbiochem; PKC
and PKC
from PanVera; PP1 and PP2A phosphatases from Upstate
Biotechnology, Inc. (Lake Placid, NY); and PP2B and CaM from Biomol.
Fluorescein-labeled peanut agglutinin (PNA) was from Vector Laboratory Inc.
5 were expressed
using baculovirus and Sf9 cells, as described previously (10,
18), and affinity-purified using either immobilized metal or
glutathione affinity chromatography. The RGS9-1 membrane anchor
protein, R9AP, with an N-terminal His6 tag, was expressed
in Escherichia coli and purified, as described
elsewhere.2
-33P]ATP to a final concentration of 0.1 mM with a specific activity of 100,000 dpm/nmol and a total
volume of 75 µl. Samples were incubated for 10 min or a longer time
as indicated, at 30 °C. The reaction was stopped by the addition of
20 µl of 100 mM H3PO4. 33P incorporation into the RGS peptide was determined by a
phosphocellulose filter paper method (21).
5-subunit complex as a substrate, and phosphorylation was detected using the A4
antibody specific for Ser475 phosphorylation.
5L
was affinity-purified from insect Sf9 cells as described (10).
In vitro phosphorylation assays were performed in kinase
buffer (20 mM MOPS, pH 7.2, 1 mM NaF, 1 mM MgCl2, 1 mM
Na3VO4, 1 mM dithiothreitol, 500 µM ATP) containing either 1 mM
CaCl2 for PKC
or 1 mM EDTA for PKC
. PS
(0.1 mg/ml) and DAG (0.01 mg/ml, Upstate Biotechnology) were added
after sonication on ice for 2 min to resuspend the lipid micelles.
Either PKC
(60 ng) or PKC
(60 milliunits) was used in the assay,
unless otherwise indicated. For autoradiography,
[
-32P]ATP was added with a working concentration of
200 Ci/mol. The reactions were performed at 30 °C for 60 min or as
indicated, and then either diluted directly into GAP assay reactions
for GAP activity measurement or quenched by SDS sample buffer for SDS-PAGE. Incorporation of 32P into RGS9-1 after kinase
assays was determined by autoradiography after separating the proteins
by SDS-PAGE, and the phosphorylation on Ser475 was
determined by immunoblotting using Ser475
phosphorylation-specific A4 antibody. Reactions without PKC kinases were used as negative controls.
, and [
-32P]GTP to measure GAP activity. Final
concentrations of the proteins in the assays were as follows: 0.5 µM RGS9-1/G
5, 15 µM rhodopsin, 1 µM Gt, and 2 µM PDE
. The first
order rate constant for GTP hydrolysis was calculated by fitting
data with PSI-Plot.
-phosphatidylserine (PS; brain, porcine-sodium salt),
L-
-phosphatidylcholine (egg, chicken), L-
-phosphatidylethanolamine (egg, chicken) (Avanti Polar
Lipids) at a ratio (w/w/w) of
L-
-phosphatidylcholine/L-
-phosphatidylethanolamine/PS = 50:35:15 as described. Rhodamine-labeled
L-
-phosphatidylethanolamine (N-(6-
tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; Molecular Probes, Inc., Eugene, OR) was added in the lipid mixture to a final concentration of 0.5% (w/w) to
facilitate the visualization of the lipids. Purified glutathione S-transferase-RGS9-1/G
5 proteins (in 10 mM
HEPES, pH 7.4, containing 100 mM NaCl and 2 mM
MgCl2) were first incubated with buffer only or
phosphorylated by commercial PKC
or PKC
at room temperature for
60 min and then centrifuged at 4 °C at 88,000 × g
for 40 min to remove aggregates. Supernatants were diluted to desired
concentrations in 10 mM HEPES, pH 7.4, containing 100 mM NaCl, 2 mM MgCl2 and 0.2 mg/ml
ovalbumin, mixed with R9AP vesicles in a volume of 150 µl, and
incubated with gentle vortexing at 4 °C for ~2 h. An aliquot (100 µl) of the reaction mixture was then transferred to a new polypropylene (Beckman) tube to separate unbound proteins from bound
proteins by pelleting the vesicles at 88,000 × g for
40 min. The pelleted vesicles were clearly visible as a pink pellet at
the bottom of the tube due to the rhodamine-labeled
phosphatidylethanolamine. The final concentrations in the binding
reactions were as follows: RGS9-1, 0.1 µM; R9AP (total
concentration), 1.0 µM. Equal proportions of the starting
binding reactions, the supernatant after pelleting the vesicles, and
the pelleted vesicles were loaded on SDS-PAGE, and the RGS9 proteins
were detected by immunoblotting using anti-RGS9-1c antiserum.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Localization of RGS9-1 in rod and cone
photoreceptors and its phosphorylation in vivo.
A, flat mount immunolocalization of RGS9-1 in bovine retina
using the D7 monoclonal antibody. Lower (a) and higher
(b) magnifications, respectively, and a cross-section of the
bovine retina (c) are shown. Cones (more prominently) and
rods were immunolabeled. B, phosphorylation of RGS9-1 in
dark- and light-adapted wild type and Coneless mouse
retinas. RGS9-1 was immunoprecipitated from dark-adapted
(Dark) or light-adapted (Light) mouse retinas and
immunoblotted with Ser475 phosphate-specific antibody (A4
antibody). RGS9-1 phosphorylation under different conditions was
compared as described under "Materials and Methods."
5, followed by separation of
the phosphorylated products on SDS gel and identification and quantification of the phosphorylation by monoclonal
anti-Ser475-phosphate-specific antibody (A4) (18). The
advantage of this method is its ability to recognize the specific
phosphorylation on the in vivo phosphorylation site
Ser475, regardless of other nonspecific
phosphorylations that could result from high kinase concentrations
or missing regulatory components in in vitro assays. The
second method involves phosphorylation of a synthetic peptide that
encompasses the RGS9-1 phosphorylation site identified in
vivo, KLDRRS475QLKKGLPPK (18), using
[
-33P]ATP as a phosphoryl donor. The phosphorylation
product is trapped on phosphocellulose filter paper and then quantified
by scintillation counting. Advantages of this filter paper assay over
the immunoassay include the ability to quantitatively measure kinase
activity in a large number of samples. In our study, we measured RGS9-1 kinase activity in ROS extracts using both of the two methods.
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Fig. 2.
Schematic representation of RGS9 and
extraction of RGS9-1 kinase and the DEAE-Sepharose chromatography of
the NH4Cl extract from bovine ROS. A,
flowchart of RGS9-1 kinase purification from ROS. Hydroxyapatite
chromatography is not included, since it did not separate RGS9-1 kinase
activities. B, the scheme represents DEP (protein
interaction), G-protein -subunit-like (GGL), and RGS
domains of RGS9. The C-terminal region of human RGS9-1 and human RGS9-2
are aligned, and the unique phosphorylation sequence in RGS9-1 is
highlighted. The sequence of human RGS9-1 was previously published
(14), and the sequence of human RGS9-2 was obtained from
GenBankTM (accession number AF071475). C,
extraction of the RGS9-1 kinase activity from bovine ROS with
H2O, 0.1 M NaCl, or 1 M
NH4Cl. The RGS9-1 kinase activity was assayed using a
synthetic peptide that encompassed the identified Ser475
phosphorylation site (KLDRRS475QLKKGLPPK) by
the phosphocellulose method (graph), or by immunoblot
(inset) using Ser475 phosphate-specific antibody
(A4 antibody) as described under "Materials and Methods." The
arrow indicates phosphorylation of recombinant RGS9-1,
whereas the asterisk represents endogenous phosphorylated
RGS9-1. D, DEAE-Sepharose chromatography. The activity
profile of RGS9-1 kinase from the DEAE-Sepharose column was measured
using the RGS9-1 peptide assay. Inset, the RGS9-1 kinase
activity was probed with A4 antibody using recombinant RGS9-1.
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Fig. 3.
Heparin-Sepharose chromatography.
Fractions eluted from the DEAE-Sepharose column were loaded onto the
heparin-Sepharose column, and the RGS9-1 kinase activity was measured
as described under "Materials and Methods." A,
phosphorylation of RGS9-1 peptide in the presence and absence of PS.
Inset, the RGS kinase activity, eluted in three peaks, as a
percentage of total activity with or without PS. B, enzyme
activity was assayed in the absence (lane 1) and presence of
cGMP (lane 2); cAMP (lane 3); DAG, PS, and
Ca2+ (lane 4); PS, PMA, and Ca2+
(lane 5); CaM and Ca2+ (lane 6);
Ca2+ (lane 7); EGTA (lane 8) or
peptide (lane 9) as described under "Materials and
Methods." Lane 9, the reaction without the
addition of any fraction. The RGS9-1 kinase activity was probed with A4
antibody using recombinant RGS9-1
5 was
used as substrate, the activity of all three kinase peaks could be enhanced similarly by combination of PS, PMA, Ca2+ or PS,
DAG, and Ca2+ (Fig. 3B). Previously, we
suggested that the RGS9-1 kinase(s) may belong to the PKC family based
on our finding that RGS9-1 phosphorylation can be inhibited by a PKC
inhibitor, bisindolylmaleimide I (18). It is known that the
conventional PKCs (
,
, and
) exhibit
Ca2+/PS/DAG-stimulated kinase activity, whereas novel PKCs
(
,
,
, and
) exhibit only PS/DAG-stimulated activity, and
atypical PKCs (
and
/
) are Ca2+- and
DAG-insensitive (29-31). The above data suggested that the kinases
involved in RGS9-1 phosphorylation at Ser475 could be
members of the Ca2+/phospholipid-dependent PKC subfamily.
, PKC
, and PKC
are expressed in the
ROS (Fig. 4B). In addition,
PKC
and PKC
were detected in heparin-Sepharose isolated fraction
24 and 17, respectively (Fig. 4C). PKC
was previously
implicated in phototransduction processes (32, 33). However, similar to
results reported by Williams at al. (34), we were not able
to localize PKC
to photoreceptor cells by immunocytochemical methods
(data not shown). Immunostaining of the retina with a polyclonal
antibody against PKC
indicated immunoreactivity in the cones, which
were identified with peanut agglutinin staining (Fig. 4A).
Preincubation with immunoreactive peptide prior to staining abolished
the PKC
signal. The immunoblotting of ROS proteins also revealed the
presence of PKC
(Fig. 4B). However, this antigen was not detected in any fraction separated on the
heparin-Sepharose column. This result was supported by the finding that
PKC
could not be extracted with high efficiency from the ROS under
our conditions even by 1 M NH4Cl (data not shown). Along with previously published data (35-37), this result indicated that ROS PKC
may be anchored to cytoskeleton proteins.
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Fig. 4.
Immunofluorescence localization and
immunoblotting analysis of PKC isozymes in the retina.
A, immunofluorescence localization of the PKC
isozyme in mouse retina. Cones, but not rods, were strongly
immunolabeled with anti-PKC
-specific antibody (a,
red), and anti-peanut agglutinin antibody labeled
cone extracellular matrix (b, green). Both
immunolabelings coincide as shown by double labeling (c,
yellow). PKC
immunolabeling is abolished by preadsorbing
antibody on the PKC
peptide antigen (d). Scale
bars, 20 µm. The retinal layers are as follows:
OS, outer segment layer; IS, inner segment layer;
ONL, outer nuclear layer; OPL, outer plexiform
layer; INL, inner nuclear layer; IPL, inner
plexiform layer; GCL, ganglion cell layer. B,
identification of PKC isozymes in the bovine ROS preparations. The
specificity of each antibody was tested using recombinant PKC isozymes
(rPKC). C, immunoblot of fractions 14, 17, and 24 with anti-PKC
and anti-PKC
antibodies.
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Fig. 5.
RGS9-1 phosphorylation by kinases present in
fractions obtained from the heparin-Sepharose column and by recombinant
PKC isozymes. A, time course of RGS9-1 phosphorylation
by kinase present in fraction 14 and by recombinant PKC . The RGS9-1
kinase activity was assayed by immunoblot using Ser475
phosphate-specific antibody and recombinant RGS9-1 (upper immunoblot)
or using a synthetic peptide that encompassed the identified
Ser475 phosphorylation site
(KLDRRS475QLKKGLPPK) (graph) as
described under "Materials and Methods." Lower panels,
the enzyme activities were assayed in the absence (lane
1) and presence of DAG and Ca2+ (lane
2); PS and Ca2+ (lane 3);
and DAG, PS, and Ca2+ (lane 4) by
immunoblot using Ser475 phosphate-specific antibody and
recombinant RGS9-1 (see "Materials and Methods"). B,
time course of RGS9-1 phosphorylation by kinase present in fraction 17 and by recombinant PKC
. C, time course of RGS9-1
phosphorylation by kinase present in fraction 24 and by recombinant
PKC
. Lower bands in the upper
panels represent RGS9-1 immunoblot with D7 monoclonal
antibody as a control of the loading on the gel.
and PKC
in the kinase activity peaks, we then
compared the phosphorylation of RGS9-1 by the partially purified kinase
peak fractions and by their corresponding recombinant PKC isozymes.
Phosphorylation of RGS9-1 peptide by fraction 14 (possibly, in part,
containing PKC
), fraction 17 (containing PKC
), and fraction 24 (containing PKC
) showed similar kinetics to those of PKC
, PKC
,
and PKC
, respectively (Fig. 5). Phosphorylation of full-length
RGS9-1 by both the kinase peaks and the corresponding recombinant PKCs
showed similar enhancement upon PS and Ca2+ stimulation.
Therefore, these data suggest that PKC
, PKC
, and PKC
are
probably the kinases responsible for RGS9-1 phosphorylation.
and PKC
phosphorylate the S475A
mutant of RGS9-1 almost as well as wild type RGS9-1 (data not shown).
Thus, there are at least two potential sites for phosphorylation of
RGS9-1 by PKC isozymes, and selection of these may be determined by the
nature of association with membranes and other ROS proteins.
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Fig. 6.
RGS9-1 phosphorylation by recombinant protein
kinases. A, time course of RGS9-1 phosphorylation by
PKG (30 units/ml), PKA (50 units/ml), and Ca2+/CaM II
kinase (0.8 µg/ml). The RGS9-1 kinase activity was assayed by
immunoblot using Ser475 phosphate-specific antibody (A4) or
a phosphocellulose method (graph) using a synthetic peptide
that encompassed the identified Ser475 phosphorylation site
(KLDRRS475QLKKGLPPK) as described under
"Materials and Methods." B, PKG, PKA, and
Ca2+/CaM II kinase activities were measured by synthetic
control peptides as described under "Materials and Methods."
C, recombinant PKGI, PKGII, and ROS were immunoblotted with
PKGI- and PKGII-specific antibodies. PKGI and PKGII are not present in
bovine ROS.
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Fig. 7.
Dephosphorylation of endogenous
phosphorylated RGS9-1. A, dephosphorylation was carried
out without (lane 1) or with the addition of PP2B
(lane 2); PP2B with CaM and Ca2+
(lane 3); PP1 (lane 4); or
protein phosphatase 2A (lane 5). B,
dephosphorylation time course of phosphorylated RGS9-1 by protein
phosphatase 2A. Upper and lower
immunoblots in both panels show P-RGS9-1
(A4) and RGS9-1 (D7), respectively.
, and its GAP activity can be
regulated by its association with ROS membranes through the interaction
with its membrane anchor R9AP. To study the functional consequences of
RGS9-1 phosphorylation, we therefore tested whether phosphorylation
modulates the function of RGS9-1 by direct regulation of its GAP
activity or by regulation of its R9AP-mediated membrane binding. When
the GAP activities of RGS9-1 before and after phosphorylation by
recombinant PKCs (PKC
or PKC
) were compared in the presence of
the effector subunit PDE
, we found that phosphorylation slightly
inhibited the GAP activity of RGS9-1 (Fig.
8A). These results were
obtained in the absence of the membrane anchor for RGS9-1, R9AP. A more
significant effect of PKC phosphorylation was found on binding to R9AP.
We found that phosphorylation caused RGS9-1 to bind the R9AP vesicles
much less tightly (Fig. 8B). Since membrane tethering of
RGS9-1 by R9AP dramatically enhances the GAP activity
RGS9-1,2 this result indicated that phosphorylation of
RGS9-1 may decrease its catalytic activity toward Gt
through
dissociating the membrane complex between RGS9-1 and R9AP. Therefore,
by modulating RGS9-1 GAP activity both directly and indirectly,
phosphorylation of RGS9-1 by PKC isoforms could provide a regulatory
mechanism for modifying the inactivation kinetics in
phototransduction.
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Fig. 8.
Functional consequences of RGS9-1
phosphorylation. A, GAP activities of RGS9-1 (RGS9) and
PKC-phosphorylated RGS9 (RGS9 + PKC , RGS9 + PKC
) were measured by
single turnover assays in the presence of PDE
. B, binding
of RGS9-1 (untreated), RGS9-1 incubated with kinase assay buffer and
ATP (buffer), or RGS9-1 phosphorylated by PKC
or PKC
to R9AP
vesicles was measured as described under "Materials and Methods."
Equal proportions of the binding reaction (input), supernatant after
pelleting R9AP vesicles (sup't), and pelleted vesicles
resuspended in SDS-sample buffer were loaded, and RGS9-1 in each
fraction was detected by immunoblotting using polyclonal anti- RGS9-1c
antiserum.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
, and
genes exhibit Ca2+/PS/DAG-stimulated
kinase activity, whereas novel PKC
, -
, -
, and -
exhibit
only PS/DAG-stimulated activity. The atypical PKC subfamily comprises
the
and
/
isotypes, which are Ca2+- and
DAG-insensitive.
, -
, and -
were present in bovine ROS, and at least two of
these kinases were found to be involved in RGS9-1 phosphorylation. They
could be extracted with 1 M NH4Cl (Fig.
2A) from ROS membranes and separated on a heparin-Sepharose
column (Fig. 3). Since PKCs are dependent upon PS for activity (46), it
was important to test the heparin-separated activity profile in the
presence of this cofactor (Fig. 3A). A combination of DAG,
PS, and Ca2+ or of PS, PMA, and Ca2+
significantly elevated activity in fraction 24, as judged by rRGS9-1
(Fig. 3B) and RGS9-1 peptide phosphorylation (data not shown). Two other fractions did not show significant changes in RGS9-1
peptide phosphorylation, but a higher level of phosphorylation was
detected when full-length rRGS9-1 was used. These data, combined with
the fact that recombinant PKC
, -
, and -
effectively catalyze phosphorylation of RGS9-1 on Ser475 (Fig. 5), support the
idea that these PKC isoforms are the enzymes involved in RGS9-1 phosphorylation.
in ROS of
several species (34, 47-55), PKC
has been detected and purified
from ROS (34, 56-58). For PKC
, we found that it specifically
localizes to COS (Fig. 4). Therefore, it is possible that the observed
kinase activity in peak 2 resulted from the contamination of ROS by
COS. Alternatively, it is also possible that immunoreactivity toward
PKC
is masked in ROS and that the enzyme is present in both COS and
ROS or simply that PKC
is present in much higher amounts in cones
than in rods, as is the RGS9-1 complex. Moreover, the levels of peak 1 varied from preparation to preparation, suggesting variable extraction efficiency between experiments or contamination of ROS preparation by
kinases from other retinal cells. Peak 1 could, in part, be due to the
presence of PKC
. It appears that PKC
is tightly bound to
membranes in ROS. Because commercially available antibody, although
effective in immunoblotting, was not useful for immunolocalization, we
were not able to localize this isoform in mouse retina sections. These
findings are also consistent with a report suggesting that PKC
,
-
I, -
II, -
, -
, -
, and -
and another structurally
unknown PKC subspecies are expressed in the retina (54).
. Although the GAP activity of RGS9-1 originates from its
RGS domain (1) and the R9AP-mediated membrane targeting originates from
its N-terminal domain (17), neither of which are adjacent in the linear
amino acid sequence to Ser475, it is known that both the C-
and N-terminal domains are important for regulation of RGS domain GAP
activity by PDE
(10, 65), and both have been implicated along with
R9AP in RGS9-1 membrane association (17, 64, 66). Therefore, our
observation that phosphorylation of Ser475 further affected
the function of RGS9-1 supports the idea that the function of RGS9-1 is
tightly controlled on multiple levels. Furthermore, RGS9-1
phosphorylation was found to be regulated by light in vivo,
with the highest phosphorylation levels detected in dark-adapted
animals and the lowest in light-adapted animals. If phosphorylation
indeed reduces RGS9-1 GAP activity in vivo, then light would
be expected to stimulate the activity of RGS9-1, potentially providing
a novel mechanism contributing to light adaptation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Nathans (Johns Hopkins University) and Dr. J. Chen (Southern California University) for Coneless mice and GCAP1/GCAP2 knockout mice, respectively, Dr. M. Uhler (University of Michigan) for the PKGII kinase construct and anti-PKGII antibody, Jing Huang for flat mount immunocytochemistry, and Dr. Volker Gerke for comments on the manuscript. We are grateful to Yunie Kim and Matthew Batten for help during the manuscript preparation.
![]() |
FOOTNOTES |
---|
* This research was supported by National Institutes of Health Grants EY13385 and EY11900, Training Grant EY07001, and Core Grant P30EY0173; the Welch Foundation; a grant from Research to Prevent Blindness, Inc. (RPB) (to the Department of Ophthalmology at the University of Washington); 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.
** An RPB Senior Investigator and a recipient of the Humboldt Research Award for Senior United States Scientists. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Washington, Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail: palczews@u.washington.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M211782200
2 Hu, G., Zhang, Z., and Wensel, T. G. (January 30, 2003) J. Biol. Chem. 10.1074/jbc.m212046200.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Gt, G-protein transducin; BTP, 1,3-Bis[tris(hydroxymethyl)methylamino]propane; CaM, calmodulin; DAG, diacylglycerol; GAP, GTPase-accelerating protein(s); MOPS, 4-morpholinepropanesulfonic acid; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; ROS, rod outer segment(s); RGS, regulator(s) of G-protein signaling; R9AP, RGS9-1 anchor protein; PP1, PP2A, and PP2B, protein phosphatase 1, 2A, and 2B, respectively.
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