(Received for publication, January 28, 1997, and in revised form, February 20, 1997)
From the Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640
The protein kinase C phosphorylation sites on bovine rhodopsin were identified using proteolytic, phosphoamino acid, mass spectrometric, and peptide sequencing analyses. Tryptic removal of the 9 carboxyl-terminal residues of rhodopsin revealed that a major fraction of the phosphates incorporated by protein kinase C are in a region containing Ser334, Thr335, and Thr336. Phosphoamino acid analysis of the tryptic product established that Ser334 accounts for approximately 65% of the phosphorylation in this region. Analysis of the endoproteinase Asp-N-generated carboxyl terminus of rhodopsin by mass spectrometry and peptide sequencing revealed that Ser338 is also a primary phosphorylation site, with minor phosphorylation of Ser343. Quantitation of high pressure liquid chromatography-separated phosphopeptides, taken together with phosphoamino acid analysis of the tryptic product, revealed that Ser334 and Ser338 were phosphorylated equally and each accounted for approximately 35% of the total phosphorylation; Thr335/336 accounted for just under 20% of the phosphorylation, and Ser343 accounted for 10%. Thus, the primary protein kinase C sites are Ser334 and Ser338, with minor phosphorylation of Thr335/336 and Ser343. Ser334 and Ser338 have recently been identified as the primary sites of phosphorylation of rhodopsin in vivo (Ohguro, H., Van Hooser, J. P., Milam, A. H., and Palczewski, K. (1995) J. Biol. Chem. 270, 14259-14262). Of these sites, only Ser338 is a significant substrate for rhodopsin kinase in vitro. Identification of Ser334 as a primary protein kinase C target in vitro is consistent with protein kinase C modulating the phosphorylation of this site in vivo.
Phosphorylation plays a pivotal role in the regulation of G protein-coupled receptors, where phosphorylation by both G protein receptor kinases (GRKs) and second messenger-regulated kinases mediate receptor desensitization (1). In phototransduction, phosphorylation of the visual receptor, rhodopsin, is the first step in desensitization of this archetypal G protein-coupled receptor (2-4). The photoexcited conformation of rhodopsin serves as a substrate for rhodopsin kinase (also called GRK1; Ref. 5).
A number of laboratories have established that Ser338 and Ser343 on the carboxyl terminus of bovine rhodopsin are the primary phosphorylation sites of this kinase in vitro (6-8). Recently, Palczewski and co-workers showed that mouse rhodopsin is phosphorylated on Ser338 and also on a novel site, Ser334, in vivo (same numbering in mouse and bovine rhodopsin) (9). Although rhodopsin kinase has now been reported to phosphorylate Ser334 in vitro (10), this phosphorylation is minor relative to that of the primary sites and has not been consistently observed (6-8). Thus, a kinase other than rhodopsin kinase likely regulates rhodopsin at Ser334.
One candidate for modulating rhodopsin phosphorylation at the nonrhodopsin kinase site is protein kinase C. The enzyme phosphorylates the carboxyl terminus of dark-adapted rhodopsin, photoexcited rhodopsin, and opsin with equal affinity in vitro (11, 12), suggesting that its phosphorylation site(s) is equally exposed in all three receptor conformations.1 Evidence that this phosphorylation is physiologically relevant was first supported by the finding that phorbol esters, potent activators of protein kinase C, modulate the phosphorylation of rhodopsin in intact retinas (14, 15). Recently, the extent of protein kinase C's involvement in rhodopsin phosphorylation in situ was established using a highly selective protein kinase C inhibitor, calphostin C; treatment of retinas with calphostin C was shown to cause a 50% reduction in the light-dependent phosphorylation of rhodopsin in intact frog retinas (16).
This contribution shows that Ser334 and Ser338 are the major protein kinase C phosphorylation sites on bovine rhodopsin and Ser343 and Thr335 and/or Thr336 are minor sites. The finding that protein kinase C phosphorylates Ser334, coupled with the finding that protein kinase C contributes to approximately half the phosphorylation of rhodopsin in situ (16), suggests that this is the kinase that is responsible for the physiological regulation of rhodopsin at Ser334.
ATP, leupeptin, phosphoserine, phosphothreonine,
phosphotyrosine, trypsin (1.14 × 104
benzoyl-L-arginine ethyl ester units mg1),
1,3-bis[tris(hydroxymethyl)-methylamino]propane
(BTP)2 and phorbol myristate acetate were
purchased from Sigma. [
-32P]ATP (3000 Ci
mmol
1) was from DuPont NEN. Horseradish
peroxidase-conjugated goat anti-rabbit IgG, Tween 80, and
endoproteinase Asp-N (sequencing grade) were supplied by Calbiochem.
Chemiluminescence developing reagents were from Pierce. Avicel thin
layer chromatography plates were purchased from Analtech. Immobilon-P
was from Millipore, and nitrocellulose was from Schleicher & Schuell.
DEAE-Sephacel resin, heparin-Sepharose Hi-Trap, and Mono Q columns were
from Pharmacia Biotech Inc. Protein kinase C
was purified from the baculovirus expression system as described previously (17); protein
kinase C was also purified from bovine retinas as described previously
(12). The cDNA for bovine rhodopsin kinase was a generous gift from
Dr. Jim Inglese. A rabbit polyclonal antibody against opsin was
generated as described (15). Rod outer segment membranes were isolated
from bovine retinas and urea-stripped as described (12). 11-cis retinal
was kindly provided by the National Eye Institute. All other chemicals
are reagent grade.
The cDNA for bovine
rhodopsin kinase was subcloned from pBluescript into the baculovirus
transfer vector pVL1393 (Invitrogen) and recombined with baculoviral
DNA using the BaculoGold kit (Pharmingen). Sf-21 cells (2 × 106 ml1; 400 ml) were infected with
recombinant virus (109 plaque-forming unit
ml
1) and harvested 72 h post-infection. Washed cells
were homogenized in buffer containing 10 mm BTP, 10 mM EGTA, 2 mM EDTA, 0.4% Tween 80, 1 mM dithiothreitol, 20 µg ml
1 leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM
benzamadine. The homogenate was centrifuged at 245,000 × g for 45 min at 4 °C and rhodopsin kinase purified from
the supernatant by sequential chromatography on DEAE-Sephacel, heparin
Hi-Trap, and Mono Q columns. Fractions containing rhodopsin kinase were
stored at
20 °C in buffer containing 50% glycerol, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM BTP, pH
7.5, 250 mM KCl.
Urea-stripped membranes were
phosphorylated by protein kinase C purified from bovine retinas,
baculovirus-expressed protein kinase C , or baculovirus-expressed
rhodopsin kinase under dim red light or under room light, as stated in
the figure legends. Reaction mixtures (80 µl) containing 10 pmol of
rhodopsin and 2 pmol of protein kinase C or 1 pmol of rhodopsin kinase
were incubated in buffer containing 50 µM
[
-32P]ATP (3 Ci mmol
1), 1 mM
MgCl2, 1 mM dithiothreitol, and 20 mM HEPES, pH 7.5, at 30 °C unless otherwise noted.
CaCl2 (500 µM) and phorbol myristate acetate
(0.5-1 µM) were included in protein kinase C reaction mixtures; 100 µM EDTA and 100 µM EGTA were
included in rhodopsin kinase reaction mixtures. Reactions proceeded for
60 min at 30 °C, followed by centrifugation at 500,000 × g for 20 min at 4 °C. Membranes were resuspended in
buffer and analyzed as described in appropriate sections below.
Rhodopsin (70 pmol)
phosphorylated by protein kinase C or rhodopsin kinase was resuspended
in 700 µl of 20 mM HEPES, pH 7.5, containing 50 µM CaCl2; 10 pmol (100 µl) were removed as
the zero time point. Proteolysis was initiated by the addition of 145 µl of 57 units ml1 trypsin, and samples were incubated
at 30 °C for 2-60 min. Aliquots (125 µl) were removed into
SDS-PAGE buffer (40 µl; 8% SDS, 40% glycerol, 0.008% bromphenol
blue, 20%
-mercaptoethanol, and 0.25 M Tris, pH 6.8).
Proteins were separated by SDS-PAGE (10% acrylamide), electrophoretically transferred to nitrocellulose, labeled with antibodies to opsin via incubation with horseradish
peroxidase-conjugated IgG, and labeling detected by chemiluminescence.
Antibodies were removed by incubation of blots in 100 mM
-mercaptoethanol, 2% SDS, and 62.5 mM Tris, pH 6.8, for
60 min at 55 °C, and blots were analyzed by autoradiography or
PhosphorImager analysis (Molecular Dynamics).
Rhodopsin (70 pmol)
phosphorylated by protein kinase C or rhodopsin kinase was proteolyzed
by trypsin (17 units ml1) as described above. Intact
rhodopsin and cleavage products were separated by SDS-PAGE (12%
acrylamide) and electrophoretically transferred to Immobilon-P. Bands
corresponding to intact (36 kDa) and proteolyzed (35 kDa) opsin were
separately excised from the Immobilon-P membrane, hydrolyzed in 500 µl of 6 M HCl for 1 h at 110 °C, and subjected to
phosphoamino acid analysis as described (18). Hydrolyzed sample was
mixed with 1 µg each of phosphoserine, phosphothreonine, and
phosphotyrosine, spotted onto a thin layer chromatography plate, and
electrophoresed horizontally (2000 V for 30 min) in pH 3.5 buffer (5%
acetic acid, 0.5% pyridine). Amino acid standards were visualized with
0.2% ninhydrin in acetone and 32P comigrating with
standards detected by autoradiography.
1
nmol of rhodopsin was phosphorylated by protein kinase C as described
above except the ATP concentration was 250 µM. After centrifugation, the pellet was resuspended in 1 ml of 20 mM
HEPES, pH 7.5, at 30 °C. The carboxyl terminus was released by the
addition of 200 µl of a 20 µg ml1 solution of
endoproteinase Asp-N in 10 mM HEPES, 50% glycerol and
incubation at 30 °C for 60 min. The solution was centrifuged again
for 20 min at 500,000 × g at 4 °C to separate the
19-amino acid carboxyl-terminal tail from the membrane-bound fragment
of rhodopsin. The supernatant containing the carboxyl-terminal peptide was applied to a reverse phase HPLC C-18 column. Masses of peaks of
interest were determined by laser desorption mass spectrometry; peptides with masses corresponding to the phosphorylated tail of
rhodopsin were sequenced by automated Edman degradation or tandem mass
spectrometry.
In determining the protein kinase C phosphorylation site(s) on
rhodopsin, we took advantage of the finding that phosphorylation on
Ser338 prevents cleavage by trypsin at the adjacent
Lys339 (8, 19) (see Fig. 4A). Thus, cleavage of
rhodopsin at this site serves as a diagnostic for whether
Ser338 has been phosphorylated and provides a measure for
what fraction of the phosphorylation occurs amino-terminal to
Ser338.
Fig. 1A (left) shows that trypsin
treatment of rhodopsin phosphorylated by protein kinase C resulted in
the appearance of a 32P-labeled product that migrated
slightly faster (35 kDa) than native rhodopsin (36 kDa); this product
corresponds to rhodopsin cleaved at Lys339 to release a
9-amino acid peptide. Fig. 1C (left) reveals that at least 40% of the radioactivity incorporated on rhodopsin was recovered in the 35-kDa cleaved product (the product is a transient intermediate, setting a lower limit on the fraction of rhodopsin modified amino-terminal to Ser338). Approximately 15% of
the radioactivity was associated with uncut rhodopsin after 60 min of
trypsin treatment, consistent with additional phosphorylation on
Ser338 to yield a population of receptor that was
proteolyzed more slowly (presumably at sites other than
Lys339). Similar data were obtained regardless of whether
light-adapted (Fig. 1) or dark-adapted (not shown) rhodopsin served as
the substrate and whether protein kinase C (Fig. 1) or retinal
protein kinase C was used.
In contrast to protein kinase C-phosphorylated rhodopsin, only one radioactive band was detected when rhodopsin kinase-phosphorylated rhodopsin was treated with trypsin under the same conditions; this band corresponds to uncut rhodopsin (Fig. 1A, right). The 35-kDa product, although present (Western blot in Fig. 1B, right), was not radioactive. Fig. 1C shows that the half-time for cleavage of rhodopsin kinase-phosphorylated rhodopsin was approximately 30 min; this is at least ten times slower than the half-time for cleavage of protein kinase C-phosphorylated rhodopsin (Fig. 1C, right), consistent with phosphorylation on Ser338 preventing proteolysis at Lys339.
To identify the residue on the tryptic fragment that is phosphorylated
by protein kinase C, the 35-kDa product was subjected to phosphoamino
acid analysis (Fig. 2). Compilation of data from 13 independent experiments, with stoichiometries varying from 0.2 to 1.0 mol phosphate/mol rhodopsin, revealed an average of 65 ± 9%
phosphoserine on the 35-kDa tryptic product; the remaining radioactivity was on phosphothreonine with no detectable
phosphotyrosine. Simlar results were obtained whether rhodopsin was
phosphorylated by recombinant protein kinase C from baculovirus
(Fig. 2) or protein kinase C purified from bovine retinas (not shown).
Thus, Ser334 is the primary phosphorylation site on the
tryptic fragment of rhodopsin, with some phosphorylation observed on
one or both of the adjacent Thr residues.
Additional phosphorylation sites (i.e. not present in the
tryptic product) were identified by analysis of a carboxyl-terminal peptide, released by endoproteinase Asp-N (20), which contains all the
in vivo (15) and protein kinase C in vitro (12)
phosphorylation sites. Reverse phase HPLC resolved the sample of the
carboxyl-terminal fragment into three peaks labeled 1, 2, and 3, respectively, in Fig. 3. Peak 3 had a more quickly
eluting shoulder that was not resolved into a separate peak; this
shoulder was collected for analysis (referred to as Peak 3 shoulder).
Electrospray mass spectrometry revealed the presence of two species in
Peak 3 shoulder with masses corresponding to unphosphorylated peptide
and monophosphorylated peptide (Table I). Peak 1 and
Peak 2 each contained a single peptide with a mass corresponding to
monophosphorylated peptide (Table I). Multiply phosphorylated species,
which elute earlier than monophosphorylated species (6), were not
detected and could account for no more than 3% of the sample based on
the sensitivity of the detection. Similar results were obtained whether
dark-adapted (Fig. 3) or light-adapted (not shown) rhodopsin was
phosphorylated by protein kinase C. The carboxyl-terminal fragment of
unphosphorylated rhodopsin eluted as a single peak with the same
retention time as Peak 3 above; it had a mass of 1938.1, establishing
no phosphorylation prior to protein kinase C treatment (not shown).
|
Sequencing by Edman degradation identified the phosphorylated residues as Ser343 and Ser338 in Peaks 1 and 2, respectively. The order of elution of these peptides relative to unphosphorylated peptide was as reported previously (6). The presence of unphosphorylated peptide in Peak 3 shoulder confounded the unambiguous identification of the phosphorylation site on the monophosphorylated peptide; the signal for detection of released derivatized amino acid dropped from 3.2 to 0.2 pmol in the cycle from residues 333 to 334, consistent with modification at Ser334. Further analysis of Peak 3 shoulder by tandem mass spectroscopy confirmed that phosphate was present at one of the 3 adjacent residues Ser334-Thr336.
The stoichiometry of the analyzed phosphorhodopsin was 0.6 mol phosphate/mol rhodopsin. Based on this and the areas of the three peaks, we calculated the relative distribution of phosphates on the identified phosphorylation sites. Peaks 1, 2, and 3 represented 6, 23, and 71%, respectively, of the total peak area in the chromatogram in Fig. 3. Given that 60% of the sample was phosphorylated, we calculated that the phosphopeptide component in Peak 3 (Ser334-Thr335) accounted for 31% of the total peak area. Expressed relative to the total phosphorylation, Ser343 (Peak 1) incorporated 10% of the phosphate in the sample, Ser338 (Peak 2) incorporated 38% of the phosphate, and Ser334-Thr335-Thr336 (phosphorylated component of Peak 3) accounted for 52% of the phosphorylation. Phosphoamino acid analysis revealed that 65 ± 9% of the phosphate in the Ser334-Thr336 region was on Ser334 (Fig. 2), so that phosphate on Ser334 accounted for 34% of the total phosphate and phosphate on Thr335 and/or Thr336 accounted for 18%. Thus, the phosphorylation preference was: Ser334 = Ser338 > Thr335/336 > Ser343.
The foregoing results reveal that 1) the primary in vitro protein kinase C phosphorylation sites of bovine rhodopsin are Ser334 and Ser338 (Fig. 4A), with minor phosphorylation on Ser343 and Thr335 and/or Thr336, 2) a single phosphate is incorporated per molecule of rhodopsin, and 3) light has no significant effect on the sites phosphorylated.
Importantly, Ser334 and Ser338 are the major in vivo phosphorylation sites of rhodopsin (9). Furthermore, of these two sites, only Ser338 is phosphorylated significantly by rhodopsin kinase in vitro (6-8). This suggests that protein kinase C is the primary kinase that modulates Ser334 in vivo. This possibility is also supported by kinetic data; the contribution of protein kinase C to the phosphorylation of rhodopsin in intact retinas is slower than that of rhodopsin kinase (16), and Ser334 is phosphorylated more slowly than Ser338 in vivo (9).
Recent elucidation of the solution structure of a domain of rhodopsin encompassing the seventh transmembrane span and carboxyl terminus (21) reveals that Ser334 and Ser338 are surface exposed (Fig. 4B). Furthermore, the secondary protein kinase C sites Thr335 and Thr336 are less exposed; and the least favored site, Ser343 is on the opposite face of the surface containing the primary sites. Because the protein kinase C phosphorylation is unaffected by light (12), Ser334 is likely to be equally exposed in all conformations of the receptor. Although protein kinase C has a preference for basic residues near the phosphoacceptor site (22), accessibility, rather than a defined consensus sequence, appears to determine substrate specificity (23). Thus, orientation and proximity to the membrane interface, where protein kinase C binds (23), may be the driving force in promoting rhodopsin phosphorylation.
The identification of the two in vivo phosphorylation sites on rhodopsin as protein kinase C phosphorylation sites underscores the importance of this second messenger-regulated kinase in regulation of rhodopsin function. The phosphorylation by rhodopsin kinase and protein kinase C at distinct sites, in addition to the shared phosphorylation of Ser338, should allow mechanistic insight into what promotes the activity of each kinase under specific conditions.
We thank Dr. William Lane and colleagues at the Harvard Microchemistry Facility for performing the HPLC, mass spectrometry, and peptide sequencing analysis, Dr. Bruce Grant for assistance in phosphoamino acid analysis, Maritess Jasmosmos for assistance in kinase expression and purification, and Dr. Philip Yeagle for kindly providing the coordinates for the carboxyl-terminal domain of rhodopsin.