(Received for publication, August 17, 1994; and in revised form, November 22, 1994)
From the
Protein kinase C isolated from retina catalyzes the
stoichiometric phosphorylation of bovine rhodopsin. Enzymological
studies using receptor in rod outer segment membranes stripped of
peripheral proteins reveal that the phosphorylation is independent of
receptor conformation or liganded state; the half-time for
phosphorylation of unbleached (dark-adapted) rhodopsin, bleached
(light-activated) rhodopsin, and opsin (chromophore removed) is the
same. The phosphorylation by protein kinase C is Ca and lipid regulated; the K
for
Ca
decreases with increasing concentrations of
membrane, consistent with known properties of
Ca
-regulated protein kinase Cs. The K
for ATP is 27 µM, with an
optimal concentration for MgCl
of approximately 1
mM. The phosphorylation of rhodopsin by protein kinase C is
inhibited by the protein kinase C-selective inhibitor sangivamycin.
Proteolysis by Asp-N reveals that all the protein kinase C
phosphorylation sites are on the carboxyl terminus of the receptor.
Cleavage with trypsin indicates that Ser
, the primary
phosphorylation site of rhodopsin kinase, is not phosphorylated
significantly; rather, the primary phosphorylation site of protein
kinase C is on the membrane proximal half of the carboxyl terminus. The
protein kinase C-catalyzed phosphorylation of rhodopsin is analogous to
the ligand-independent phosphorylation of other G protein-coupled
receptors that is catalyzed by second messenger-regulated kinases.
Second messenger-regulated kinases and substrate-regulated
kinases provide two desensitizing mechanisms in the regulation of G
protein-coupled receptors. Most notably, the adrenergic receptor
is desensitized at low ligand levels primarily by phosphorylation
catalyzed by protein kinase A, and possibly C, and at high ligand
levels by phosphorylation mediated by the
adrenergic receptor
kinase(1) . Recent evidence implicates both types of kinases in
regulation of olfactory receptors (2, 3) and
muscarinic acetylcholine receptors(4) . For these signaling
pathways, phosphorylation by two differently regulated kinases allows
exquisite fine tuning of receptor function.
In phototransduction, it
has been clearly established that a G protein-coupled receptor kinase,
rhodopsin kinase, phosphorylates and deactivates light-activated
rhodopsin(5, 6) . In this pathway, absorption of a
photon induces isomerization of the receptor's covalently bound
ligand, 11-cis retinal, thus effecting a conformational change that
exposes cytoplasmic surfaces on the receptor to allow interaction with
transducin(6) . These exposed surfaces also promote binding of
rhodopsin kinase and subsequent phosphorylation on the receptor's
carboxyl terminus. The primary phosphorylation sites by rhodopsin
kinase in vitro have been recently identified as Ser and Ser
(7, 8, 9) .
Phosphorylation on the carboxyl terminus decreases the interaction with
transducin, an interaction that is effectively quenched when arrestin
binds the polyphosphorylated carboxyl tail(10) . Similar to
other G protein-coupled receptor kinases, rhodopsin kinase displays
strict specificity for the active conformation of the receptor.
Mounting evidence implicates phosphorylation by an additional
kinase, protein kinase C, in the phosphorylation of rhodopsin. First,
hyperactivation of protein kinase C in the intact retina, by treatment
with phorbol esters, alters the phosphorylation of rhodopsin in a
light-dependent manner(11, 12) . Second, rhodopsin is
phosphorylated by protein kinase C in
vitro(11, 13) . This phosphorylation has been
shown to uncouple the receptor from transducin(13) . Third, the
allosteric activator of protein kinase C, diacylglycerol, has been
shown by many groups to be produced in response to
light(14, 15, 16, 17, 18) .
Phospholipase Cs have been biochemically isolated from and
immunolocalized to photoreceptors(19, 20) , and, more
recently, cDNAs encoding 4 phospholipase Cs with high homology to
the Drosophila norpA gene (which encodes the phospholipase C
involved in invertebrate phototransduction) have been
identified(21) . The sensitivity of rhodopsin phosphorylation
to protein kinase C activators in situ and the light-dependent
generation of diacylglycerol in rod outer segments support a role for
protein kinase C in visual transduction.
This contribution provides a kinetic and structural analysis of the phosphorylation of rhodopsin by protein kinase C. Our data reveal that the phosphorylation of rhodopsin by protein kinase C is mechanistically similar to the phosphorylation of other G protein-coupled receptors by second messenger-regulated kinases. Most importantly, the phosphorylation is independent of the liganded state of the receptor, and the primary phosphorylation site, although on the carboxyl terminus, differs from that of the G protein-coupled receptor kinase.
Figure 2:
Time course of phosphorylation of
unbleached rhodopsin (), bleached rhodopsin (
), and opsin
(
) by protein kinase C. Receptor (275 nM) in
urea-stripped rod outer segment membranes was incubated with retinal
protein kinase C (40 nM) and 100 µM CaCl
for the indicated times at 30 °C. Data represent the mean
± S.E. of a triplicate assay; lines drawn are those
predicted from the Michaelis-Menten
equation.
For
the experiment shown in Fig. 4, the rate of phosphorylation of a
protein kinase C selective peptide (FKKSFKL-NH; (22) ) was measured as described(30) , except that rod
outer segment membranes provided the lipid to stimulate protein kinase
C rather than phosphatidylserine/diacyglycerol vesicles.
Figure 4:
Ca-dependence of retinal
protein kinase C in the presence of two membrane concentrations. A, the initial rate of phosphorylation of bleached rhodopsin
(0.35 µM) catalyzed by retinal protein kinase C (10
nM) was examined in the presence of 0-250 µM free Ca
and stripped rod outer segment membranes
resulting in a lipid concentration in the assay of 32 µM (
). Data represent the weighted average ± S.D. of two
or three experiments, each in hextuplicate. B, The initial
rate of phosphorylation of a protein kinase C-selective peptide,
stimulated by 32 µM lipid (
) or 320 µM lipid (
) in rod outer segment membranes, was measured
under the exact conditions described in A except that 50
µg ml
of the synthetic peptide were included in
the assay. Data represent the weighted average ± S.D. of two
experiments, each in hextuplicate. Lines drawn are those
predicted from the modified Hill equation(33) . All assays
included 525 µM EGTA.
Figure 1:
Phosphorylation of unbleached
rhodopsin, bleached rhodopsin, and opsin by protein kinase C or
rhodopsin kinase. A, autoradiogram of unbleached rhodopsin (U, lanes3-8), bleached rhodopsin (B,
lanes9-14), or opsin (O lanes15-20) (275 nM receptor in all lanes), in
urea-stripped rod outer segment membranes, incubated in the presence (lanes3-5, 9-11, and 15-17) or absence (lanes6-8, 12-14, and 18-20) of 40 nM retinal protein kinase C, 5 mM MgCl, and 50
µM ATP for 30 min at 30 °C. CaCl
(100
µM) or PMA (1 nM) was included as indicated.
Receptor samples were prepared as described under
``Methods.'' In addition to rhodopsin phosphorylation,
autophosphorylation of protein kinase C is evident. Coomassie
Blue-stained gel corresponding to the autoradiogram of lanes5 and 6, showing rhodopsin and protein kinase C,
is presented in lanes1 and 2. Panel
B, as in A except that rhodopsin kinase (10 nM)
replaced protein kinase C.
In contrast to the phosphorylation by
protein kinase C, the phosphorylation catalyzed by rhodopsin kinase was
specific for the bleached conformation of rhodopsin. Fig. 1B shows that unbleached rhodopsin (lanes3-5) and opsin (lanes15-17) were not substrates of rhodopsin kinase,
whereas bleached rhodopsin was (lanes9-11).
The phosphorylation of bleached rhodopsin by rhodopsin kinase was not
sensitive to Ca (lane10) or
phorbol esters (lane11) under the conditions of the
experiment. Note that less than 0.1 µM recoverin was
present in the reaction mixture, based on Coomassie staining of gels,
and thus recoverin-dependent Ca
effects would not be
expected(38) .
Fig. 2compares time courses for
phosphorylation of unbleached rhodopsin, bleached rhodopsin, and opsin.
For the concentrations of protein kinase C and receptor examined in
this figure, the half-time of phosphorylation was approximately the
same for all species of receptor: 57 ± 9 min for unbleached
rhodopsin and opsin (weighted average ± S.E. for both sets of
data) and 52 ± 3 min for bleached rhodopsin. The final
stoichiometry for phosphorylation of bleached rhodopsin by retinal
protein kinase C varied from 0.6 to 1.2 mol of phosphate/mol of
receptor, depending on membrane preparation, and was typically 4-fold
less than the maximal phosphorylation catalyzed by rhodopsin kinase
(data not shown). In general, unbleached rhodopsin and opsin
incorporated 80-100% of the phosphate incorporated by bleached
rhodopsin. Addition of fresh protein kinase C to the reaction mixture
after phosphate incorporation had plateaued did not result in a
significant increase in phosphorylation, revealing that P
incorporation had plateaued because phosphorylation sites were no
longer available. The receptor present in these experiments was
quantitatively proteolyzed by endoproteinase Asp-N to generate a 32-kDa
truncated form (see Fig. 7B). Because this protease
cleaves the cytoplasmic (carboxyl) terminal tail of rhodopsin, all
receptor molecules were oriented with their cytoplasmic surface exposed
to the solution.
Figure 7: Partial proteolysis to identify the domain of rhodopsin phosphorylated by protein kinase C. A, Cartoon representation of bovine rhodopsin showing sites of cleavage on the carboxyl terminus catalyzed by endoproteinase Asp-N and trypsin. B, Western blot (panels on left) and corresponding autoradiogram (panels on right) of unbleached rhodopsin (U), bleached rhodopsin (B), or opsin (O) phosphorylated with protein kinase C (lanes1-6) or rhodopsin kinase (lanes7-12) and then treated with endoproteinase Asp-N for 30 min at 30 °C. The receptor phosphorylated by protein kinase C incorporated 0.2 mol of phosphate/mol of receptor; the bleached rhodopsin phosphorylated by rhodopsin kinase incorporated 0.4 mol of phosphate/mol of receptor. C, Western blot (panels on left) and corresponding autoradiogram (panels on right) of unbleached rhodopsin (U), bleached rhodopsin (B), or opsin (O) phosphorylated with protein kinase C (lanes1-12) or rhodopsin kinase (lanes13-24) and then treated with the indicated concentration of trypsin for 30 min at 30 °C. All receptor species incubated with protein kinase C, and bleached rhodopsin incubated with rhodopsin kinase, incorporated 0.4 mol of phosphate/mol of receptor.
Fig. 3shows the rate of rhodopsin
phosphorylation catalyzed by protein kinase C or rhodopsin kinase as a
function of increasing amounts of stripped rod outer segment membranes.
Because the relative concentration of substrate in the membrane was the
same for all rhodopsin concentrations (i.e. same
rhodopsin/lipid ratio), a K for rhodopsin cannot
be interpreted from these data (i.e. because of the reduction
in dimensionality, once the kinase binds the first substrate, the local
concentration of substrate near the kinase is the same for all
conditions). However, the data do reveal that 1) protein kinase C has a
higher affinity for rod outer segment membranes than does rhodopsin
kinase and 2) under the conditions of these assays, 70 ± 2
nM rhodopsin (approximately 6 µM phospholipid)
resulted in half-maximal activation of protein kinase C.
Figure 3:
Dependence of rhodopsin phosphorylation on
concentration of stripped rod outer segment membranes. The initial rate
of phosphorylation of bleached rhodopsin catalyzed by retinal protein
kinase C (40 nM) () or rhodopsin kinase (2 nM)
(
) was examined in the presence of increasing amounts of
stripped rod outer segment membranes (1-800 nM rhodopsin). CaCl
(150 µM) was included in
the protein kinase C incubation. Data represent the mean ± S.E.
triplicates.
The
Ca dependence for the activation of retinal protein
kinase C was measured for two substrates and as a function of membrane
concentration. The concentration of Ca
resulting in
half-maximal activity toward rhodopsin phosphorylation (Fig. 4A) or phosphorylation of a synthetic peptide (Fig. 4B, opencircles) was the same
(14 ± 3 µM or 14 ± 2 µM,
respectively) in the presence of 32 µM lipid. Note that
this concentration of lipid and rhodopsin (0.35 µM) result
in stimulation of protein kinase C to 90% of its maximal rate (see Fig. 3). The identical Ca
requirement for
phosphorylation of two different substrates is consistent with this
cation allosterically modulating the membrane binding and catalytic
activity of protein kinase C (39, 40) rather than
substrate binding.
Fig. 4B shows that the K for Ca
decreased to 4.6
± 0.3 µM when the concentration of rod outer
segment membranes was increased to 320 µM lipid. The drop
in K
that occurred upon a 10-fold increase in
lipid concentration reflects an increase in affinity of protein kinase
C for these membranes. Mosior and Epand (40) showed that the
apparent binding constant of conventional protein kinase Cs for
membranes is linearly proportional to Ca
concentration between 100 nM and 0.5 mM Ca
. Because the binding of Ca
to protein kinase C depends on the total lipid concentration, mol
% phosphatidylserine, and mol %
diacylglycerol(39, 40, 41) , the K
for Ca
measured in vitro is relative to the specific assay conditions. (
)The
Ca
requirement for phosphorylation of unbleached
rhodopsin or opsin was not significantly different from that of
bleached rhodopsin (not shown).
The K for ATP
for the phosphorylation of bleached rhodopsin by protein kinase C was
27 ± 3 µM ATP (Fig. 5A). This
number is similar to the K
for ATP of known
isozymes when measured using phosphorylation of synthetic peptides, (
)and slightly higher than the reported K
for phosphorylation of histone (5-10 µM ATP,(42) ). A concentration of 50 µM ATP was
included in subsequent phosphorylation assays.
Figure 5:
Dependence of the rate of rhodopsin
phosphorylation catalyzed by retinal protein kinase C on ATP and
Mg. A, bleached rhodopsin in stripped rod
outer segment membranes (275 nM receptor) was incubated with
retinal protein kinase C (25 nM), 100 µM CaCl
, 5 mM MgCl
, and 0-50
µM ATP for 10 min at 30 °C. Data represent the mean
± S.D. of an experiment in triplicate. The line drawn
is that predicted from the Michaelis-Menten equation. B,
phosphorylation reactions were as described in A except that
the ATP concentration was 50 µM and the MgCl
concentration was varied from 0-50 mM. The solidcircle represents activity in the absence of
CaCl
. Data represent the mean ± S.E. of an
experiment in triplicate.
The dependence on
Mg for the phosphorylation of bleached rhodopsin by
protein kinase C is presented in Fig. 5B. The maximal
rate of phosphorylation of rhodopsin required 1-5 mM MgCl
, with higher concentrations resulting in
inhibition of the kinase. This stimulation is consistent with the
Mg
requirements reported for the known
Ca
-dependent protein kinase Cs; Burns and Bell (42) reported that protein kinase C
,
II and
are half-maximally stimulated by 0.9, 0.6, and 0.7 mM Mg
, respectively. Subsequent phosphorylation
assays were conducted in the presence of 5 mM MgCl
.
Fig. 6shows that sangivamycin
selectively inhibited the phosphorylation of bleached rhodopsin
catalyzed by protein kinase C compared with the phosphorylation
catalyzed by rhodopsin kinase. Although this nucleoside analogue
competes with ATP for the ATP-binding site, it displays significant
selectivity for protein kinase C's active site compared with that
of other kinases(43) . The phosphorylation catalyzed by protein
kinase C was half-maximally inhibited by 13.4 ± 0.7 µM sangivamycin in the presence of 50 µM ATP; the
IC for this concentration of ATP agrees with the K
of 11 µM reported for
Ca
-dependent protein kinase Cs(43) . In
contrast, rhodopsin kinase was considerably less sensitive to
sangivamycin, with only 35 ± 1% inhibition observed in the
presence of 100 µM inhibitor. Because rhodopsin kinase has
a higher affinity for ATP than protein kinase C (K
= 2 µM with 1 mM Mg
(44) ), it may be less sensitive to
inhibition by sangivamycin even though the K
for
interaction with sangivamycin may be similar. Inhibition of
autophosphorylation of both kinases followed similar kinetics as
inhibition of rhodopsin phosphorylation (data not shown), indicating
that sangivamycin was inhibiting the intrinsic catalytic activity of
each kinase rather than preventing substrate interaction.
Figure 6:
Sangivamycin selectively inhibits
rhodopsin phosphorylation catalyzed by retinal protein kinase C. The
initial rate of rhodopsin phosphorylation was measured in the presence
of 50 µM ATP, 10 mM MgCl, and
0-100 µM sangivamycin. Phosphorylation was catalyzed
by 20 nM retinal protein kinase C (in which case 330
µM CaCl
, 0.12 mM EDTA, and 0.12
mM EGTA were included in the reaction mixture) or 8 nM of rhodopsin kinase (in which case 0.12 mM EDTA and 0.12
mM EGTA were included in the reaction mixture; similar data
were obtained in the presence of 330 µM CaCl
(not shown)). Data represent the mean ± S.D. of an
experiment in triplicate.
Proteolysis with trypsin, which cleaves after Lys to
release the carboxyl-terminal 9 residues(46) , was used to
further narrow down the domain phosphorylated by protein kinase C (Fig. 7A). Unbleached rhodopsin, bleached rhodopsin, or
opsin were phosphorylated by protein kinase C or rhodopsin kinase and
then treated with trypsin; 0.4 mol of phosphate were incorporated per
mol of bleached rhodopsin for both kinases (the substoichiometric
phosphorylation allowed determination of the initial phosphorylation
domain). The Western blot in Fig. 7C shows that limited
proteolysis by trypsin resulted in the formation of a fragment
migrating with an apparent molecular mass 1 kDa smaller than the native
enzyme, consistent with cleavage at Lys
. The
susceptibility of all three forms of protein kinase C-phosphorylated
receptor to cleavage at Lys
was similar (e.g. same amount of cleaved rhodopsin in lanes2, 6, and 10). Furthermore, the sensitivity to
trypsin of the protein kinase C-phosphorylated receptor (e.g.lanes1-4) was similar to that of
nonphosphorylated receptor (approximately 50% proteolysis in the
presence of 2 µg ml
trypsin); however, note that
approximately 20% of the protein kinase C-phosphorylated receptor, but
not unphosphorylated receptor, was resistant to proteolysis at the
highest trypsin concentration. Thus, phosphorylation by protein kinase
C did not affect the accessibility of Lys
to trypsin for
the majority of the receptor population. Ohguro et al.(8) have shown that phosphorylation at the adjacent
Ser
, the primary phosphorylation site by rhodopsin
kinase, inhibits proteolysis at Lys
. The autoradiogram in Fig. 7C shows that rhodopsin phosphorylated by protein
kinase C had significant
P associated with it after
cleavage at Lys
(e.g.lanes4, 8, and 12). In contrast, bleached rhodopsin
phosphorylated by rhodopsin kinase was significantly less sensitive to
proteolysis by trypsin (lanes17-20).
Importantly, any cleaved rhodopsin from the sample phosphorylated with
rhodopsin kinase was not radioactive (lane20). The
inability of trypsin to cleave receptor phosphorylated at Ser
indicates that the primary phosphorylation site of protein kinase
C is not Ser
; protein kinase C-phosphorylated receptor is
cleaved at Lys
. Rather the primary phosphorylation site
is on the carboxyl terminus between residues 330 and 337. Some
phosphorylation on the trypsin-sensitive domain is also catalyzed by
protein kinase C as well as minor phosphorylation at Ser
(based on insensitivity of approximately 20% of the
phospho-rhodopsin to proteolysis at Lys
).
Table 1compares kinetic and structural parameters for the phosphorylation of rhodopsin by protein kinase C and by rhodopsin kinase. The most striking difference in the phosphorylation by the two kinases is that protein kinase C does not discriminate between receptor conformations or liganded state, whereas rhodopsin kinase phosphorylates only bleached rhodopsin(5) . A second important difference is the regulation of the two kinases; protein kinase C is regulated by a second messenger, whereas rhodopsin kinase is regulated by the conformation of its substrate. The most important similarity in the effects of the two kinases is that both catalyze a desensitizing phosphorylation of the visual receptor. Because both kinases have similar consequences on rhodopsin function, the advantage of having two different kinases would be if each were dominant under different conditions(47) , perhaps allowing rhodopsin to be regulated under a much broader range of illumination as well as by a heterologous pathway.
Identification of the major phosphorylation site by protein kinase C as being on the amino-terminal half of the carboxyl tail supports in situ findings. Hyperactivation of protein kinase C in the intact rat retina by treatment with phorbol esters results in an increase in the phosphorylation of rhodopsin exposed to a brief flash of light(11, 12) . Proteolytic digests of receptor phosphorylated in situ revealed that the increased phosphorylation resulting from phorbol ester treatment occurred on a trypsin-resistant (11) but Asp-N-sensitive (12) domain. Thus, protein kinase C modifies the same domain of rhodopsin in situ and in vitro.
The phosphorylation of rhodopsin by protein
kinase C is analogous to the heterologous phosphorylation of other G
protein-coupled receptors. Notable similarities are as follows. 1) The
phosphorylation by protein kinase C is independent of receptor
conformation, similar to the ligand-independent phosphorylation of the
adrenergic receptor by protein kinases A or C or the
ligand-independent phosphorylation of the muscarinic acetylcholine
receptor by protein kinase C. This contrasts with the ligand-dependent
homologous phosphorylation catalyzed by G protein-coupled receptor
kinases. 2) One mol of phosphate is incorporated per mol of receptor,
similar to the phosphorylation of only one or two sites of the
adrenergic receptor by protein kinases A or C, and contrasting with the
multiple phosphorylations catalyzed by G protein-coupled receptor
kinases. 3) The phosphorylation catalyzed by protein kinase C is
regulated by a second messenger, diacylglycerol; the heterologous
phosphorylation of the
adrenergic receptor is also stimulated by
a second messenger, cAMP. This contrasts with the regulation of G
protein-coupled receptor kinases by substrate conformation. 4) The
major phosphorylation site by protein kinase C differs from that of
rhodopsin kinase, although it is also on the carboxyl terminus. Protein
kinases A and C modify the
adrenergic receptor on the carboxyl
tail of this receptor (as well as on the third cytoplasmic loop), but
on a residue proximal to the membrane span that is not a
phosphorylation site by G protein-coupled receptor kinases.
Phosphorylation of rhodopsin by two differently regulated kinases may allow rhodopsin to respond to a much broader range of stimuli. Whether protein kinase C is activated directly in response to light on the photoreceptor cell or whether its activation arises heterologously from activation of another signaling pathway remains to be determined.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L39909[GenBank].