(Received for publication, November 6, 1996)
From the Departments of Pharmacology and § Neurosciences, University of California at San Diego School of Medicine, La Jolla, California 92093-0983
Similar to other G protein-coupled receptors, the visual receptor, rhodopsin, is phosphorylated by both a substrate-regulated kinase, rhodopsin kinase, and a second messenger-regulated kinase, protein kinase C. In the present study, the extent of involvement of protein kinase C in the light-dependent phosphorylation of rhodopsin in intact retinas was assessed using a specific activator (phorbol ester) and specific inhibitor (calphostin C) of protein kinase C. Kinetic analysis of rhodopsin phosphorylation following different illumination conditions revealed that hyperactivation of protein kinase C with phorbol ester resulted in a relative increase in rhodopsin phosphorylation that peaked 10-15 min after the onset of illumination. Following this period, the rate of rhodopsin dephosphorylation was increased in the phorbol ester-treated retinas, so that by about 30 min the amount of phosphorylation was similar to that in control retinas. Treatment of retinas with calphostin C, a potent regulatory domain-directed inhibitor of protein kinase C, resulted in an approximately 50% reduction in the light-dependent phosphorylation of rhodopsin. This inhibitor had no effect on the activity of rhodopsin kinase in vitro. Last, we show that frog rhodopsin is phosphorylated in vitro by protein kinase C from frog rod outer segments, indicating that this kinase could directly modulate rhodopsin in vivo. In conclusion, the present results reveal that the kinetics of rhodopsin phosphorylation/dephosphorylation differ markedly, depending on whether protein kinase C or rhodopsin kinase activity dominates, and that, under the conditions studied, protein kinase C contributes to approximately half of the phosphorylation of rhodopsin in intact frog retinas.
The involvement of multiple kinases in the regulation of specific signaling pathways is a common cellular theme. For G protein-coupled pathways, two classes of kinases phosphorylate receptors: substrate-regulated G protein-coupled receptor kinases and second messenger-regulated kinases such as protein kinase A and protein kinase C (1). In the examples studied to date, phosphorylation by G protein-coupled receptor kinases is typically involved in homologous desensitization (the phosphorylation follows direct stimulation of the receptor), and phosphorylation by second messenger-regulated kinases is involved in heterologous desensitization (the phosphorylation is independent of the liganded state of the receptor and may be initiated by activation of the kinase by a separate receptor) (1, 2).
Phototransduction serves as the archetypal transduction pathway, and extensive studies with photoreceptor proteins have provided invaluable insight into the regulation of not only this pathway but numerous other G protein-coupled receptor signaling pathways as well (3-5). Activation of the heptahelical receptor, rhodopsin, by a photon of light results in activation of the G protein transducin, which in turns activates a cGMP phosphodiesterase. The ensuing drop in cGMP levels in photoreceptor outer segments causes hyperpolarization of the plasma membrane by closing cGMP-gated channels, thus converting a biochemical signal into an electrophysiological response (6). Phosphorylation of the light-activated receptor by rhodopsin kinase, followed by binding of arrestin, effectively quenches the signal. Ca2+ levels, which are lowered when the cGMP-gated channels close, regulate deactivation and recovery stages of phototransduction. Two targets of Ca2+ have been identified in photoreceptors: guanylate cyclases, which are responsible for restoring cGMP levels, are activated at low Ca2+ concentrations by calmodulin-like proteins (7), and the activity of rhodopsin kinase is inhibited by another Ca2+-binding protein, recoverin, at high concentrations of Ca2+ (8, 9).
Several reports over the past decade have implicated a role for protein
kinase C in photoreceptor function; its presence in rod outer segments
(10-12) and its phosphorylation of rhodopsin (13, 14), transducin
(15), the subunit of the cGMP-phosphodiesterase (16), and arrestin
(17) have been described. Protein kinase C is activated by the lipid
second messenger diacylglycerol through binding of this ligand to a
membrane-targeting domain (C1) that also binds phorbol esters (18, 19).
Some isozymes of protein kinase C are sensitive to Ca2+,
which binds to a separate membrane-targeting domain (C2) and thus also
increases the enzyme's affinity for membranes (18). Generation of
diacylglycerol, the key activator of protein kinase C, has been
reported to be stimulated by light in photoreceptors (20-23),
consistent with a potential role for this kinase in
phototransduction.
Evidence that the phosphorylation of rhodopsin by protein kinase C is physiologically relevant comes from the finding that hyperactivation of protein kinase C in intact retinas, by treatment with phorbol esters, alters the light-dependent phosphorylation of rhodopsin (24, 25). In vitro studies reveal remarkable similarities between the enzymology of phosphorylation of rhodopsin by protein kinase C and the phosphorylation of other G protein-coupled receptors by second messenger kinases (26). Most notably, phosphorylation is independent of the activation state of the receptor (13, 14) and results in decreased coupling of the receptor to G protein (13). Taken together, the in situ and in vitro data are consistent with direct modulation of rhodopsin by protein kinase C. Nonetheless, the extent of involvement of protein kinase C in rhodopsin phosphorylation in vivo remains to be established.
In the present study, specific inhibition of protein kinase C by calphostin C was used to determine the contribution of protein kinase C in the light-dependent phosphorylation of rhodopsin in intact frog retinas. We found that this kinase contributes to approximately 50% of the phosphorylation of rhodopsin. Furthermore, using phorbol esters to hyperactivate the kinase, we show that the kinetics of rhodopsin phosphorylation and dephosphorylation differ markedly, depending on whether protein kinase C or rhodopsin kinase activities dominate.
Phorbol 12-myristate 13-acetate
(PMA)1 and 4--phorbol 12-myristate
13-acetate (4-
-PMA) were purchased from Research Biochemicals International. sn-1-Palmitoyl-2-oleoylphosphatidylserine and
sn-1,2-dioleoylglycerol were from Avanti Polar Lipids, Inc.
Phenol red (free acid) was obtained from Mallinckrodt Inc., casamino
acids from Difco, and calphostin C from Calbiochem.
-D-Glucose, HEPES, dithiothreitol (DTT), EGTA,
hydroxylamine, and ATP were from Sigma.
[32P]Orthophosphoric acid (8,500-9,120
Ci·mmol
1) and [
-32P]ATP (3000 Ci·mmol
1) were supplied by DuPont NEN. A protein kinase
C-selective peptide, Ac-FKK
FKL-NH2 (27), was
synthesized by the Indiana University Biochemistry Biotechnology
Facility, Indiana University Medical School. Q-Sepharose Fast Flow,
phenyl-Superose HR 5/5, and DEAE Sephacel were purchased from Pharmacia
Biotech Inc. All other chemicals were reagent grade. Rat protein kinase
C
from a baculovirus expression system was purified as described
(28) and stored at
20 °C in 10 mM Tris-HCl (pH 7.5 at
4 °C), 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 50-100 mM KCl, 10 µg·ml
1 leupeptin, and 50% glycerol. Rhodopsin kinase
(bovine) was cloned into baculovirus, and the cytosolic fraction of
infected Sf-21 cells was used as the source of
enzyme.2 This fraction was stored in 20 mM HEPES, pH 7.5, 1 mM EDTA, 5 mM
EGTA, 1 mM DTT, 0.25% Triton X-100, 10 µg·ml
1 leupeptin, and 50% glycerol at
20 °C.
Northern grass frogs (Rana pipiens), weighing 20-30 g, were
purchased from Carolina Biological Supply Company.
Freshly dissected retinas
from dark-adapted (12-14 h) frogs were incubated individually in 1 ml
of amphibian culture medium (ACM; 65 mM NaCl, 35 mM NaHCO3, 3 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM Na-HEPES (pH 7.3), 10 µg·ml1 phenol
red, 1 mg·ml
1 casamino acids, 10 mM
-D-glucose, 0.1% bovine serum albumin) containing
20-100 µCi·ml
1 carrier-free
[32P]orthophosphate and gassed with 95% O2,
5% CO2 at room temperature (21-23 °C). After 30-120
min, 100 µl of ACM containing an 11-fold concentrate of PMA or
calphostin C was added to gently agitated tubes to yield final
concentrations of 1-1000 nM PMA and 1 µM calphostin C. An appropriate concentration (0.1%) of Me2SO
(solvent for PMA and calphostin C) was added to control tubes. One
retina from each frog served as the experimental and the other as the control. After a 15-120-min incubation with Me2SO or
pharmacophore, retinas were exposed to one of three light regimes by
flash or continuous illumination. A Vivitar flash unit (model 285) was used to provide a single flash of one of two intensities. For the lower
intensity, the flash unit was set at 1/16 maximal intensity and
filtered by 20 layers of lens tissue, which act as a neutral density
filter, with measured attenuation of 1.2 log units. The higher
intensity was effected by an unfiltered flash from the unit at
1/2 maximal intensity. For a third condition of light exposure, retinas were illuminated by 500-lux cool fluorescent lighting for 1-30
min. Some samples were not exposed to light. After the flashes, retinas
were incubated under dim red light for 1-60 min. Retinas were gently
agitated (approximately 40 rpm) during incubations. All procedures were
carried out under dim red light (15-W bulb, No. 1A Safelight Filter,
Eastman Kodak Co.) unless otherwise stated.
Rhodopsin phosphorylation from retinas was analyzed by one of two
methods. In one, 300 µl of ice-cold buffer containing 80 mM sodium phosphate (pH 7.3), 20 mM EDTA, 20 mM EGTA was added to each sample, retinas were vortexed for
10 s to break off rod outer segments, and tubes were placed on ice
for 2 min to allow retinas to settle. Aliquots (1 ml) of the rod outer
segment-enriched supernatant were centrifuged at 16,000 × g for 30 s. The sedimented rod outer segments were
quickly resuspended in 1 ml of ice-cold buffer containing 110 mM NaCl, 5 mM Na-HEPES (pH 7.3), 1 mM EDTA, 1 mM EGTA and centrifuged again, and
the pellet was resuspended in 50 µl of 1% SDS. To reduce viscosity,
samples were frozen at 80 °C, thawed, and centrifuged at
16,000 × g for 15 min. Aliquots of the supernatant
(10-20 µl) were mixed with a 5-fold concentrate of SDS-PAGE sample
buffer (250 mM Tris-HCl, pH 6.8, 5% SDS, 50 mM
-mercaptoethanol, 0.01% bromphenol blue, 50% glycerol). Samples were analyzed by SDS-PAGE (10% acrylamide) and stained with Coomassie Blue R-250; protein in each lane was quantified by densitometric analysis on a Molecular Dynamics computing densitometer, and
32P incorporation into rhodopsin was quantified by
PhosphorImager analysis (Molecular Dynamics) or scintillation counting
of excised rhodopsin bands. This method yielded good quality rod outer
segments with clean separation of rhodopsin from other proteins, but
there was concern that phosphorylation reactions were not quenched
instantly. In the other method, 100% trichloroacetic acid (125 µl)
was added to retinas in ACM. The tubes were vortexed for 3 s and
then placed on ice for 5 min. Samples were then centrifuged for 15 s at 20,000 × g. The retinal pellets were resuspended
by sonication (for 10 s with a Branson Probe Sonifer-250, power
setting on 2) in 500 µl of 50 mM Tris-HCl buffer (pH 7.5)
containing 10 mM EDTA, 10 mM EGTA, and 20 mM sodium phosphate and then centrifuged again for 20 min
at 23,000 × g, 2 °C. Pellets were resuspended in
1% SDS (200 µl) and sonicated again, and after a second
centrifugation (20 min, 23,000 × g, 22 °C), two
aliquots (40 µl) of the supernatant were mixed with 10 µl of a
5-fold concentrate of SDS-PAGE sample buffer. One sample was kept at
room temperature while the other was heated for 15 min at 100 °C to
oligomerize rhodopsin molecules (this treatment resulted in
quantitative disappearance of monomeric rhodopsin on SDS-PAGE and
served as the background for calculation of 32P
incorporation into rhodopsin). Both methods yielded qualitatively similar results.
Frog retinas in ACM (without
[32P]orthophosphate) were subjected to various flash
intensities or continuous light under the same conditions used in the
in situ experiments above. After illumination, rod outer
segment-enriched supernatants were prepared as in the first method
above, except that 20 mM hydroxylamine was included in the
vortexing mixture. Rod outer segments were isolated by sucrose density
gradient centrifugation (29) and washed twice by centrifugation at
300,000 × g, 10 min, 2 °C, in buffer containing 10 mM sodium phosphate, pH 7.3, 110 mM NaCl. Final
pellets were solubilized in 80 µl of 50 mM sodium
phosphate buffer, pH 7.0, containing 1% octylglucoside and incubated
for 15 min on ice, and insoluble material was removed by centrifugation
at 100,000 × g for 5 min, 2 °C. The
A278/A498 absorbance
ratios of supernatants were measured before and after complete
bleaching (5,000 lux, 2 min) of rhodopsin in the presence of 20 mM hydroxylamine. Fig. 1 shows the amount of bleached
rhodopsin in intact frog retinas calculated by this method as a
function of flash intensity. Arrows indicate the two
intensities used in the in situ phosphorylation experiments;
setting the flash at 1/16 maximal power and using a 1.2-log unit
neutral density filter bleached 6 ± 3% of the rhodopsin (arrow a), and an unfiltered flash set at 1/2 maximal
intensity bleached 80 ± 6% of the rhodopsin (arrow
b). Ten minutes of 500-lux continuous illumination bleached
97 ± 1% of the rhodopsin (not shown). Octylglucoside-solubilized
rhodopsin from purified dark-adapted frog rod outer segments was
illuminated under the same light regimes as intact retinas, and the
amount of bleaching, as a function of light intensity, was determined
as described above. Similar levels of bleaching resulted from
illumination of intact retinas or solubilized rhodopsin from purified
rod outer segments.
Protein Kinase C Assay
Activity of protein kinase C (0.6-3
nM) was measured in the presence of histone IIIS (200 µg·ml1) or protein kinase C-selective peptide
(Ac-FKK
FKL-NH2, 50 µg·ml
1)
in 20 µl of 10 mM Tris-HCl buffer (pH 7.5 at 30 °C)
containing 50-200 µM [
-32P]ATP (0.1 Ci·mmol
1), 5 mM MgCl2, 0.1 mg·ml
1 bovine serum albumin, 1 mM DTT,
lipid/detergent mixed micelles with 0.1% Triton X-100, 12.5 mol % phosphatidylserine (180 µM), 1.6 mol % diacylglycerol
(24 µM), and either 100 µM
CaCl2 or 1 mM EGTA for 5-8 min at 30 °C.
Reactions were quenched and analyzed as described (28).
Urea-stripped rod
outer segment membranes were prepared from 30 frog retinas, as
described (30), and stored in 20 mM Tris-HCl, pH 7.5, at
4 °C, 120 mM NaCl, 30 mM KCl, 2 mM MgCl2, 10% glycerol at 20 °C. The
concentration of rhodopsin was 30-50 µM as assessed by
its absorbance at 498 nm in 1% octylglucoside using an extinction coefficient of 40,600 M
1·cm
1
(31).
Phosphorylation of rhodopsin by protein kinase C was determined as described above except that substrate was replaced with 0.5 µM rhodopsin in urea-stripped membranes and Triton X-100/lipid mixed micelles were omitted. In some experiments, 20-100 nM PMA was present. For calphostin inhibition studies, 0-1 µM of this inhibitor was included in phosphorylation mixtures that also contained 20 nM PMA to ensure full activation of protein kinase C. Phosphorylation by rhodopsin kinase was performed under similar conditions, except that protein kinase C was replaced with rhodopsin kinase and assays did not include PMA (PMA does not affect rhodopsin phosphorylation by rhodopsin kinase in vitro (14)). Reactions were quenched by the addition of 5 µl of a 5-fold concentrate of SDS-PAGE sample buffer, and samples were analyzed by SDS-PAGE (10% acrylamide). 32P incorporation into rhodopsin was assessed as described above for in situ experiments.
Isolation of Protein Kinase C from Frog Rod Outer SegmentsProtein kinase C from rod outer segments was isolated
following the procedure of Udovichenko et al. (32). Briefly,
rod outer segments from 20 frogs were collected under dim red light,
purified by two sequential sucrose gradients (29), and lysed in 10 mM Tris, pH 7.5, at 4 °C, 1 mM
MgCl2, 2 mM DTT, 1 µg·ml1
leupeptin, 1 µg·ml
1 aprotinin, 1 µg·ml
1 pepstatin, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.1 mg·ml
1 sodium
L-ascorbate. Membranes were washed three times by
centrifugation at 300,000 × g for 10 min in this
buffer. Membranes were resuspended in buffer containing 10 mM Tris-HCl, pH 7.5, at 4 °C, 2 mM EGTA, 2 mM EDTA, 5 mM DTT, 1 µg·ml
1
leupeptin, 1 µg·ml
1 aprotinin, 1 µg·ml
1 pepstatin, 0.5 mM
phenylmethylsulfonyl fluoride (buffer A) to extract protein kinase C,
and sample was centrifuged at 100,000 × g for 1 h
at 4 °C. The supernatant, containing protein kinase C, was applied
to a 50-µl DEAE-Sephacel column equilibrated with buffer A, and the
column was washed with 2 ml of buffer A. Protein kinase C was eluted
with a step gradient (50-µl steps in a 1-ml total volume) of 0-500
mM NaCl in buffer A, in 25 mM NaCl increments. Fractions (50 µl) were collected and assayed for protein kinase C
activity.
Concentrations of free Ca2+ were calculated using a computer program kindly provided by Dr. Claude Klee that takes into account pH, Ca2+, Mg2+, K+, Na+, EGTA, EDTA, and ATP concentrations (33).
Previously we showed that PMA alters the phosphorylation of rhodopsin in intact rat retinas (24, 25). To understand better the effect of protein kinase C activation on rhodopsin phosphorylation, we undertook a systematic study of the time-dependent phosphorylation of rhodopsin after different illumination conditions. For these studies, we chose frog retinas because they have been well characterized in short term culture. They are more stable in culture medium than mammalian retinas, so that they are more suited to the present experiments, which require longer incubation periods than those of our previous studies (24, 25).
As a first step, we characterized the light-dependent
phosphorylation of rhodopsin initiated by three light treatments (Fig. 1): exposure to a filtered flash of light resulting in
photoexcitation of approximately 6% of rhodopsin, exposure to a flash
of light resulting in photoexcitation of approximately 80% of
rhodopsin, and exposure to continuous room lighting (approximately 500 lux) resulting in photoexcitation of approximately 97% of rhodopsin. Fig. 2 shows the time-dependent increase in
rhodopsin phosphorylation after treatment of intact frog retinas with
these light regimes; these data are compiled from 35 separate
experiments. All lighting conditions resulted in a rapid
phosphorylation of rhodopsin, with a half-time of approximately 2 min.
Relative to the phosphorylation resulting from photoexcitation of 97%
of the rhodopsin, approximately 12 and 50% of the rhodopsin was
phosphorylated following the two different flash intensities, which
photoexcited 6 and 80% of the rhodopsin, respectively. Thus,
conditions were established where the kinetics and extent of
phosphorylation of control retinas, in the absence of any stimulation
other than light, were characterized.
A, time course of rhodopsin phosphorylation in intact frog retinas as a function of illumination conditions. Retinas were incubated with [32P]orthophosphate under dim red light for 2 h and then subjected to different light stimuli. a, a flash of light that photoexcites 6% of the rhodopsin, followed by continued incubation under dim red light; b, a flash of light light that photoexcites 80% of the rhodopsin, followed by continued incubation under dim red light; c, exposure to continuous room lighting (approximately 500 lux), which results in photoexcitation of 97% of the rhodopsin after 10 min. At the indicated times after the onset of illumination, 32P incorporation into rhodopsin was determined as described under "Materials and Methods." Data represent the amount of 32P incorporation normalized to a constant amount of rhodopsin and are expressed relative to the maximal level of phosphorylation obtained after 10 min of continuous 500-lux illumination. Data from 15 separate experiments (n = 58), 12 separate experiments (n = 64), and 8 separate experiments (n = 34) were compiled for curves a, b, and c, respectively. B, determination of rhodopsin phosphorylation. The second of two procedures (as described under "Materials and Methods") used to determine the phosphorylation of rhodopsin relied on the property of rhodopsin to oligomerize when heated in SDS-PAGE sample buffer (see "Materials and Methods"). Coomassie Blue-stained SDS-polyacrylamide gel (lanes 1 and 2) and phosphor image showing radioactivity (lanes 3 and 4) of a heated sample (lanes 1 and 3) and a sample kept at room temperature (lanes 2 and 4) are shown. The area of the gel analyzed is indicated by the boxes; the arrow indicates the position of rhodopsin. The heated sample contains little rhodopsin in this area and serves as the background level of protein and radioactivity.
We next studied how hyperactivation of protein kinase C, resulting from
PMA treatment of retinas, altered the kinetics of rhodopsin
phosphorylation. The data in Fig. 3 show the effect of
PMA treatment on rhodopsin phosphorylation relative to that of
untreated retinas (for these experiments, one retina from each frog was
treated with PMA and the other with Me2SO, the solvent for
PMA). For all illumination conditions, the effects of phorbol esters
were biphasic. In general, PMA first caused an increase in rhodopsin
phosphorylation relative to controls; this increase was greatest at
about 10-15 min after the onset of illumination. After this time, the
relative difference in rhodopsin phosphorylation level between
PMA-treated retinas and control retinas decreased, so that by
approximately 30 min after the onset of illumination there was little
difference. What was unique to each illumination condition was the
magnitude of the phorbol ester effect. In response to the flash
photoexciting 6% of the rhodopsin, PMA caused an approximately 20%
increase in rhodopsin phosphorylation that was sustained for 20 min
(Fig. 3, B and E). This increase was followed by
a marked decrease in the relative level of rhodopsin phosphorylation in
PMA-treated retinas, such that by 60 min, it was 50% less relative to
controls. In retinas exposed to the flash of light resulting in 80%
photoexcitation of rhodopsin, PMA treatment resulted in 80% greater
rhodopsin phosphorylation by 15 min, although the initial
phosphorylation level in PMA-treated retinas was actually less (Fig. 3,
C and F). As with the lower flash intensity,
rhodopsin phosphorylation in PMA-treated retinas became progressively
less relative to control with incubation times exceeding 15 min, except that at the higher flash intensity, the amount of phosphorylated rhodopsin in PMA-treated retinas did not drop below that in control retinas. In retinas exposed to continuous illumination, the same biphasic trend was evident, with the greatest increase in rhodopsin phosphorylation due to PMA occurring after 15 min (Fig. 3, D
and G). No effect of PMA was detected in the absence of
illumination (Fig. 3A).
To test whether the PMA-induced effect resulted from direct stimulation
of protein kinase C, we determined the effect of the biologically
inactive enantiomer 4--PMA on rhodopsin phosphorylation in
situ. Although 4-
-PMA caused a slight increase in rhodopsin phosphorylation, PMA caused at least a 2-fold increase above this level, with a half-maximal effect at 2 nM (data not shown).
Thus, the major effect of PMA is stereospecific and does not arise from perturbation of rod outer segment membranes or nonspecific interactions with rod outer segment components.
As an alternative approach to studying the effect of protein kinase C
on rhodopsin phosphorylation, we investigated the effect of a specific
protein kinase C inhibitor, calphostin C, on rhodopsin phosphorylation.
Calphostin C binds the phorbol ester-binding (C1) domain of protein
kinase C (34) and at concentrations up to 1 µM has no
significant effect on other kinases (35). Fig. 4 shows
that calphostin C specifically inhibits the protein kinase C-catalyzed
phosphorylation of rhodopsin in vitro (filled
circles), with no detectable inhibition of the rhodopsin
kinase-catalyzed phosphorylation of rhodopsin (open
circles). Protein kinase C was half-maximally inhibited by 400 nM calphostin C, with 1 µM causing
approximately 70% inhibition. Note that this inhibition occurred in
the presence of 20 nM PMA, which, without calphostin, results in full activation of the kinase; greater inhibition by calphostin would be expected at subsaturating concentrations of activator. In marked contrast, 1 µM calphostin C had no
effect on rhodopsin kinase activity. Thus, calphostin C, like PMA,
serves as a specific agent for examining protein kinase C effects.
Fig. 5 shows that treatment of intact retinas with 1 µM calphostin C resulted in a marked reduction in the
light-dependent phosphorylation of rhodopsin. Compilation
of data from four separate experiments revealed a 2-fold reduction in
32P incorporation into rhodopsin from calphostin-treated
retinas relative to controls (as in the preceding experiments, one
retina from each frog served as the experimental and the other as the control). These data reveal that at least 50% of the 32P
incorporated into rhodopsin in response to continuous room illumination (97% bleach) depends on protein kinase C.
The foregoing data establish that protein kinase C is a major
contributor to the phosphorylation of rhodopsin in vivo. To test whether frog rhodopsin serves as a direct substrate for protein kinase C, urea-stripped membranes from purified frog rod outer segments
were incubated with recombinant protein kinase C and Mg2+-ATP. Fig. 6A shows that
rhodopsin in these membranes was phosphorylated effectively by protein
kinase C in a Ca2+-dependent manner. As
reported for bovine rhodopsin (14), protein kinase C phosphorylated
dark-adapted (stippled columns) and photoexcited (open
columns) rhodopsin equally well. Under the conditions of the
assay, we found that approximately 1 mol of phosphate could be
incorporated per mol of rhodopsin, although, as the curve in Fig.
6B indicates (it has not reached a plateau), this value
underestimates the maximal stoichiometry.
Last, we investigated whether protein kinase C isolated from frog rod
outer segments was also an effective kinase for frog rhodopsin. Protein
kinase C was partially purified from frog rod outer segments by ion
exchange chromatography (Fig. 7A), with Ca2+/lipid-stimulated activity eluting at approximately 200 mM NaCl. Due to the limited amount of material, we used a
microcolumn (50-µl volume) and obtained about 45 milliunits of
partially pure rod outer segment protein kinase C from 30 retinas. This
protein kinase C phosphorylated rhodopsin in a
Ca2+-dependent manner (Fig. 7B),
revealing that, like the protein kinase C in bovine outer segments (12,
13, 36), it is Ca2+-regulated.
The present results establish that 1) protein kinase C is a major contributor to the phosphorylation of rhodopsin in intact frog retinas; 2) the kinetics of rhodopsin phosphorylation/dephosphorylation differ markedly, depending on whether or not protein kinase C is hyperactivated; and 3) frog rhodopsin is phosphorylated in vitro by protein kinase C.
Protein Kinase C Contributes Significantly to the Phosphorylation of Rhodopsin in SituSpecific inhibition of protein kinase C by calphostin C provides a unique tool to evaluate the contribution of protein kinase C in the phosphorylation of rhodopsin in intact frog retinas. This inhibitor binds the phorbol ester-binding (C1) domain of protein kinase C and thus inhibits the regulation, rather the catalytic activity, of protein kinase C (34). This inhibitor was shown to have no effect on the activity of rhodopsin kinase in vitro, at concentrations causing about 70% inhibition of PMA-activated protein kinase C. It should be noted that inhibition of protein kinase C by this molecule is light-dependent and appears to be an oxidation step (37, 38). This property served an advantage in our experiments in that inhibition of protein kinase C was initiated by light exposure.
Inhibition of protein kinase C in retinas by calphostin C revealed that
this kinase contributes to about half of the phosphorylation of
rhodopsin under the conditions studied. Specifically, the extent of
rhodopsin phosphorylation in intact retinas was decreased by approximately 50% in the presence of 1 µM calphostin C. Similar use of kinase-specific inhibitors to study the -adrenergic
signaling pathway suggested that protein kinase A and
-adrenergic
receptor kinases each contribute 40-50% toward the desensitization of
the
2-adrenergic receptor in intact A431 cells at high (>70%)
receptor occupancy (39). These conditions would be analogous to the
high levels of rhodopsin photoexcitation in the our calphostin C
experiments. In the
-adrenergic system, protein kinase A
phosphorylation dominated over that of the G protein-coupled receptor
kinase at ligand concentrations resulting in <10% receptor occupancy
(39). More recently, the use of inhibitors and dominant negative kinase
mutants revealed that protein kinase C and G protein-coupled receptor
kinase 2 each contribute equally (about 40-50%) toward the
phosphorylation of a different G protein-coupled receptor, the type 1 angiotensin II receptor, in 293 cells (40). Thus, the phototransduction pathway shares in common with at least two other G protein receptor transduction pathways equal regulation of the receptor by a second messenger-regulated kinase and a G protein-coupled receptor kinase.
Previously we reported that hyperactivation of protein kinase C with phorbol esters had varying effects on the phosphorylation of rhodopsin in intact retinas. To resolve why under some conditions phorbol ester treatment caused an increase in rhodopsin phosphorylation and in other cases a decrease, we undertook here a systematic study of the kinetics of the phorbol ester effect at different levels of rhodopsin photoexcitation. Examination of the kinetics of rhodopsin phosphorylation revealed that PMA had a similar effect, irrespective of the amount of photoexcited rhodopsin. First, PMA caused a progressive increase in rhodopsin phosphorylation, which peaked around 10-15 min. Then, at later times, PMA caused a progressive decrease in rhodopsin phosphorylation relative to controls, such that by about 30 min the level of rhodopsin phosphorylation in PMA-treated retinas and control retinas was similar. Thus, the overall effect of hyperactivation of protein kinase C is a relatively short-term increase in rhodopsin phosphorylation (Fig. 3, E, F, and G).
The progressive nature of the increase in rhodopsin phosphorylation, at
earlier times, in PMA-treated retinas could arise because protein
kinase C phosphorylation is slower than that of rhodopsin kinase. In
the absence of PMA, the half-time for the light-dependent
phosphorylation of rhodopsin was approximately 2 min for the conditions
studied, irrespective of stimulus intensity. But in PMA-treated retinas
exposed to the higher intensity flash or continuous light, it was
evident that the half-time was significantly increased (by at least
2-fold) (Fig. 3, F and G). Thus, the
phosphorylation by protein kinase C appeared to display slower kinetics
than that by rhodopsin kinase. By comparison, the phosphorylation of
the -adrenergic receptor by protein kinase A is significantly slower relative to that catalyzed by the
-adrenergic receptor kinase in
permeabilized cells (41).
The progressive decrease in rhodopsin phosphorylation observed after 30 min postillumination in PMA-treated retinas could be accounted for by
several mechanisms. First, the protein kinase C phosphorylation site
could be more sensitive to dephosphorylation than the rhodopsin kinase
site. At least in vitro, the primary sites of
phosphorylation of bovine rhodopsin by protein kinase C and rhodopsin
kinase differ (14),3 so that the
phosphatase sensitivity of these sites could also differ (see further
discussion below). Second, protein kinase C activation could stimulate
phosphatase activity. Although Ser/Thr phosphatases directly regulate
protein kinase C structure and function (42), the reverse has not been
established; nonetheless, phorbol esters have been shown to indirectly
modulate the activity of protein phosphatase 1 in intact cells (43).
Third, selective binding of secondary proteins such as arrestin to
receptor phosphorylated by rhodopsin kinase (44), but not protein
kinase C, could decrease the phosphatase accessibility of the rhodopsin
kinase-phosphorylated receptor. In this regard, arrestin has been shown
to regulate -adrenergic receptor phosphorylated by G protein-coupled
receptor kinase but not protein kinase A (45). Whether or not protein kinase C regulates phosphatase activity in photoreceptors remains to be
established.
The kinetics of the light-dependent phosphorylation and dephosphorylation of rhodopsin reported here for intact frog retinas are similar to in vivo data on frogs reported by Kühn (46). Kühn found that 20-min illumination of dark-adapted frogs resulted in a level of phosphorylation that was constant between 20 and 35 min after the onset of illumination and then decayed with a half time of approximately 30 min. Although these conditions are not identical to those in this report, the kinetics of phosphorylation/dephosphorylation observed under our conditions are remarkably similar.
The kinetics of light-dependent phosphorylation of rhodopsin have been examined more recently in mouse retinas as a function of phosphorylated residue (47). Two residues were found to be phosphorylated in vivo, Ser-334 and Ser-338. In striking contrast, only one of these sites, Ser-338, is a primary phosphorylation site by rhodopsin kinase in vitro using bovine rhodopsin as the substrate; secondary phosphorylations occur in vitro at Ser-343 and Thr-336 (48-50). The novel site, Ser-334, can be phosphorylated to only 10% under appropriate pH and temperature conditions in vitro (51), suggesting that rhodopsin kinase is not the physiological regulator of this site. However, this site lies within the primary phosphorylation domain of protein kinase C, using bovine rhodopsin as the substrate (14). Interestingly, the kinetics of phosphorylation at both in vivo sites differ markedly. In response to three continuous flashes of light, both residues are phosphorylated equally; however, Ser-338 is phosphorylated and dephosphorylated significantly more rapidly compared with Ser-334. In contrast, continuous illumination results in significantly greater phosphorylation of Ser-334 than Ser-338, and both sites are dephosphorylated at comparable rates (47). Although the conditions in these in vivo experiments are clearly different from ours, it is noteworthy that in our experiments PMA caused a progressive increase in rhodopsin phosphorylation relative to the control, suggesting that phosphorylation of the protein kinase C site(s) lagged behind that of the rhodopsin kinase. The slower kinetics of the phorbol ester-simulated phosphorylation would be consistent with regulation of the more slowly phosphorylated Ser-334 site by protein kinase C. Curiously, the dephosphorylation at Ser-334 is slow relative to that of Ser-338, yet PMA appeared to accelerate the dephosphorylation of rhodopsin. If Ser-334 is the protein kinase C site, the possibility that PMA activates a rhodopsin-directed phosphatase could account for the accelerated dephosphorylation in our experiments.
ConclusionThe use of a specific activator and a specific inhibitor of protein kinase C reveals that this kinase is a major contributor to the light-dependent phosphorylation of rhodopsin. Because the phosphorylation by protein kinase C is regulated by second messengers, rather than by substrate conformation, the protein kinase C-catalyzed phosphorylation of rhodopsin may be analogous to the heterologous regulation of other G protein-coupled receptors. Whether protein kinase C in photoreceptors is activated by signals separate from rhodopsin activation, perhaps arising from feedback pathways from elsewhere in the retina, remains to be explored.
We thank Michelle Greene for expression and
purification of recombinant protein kinases. We thank Peter Parker for
the gift of the baculovirus constructs for protein kinase C and
James Inglese for the gift of the cDNA of rhodopsin kinase.