(Received for publication, July 14, 1995; and in revised form, October 4, 1995)
From the
Photoactivated rhodopsin (Rho*) is phosphorylated near the C
terminus at multiple sites, predominantly at Ser,
Ser
, and Ser
. We systematically examined
the sites of phosphorylation upon flash activation of Rho in rod outer
segment (ROS) homogenates. Addition of an inhibitory antibody against
rhodopsin kinase (RK) lowered phosphorylation at Ser
,
Ser
, and Ser
, without changing the ratio
between phosphorylation sites. In contrast, no effect of protein kinase
C was detected after stimulation (by a phorbol ester), inhibition (with
H7), or reconstitution of protein kinase C with purified ROS membranes.
The stoichiometry and the ratio between different phosphorylation sites
in purified Rho were also reproduced using RK, purified to apparent
homogeneity from ROS or from an insect cell expression system. Thus, we
conclude that light-dependent phosphorylation of Rho is mediated
primarily by RK. Depalmitoylation of Rho at Cys
and
Cys
altered the conformation of the C terminus of Rho, as
observed by phosphorylation by casein kinase I, but did not affect
phosphorylation by RK. The sites of phosphorylation were influenced,
however, by the presence of four conserved amino acids at the C
terminus of Rho. The accumulation of phosphorylated Ser
observed in vivo could result from slower
dephosphorylation of this site as compared with dephosphorylation of
Ser
and Ser
. These data provide a molecular
mechanism for the site-specific phosphorylation of Rho observed in
vivo.
G protein-coupled receptors, a large family of topologically
homologous proteins, share several characteristics that include seven
transmembrane -helical segments and two domains, extra- and
intracellular. Intracellular loops are involved in the interaction with
G proteins, G protein-coupled receptor kinases, and arrestins, while
extracellular fragments are important for protein folding and, in some
cases, for the binding of large polypeptide hormones or calcium
(reviewed by Baldwin(1994)). In addition, most of G protein-coupled
receptors are co- and/or post-translationally modified by glycosylation
at the N terminus and by palmitoylation and phosphorylation on the
cytoplasmic surface. Among this group of proteins, Rho possesses a
special status because of its high expression in retinal rod
photoreceptors and strict cellular compartmentalization to ROS, (
)the precision in its activation by light, and its rapid
physiological response (Hargrave and McDowell, 1992).
In rod photoreceptor cells, during transition from active Rho* to inactive opsin, the receptor assumes three relatively stable conformations: Meta I, II, and III (Baumann and Reinheimer, 1973). Meta II binds to and activates hundreds of photoreceptor-specific G proteins (transducins), thus initiating the signal-amplifying cascade of reactions (Hargrave et al., 1993). This activated state of Rho, along with Meta I, serve as substrates for RK (Paulsen and Bentrop, 1983; Pulvermüller et al., 1993), a major protein kinase in ROS. The dissociation of RK from Rho* is followed by the binding of p44 and/or arrestin, thereby preventing continuous G protein activation (Wilden et al., 1986; Palczewski et al., 1994b).
Studies in vitro have shown that Rho* is
phosphorylated at multiple Ser and Thr residues. Depending on the
experimental conditions, the initial sites of phosphorylation were
identified at Ser and Ser
(McDowell et
al., 1993; Ohguro et al., 1993; Papac et al.,
1993), prior to multiple phosphorylation. The binding of arrestin to
Rho* and the reduction of the photolyzed chromophore,
all-trans-retinal, by membrane-bound retinol dehydrogenase
limit the high stoichiometry of phosphorylation (Ohguro et
al., 1994b). In vivo analysis however, stands in sharp
contrast with in vitro studies, because only
monophosphorylated species were found at Ser
,
Ser
, and Ser
(Ohguro et al.,
1995). Kawamura(1994) and, subsequently, other investigators
(Gorodovikova et al., 1994a, 1994b) proposed that RK is under
a calcium mediated inhibition through recoverin, a retina-specific
calcium-binding protein (Dizhoor et al., 1991; Lambrecht and
Koch, 1991; Polans et al., 1991). PKC was also proposed to
phosphorylate Rho in a light-independent manner in a reconstituted
system (Greene et al., 1995) or in the whole retina when PKC
was hyperactivated by a phorbol ester (Newton and Williams, 1993).
To obtain additional insight into the structural and functional aspects of Rho phosphorylation, we systematically tested whether RK, PKC, or another kinase is responsible for phosphorylation of flash-activated Rho in ROS homogenates and for effects of Rho palmitoylation on the stoichiometry and selectivity of Rho phosphorylation. We also examined the role of the C-terminal four amino acids in the specificity of phosphorylation, the effect of phosphatase on accumulation of phosphorylated species of Rho, and the effect of calcium on Rho phosphorylation/dephosphorylation in vitro and in vivo.
In vitro and in vivo studies reveal that
light-dependent Rho phosphorylation occurs at the C-terminal
Ser, Ser
, and Ser
(Ohguro et al., 1993, 1994b, 1995; McDowell et al., 1993;
Papac et al., 1993). The specificity and extent of
phosphorylation could result from 1) reactions catalyzed by two or more
protein kinases, 2) different states of palmitoylation of Rho at the C
terminus, 3) dynamically altering conformations of Rho* that RK has
phosphorylated at different sites, 4) random catalysis by RK; 5)
selective phosphatase activity, or 6) metabolism of
all-trans-retinal.
Although the extent of
phosphorylation was decreased by the inclusion of a specific anti-RK
antibody (Palczewski et al., 1993) that inhibits secondary
phosphorylation of Rho*, the ratio among initial phosphorylation sites
was not changed (Table 1). Similar results were observed with H7,
a potent inhibitor of PKC (Hidaka et al., 1984) and a weak
inhibitor of RK (Palczewski et al., 1990). Active and inactive
phorbol myristoyl acetate isomers (PMA and 4--PMA) inhibited
phosphorylation of Rho* to a similar degree, most likely by perturbing
the membrane structure, rather than affecting PKC.
Purified Rho
(urea-washed ROS lacking endogenous kinase activity) was phosphorylated
by homogeneous RK, purified from ROS or from an insect cell expression
system (Fig. 1) at all three Ser residues (Table 1), as in
ROS homogenates. Minor variability in the initial sites of
phosphorylation may relate to the presence of Tween 80 in preparations
of RK. Rho purified in dodecyl--maltoside on concanavalin
A-Sepharose (Litman, 1982), was also phosphorylated by homogeneous RK
at these three Ser residues (data not shown). No phosphorylation of Rho
was detected by recombinant PKC (
-isoenzyme) or a constitutively
active form of PKC (Allen et al., 1994), despite high specific
radioactivity of [
-
P]ATP (more than 1000
cpm/pmol) and high enzymatic activities of PKCs toward myelin basic
protein (2 nmol of phosphate transferred/reaction/10 min). These data
show that Rho* in ROS homogenates is phosphorylated at different
C-terminal Ser residues exclusively by RK, but not by PKC or any other
protein kinases.
Figure 1:
Purification of RK from High Five
insect cells. A, SDS-PAGE analysis. Aliquots from each step of
purification (DEAE-cellulose, lane a; heparin-Sepharose, lane b) was analyzed for protein content using SDS-PAGE
(8 µg of total proteins) stained with Coomassie Blue (see
``Experimental Procedures''). Autophosphorylated and
dephosphorylated RK is indicated as P
-RK and RK,
respectively. B, Western blot analysis. Recombinant RK
obtained from heparin-Sepharose was identified on Western blots with a
panel of antibodies (Palczewski et al., 1993) as exemplified
using GS19 antibody. C, autoradiography. Additionally,
P-labeled RK (autophosphorylated) obtained from
heparin-Sepharose was identified using SDS-PAGE and
autoradiography.
Figure 2:
Analysis of the phosphorylation sites on
depalmitoylated or dipalmitoylated Rho. A, separation of the
depalmitoylated, monopalmitoylated, and dipalmitoylated C-terminal
peptide of Rho (Asn-Ala
) on a
pH-stable, C8 HPLC column (see ``Experimental Procedures''). a represents the absorption profile; b-d represent the
P profile for the following samples. b, the doubly palmitoylated tryptic peptide obtained from
P-labeled, carboxymethylated, acetylated opsin was eluted
at 60% acetonitrile (fractions 27-30), on a shoulder of the major
absorption peak in fractions 25-26; c, the tryptic
digest was treated with 2-mercaptoethanol (5 mM) for 30 min at
30 °C prior to injection on the column. This procedure led to
partial removal of the palmitoyl group and the elution of the
radioactive peptides in fractions 8-12 (depalmitoylated peptide; open circle), 20-23 (monopalmitoylated; closed
circle), and 25-26 (dipalmitoylated peptide, closed
triangle); d, the tryptic digest was depalmitoylated with
hydroxylamine (1 M) for 30 min at 30 °C prior to injection
on the column (fractions 8-12, depalmitoylated peptide). The
identity of the major phosphorylated component (panel A, b) was determined by a combination of Edman degradation and
laser desorption mass spectrometry as follows: the radioactive peak was
pooled, dried, and subjected to hydroxylamine treatment (see panel
A, d) and rechromatographed on the same column. The
radioactive peptide was eluted earlier in the gradient, whereas
contaminated peptides were eluted without change late in the
chromatography. Edman sequence analysis revealed that the radioactive
peptide starts with the sequence: NCMVTTL . . . , which indicates the
predicted tryptic peptide beginning at residue 315 from the C terminus
of acetylated opsin. The average molecular mass determined for this
peptide by laser desorption mass spectrometry was 3916 Da (observed),
in accordance with the calculated mass(3900) for the modified
C-terminal peptide of Rho (Asn
-Ala
). B, analysis of the phosphorylation sites of depalmitoylated
and dipalmitoylated Rho. Depalmitoylated and dipalmitoylated peptides
from partly depalmitoylated Rho were separated as described above,
individually digested with Asp-N endoproteinase, and purified on C18
HPLC (see ``Experimental Procedures''). The Asp-N proteolytic
peptides were separated into di-, mono-, and unphosphorylated peptides
by C18 HPLC. Next, the differently phosphorylated peptides were
subdigested with S. aureus V8 protease, and proteolytic
peptides were separated into
TSQVAPA and
ASTTVSK*TE (where K* is acetylated Lys) using C18 HPLC.
The peptides were monitored by absorbance at 215 nm (panel a)
and their identity confirmed by MS/MS. b,
P-radioactive profile for monophosphorylated peptide
obtained from depalmitoylated Rho (open circle) and
dipalmitoylated Rho (closed squares). Peptide TSQVAPA
phosphorylated at Ser
is marked by an open circle (fractions 25-27), while peptide ASTTVSK*TE phosphorylated
at Ser
or Ser
is marked by a closed
circle (fractions 29-32). c,
P-radioactive profile for diphosphorylated peptides
obtained from depalmitoylated Rho (open circle) and
dipalmitoylated Rho (closed
squares).
To identify phosphorylation sites, a
mixture of partially depalmitoylated Rho (Morrison et al.,
1991) was phosphorylated with purified RK. The distribution of
phosphate on di-, mono-, and depalmitoylated tryptic
Asn-Ala
peptides was analyzed by
further digestion of individual species with endoproteinase Asp-N. Di-,
mono-, and unphosphorylated Asp
-Ala
mixture of peptides were separated to individual phosphorylated
components by HPLC chromatography in the presence of heptafluorobutyric
acid (Ohguro and Palczewski, 1995). Di- and monophosphorylated peptides
were further digested with S. aureus V8. No differences in
phosphorylation for di- and depalmitoylated Rho were observed (Fig. 2B). Similar results were obtained for
monopalmitoylated Rho* (data not shown). Phosphorylation was also not
altered for depalmitoylated Rho after modification of Cys
and Cys
by carboxymethylation or S-sulfenylsulfonylation (negative charged groups), or
carboxamidomethylation (neutral groups). Importantly, neither the rate
of dephosphorylation by catalytic or holo-PrP 2A nor the binding of
arrestin was affected by depalmitoylation (data not shown).
In
contrast, casein kinase I phosphorylated depalmitoylated Rho/opsin
decisively faster than Rho*, while Rho was not phosphorylated (Fig. 3). As determined by mass spectrometric analysis and
proteolytic fragmentations, the phosphorylation was restricted to the
C-terminal region of Rho, which contains a consensus sequence for
casein kinase I, Asp-Asp-Glu-X-Ser (Agostinis et al., 1989). (
)Apparently, depalmitoylation
removed some conformational or accessibility constraints to casein
kinase I.
Figure 3:
Phosphorylation of depalmitoylated Rho but
not palmitoylated Rho by casein kinase I. Urea-washed Rho or
depalmitoylated urea-washed Rho (100 µg) was phosphorylated by
casein kinase I (1 µg) at 30 °C in the dark or under 150-watt
illumination. At each indicated time, an aliquot was precipitated with
10% trichloroacetic acid, and the stoichiometry was determined by P counting (A) or mixed with SDS-PAGE sample
buffer and analyzed by SDS-PAGE. After staining and destaining of the
gels, Rho phosphorylation was analyzed at 60 min by autoradiography (B).
Figure 4:
Analysis of the phosphorylation sites on
truncated Rho. A, C-terminal peptides were obtained from
RK-phosphorylated, Gln-truncated Rho,
Glu
-truncated Rho, and Lys
-truncated Rho by
endoproteinase Asp-N cleavage and conventional C18 HPLC. Each product
was further separated into multiply phosphorylated, monophosphorylated,
and unphosphorylated peptides by a C18 HPLC microbore column using
heptafluorobutyric acid (see ``Experimental Procedures''). Panel A illustrates HPLC of the monophosphorylated peptides
during rechromatographed. B, MS/MS spectra of
monophosphorylated peptides a-e from panel A. a, MS/MS spectrum of
DDEASTTVSKTETSQ, the singly
phosphorylated (M+2H)
ion at m/z 839.8. b and c, MS/MS spectra of singly phosphorylated
(M+2H)
ions at m/z 681.7 from
corresponding to
DDEASTTVSKTE. d and e,
MS/MS spectra of singly phosphorylated (M+2H)
ions at m/z 567.2 corresponding to
DDEASTTVSK. The
phosphorylation site (closed circle above sequence) was
confirmed by fragment b and y ions in each panel. The
ions designated with open circles indicate
-elimination
of phosphate group.
Figure 5:
Dephosphorylation of Rho* in ROS
homogenates. A, the time course of Rho phosphorylation after
flash was performed as described under ``Experimental
Procedures'' in the presence of KF (50 mM), CoCl (0.2 mM), or PrP 2A (620 pmol of released
phosphate/min/phosphatase activity). At 5-, 20-, 40-, and 60-min time
points, the reaction was quenched and the membranes were collected by
centrifugation, washed sequentially with 200 mM KCl and 10
mM Hepes buffer, pH 7.5, and digested with endoproteinase
Asp-N overnight at room temperature. Rho phosphorylation was determined
by
P radioactivity associated with the HPLC-purified
C-terminal peptide. B, percent of monophosphorylated Rho at
different sites, Ser
, Ser
, or Ser
was determined by radioactive profiles of the trypsin digests of
the C-terminal peptides as described under ``Experimental
Procedures.''
Figure 6:
Activation of PrP 2A by recoverin and
Ca. The effects of recoverin and/or Ca
on PrP 2A catalytic subunit and holoenzyme was studied in 60
µl of 50 mM Tris buffer, pH 7.0, containing 50 mM 2-mercaptoethanol, 0.1 mM EDTA, 1 mg/ml bovine serum
albumin, and 0.8 mg/ml
P-labeled opsin). The final
concentrations were as follows: 100 µM Ca
, 16 µM calmodulin, 1 mM (free calcium 50 nM) EGTA, 1 mM MgCl
; 1.37 µM recoverin; 100
nMPrP inhibitor I (Inh-1); 0.4 mg/ml poly-Lys. The reaction
was carried out for 15 and 8 min for holoenzyme and catalytic subunit,
respectively, at 30 °C (maximum dephosphorylation was less than 10%
of total phospho-opsin). Experiments were performed in
triplicate.
No evidence was found for
involvement of PKC in Rho phosphorylation, as an activator or an
inhibitor; addition of purified constitutive active or recombinant
-isoforms of PKCs did not produce any significant effect. In
previous studies, the site(s) of PKC phosphorylation was not
identified; however, it was confined to the C-terminal region of Rho
phosphorylated by RK (Greene et al., 1995). It is worth noting
that this region lacks, even loosely defined, a consensus sequence for
PKC (Kennelly and Krebs, 1991), and that phosphorylation described in
previous reports was performed with Rho at low nanomolar concentrations
(Kelleher and Johnson, 1986; Newton and Williams, 1991), which may
produce highly fragmented forms of membrane vesicles. If the sites of
phosphorylation by PKC are identical to those modified by RK, based on
the amounts of enzymatic activities, the contribution of PKC would be
200-fold less than of RK (Palczewski, 1993; Newton, 1993). It is
unlikely that our preparations of ROS lost PKC, because they retained
soluble proteins such as arrestin, and they were prepared without
buffering calcium, conditions that should promote association of PKC to
ROS membranes. It should be noted that electrophysiological experiments
with intact Gecko ROS to which several different inhibitors of
PKC, including H7, H8, staurosporin, or chelerythine, were introduced,
did not affect electrical light responses. (
)We conclude
that it is not necessary to implicate any protein kinase other than RK
in the phosphorylation of Rho.
Arguments against these suppositions are
based on evidence that dephosphorylation of Rho is a slow process in vitro and in vivo (minutes) (Palczewski et
al., 1989a, 1989b; Fowles et al., 1989; King et
al., 1994; Kühn, 1974; Ohguro et al.,
1995). Prerequisites to dephosphorylation include binding of
p44/arrestin (Hofmann et al., 1992) and reduction of
photolyzed chromophore by retinol dehydrogenase (Palczewski et
al., 1994a) to remove arrestins (Palczewski et al.,
1989b). In addition, slow decay of Meta II (t= 16 s in frog at 30 °C, and even slower
release of all-trans-retinal) (Baumann and Reinheimer, 1973)
and accumulation of all-trans-retinal in the first minutes
following illumination (Zimmerman, 1974), suggest that
dephosphorylation occurs on a time scale of minutes, rather than
seconds. Finally, the minimum estimated enzymatic activity of RK in
vivo would predict the introduction of 3 P
/Rho* during
3 s after a bleach that generated 0.4% Rho*, well within the detection
limit of our methods. A single phosphorylation is sufficient for the
quenching of phototransduction (Bennett and Sitaramayya, 1988), and a
prolonged, low intensity illumination (3 h) did not produce multiply
phosphorylated species at detectable levels in the mouse retina. The
multiple phosphorylation of Rho* could be an artifact of biochemical
procedures that uses very dilute ROS preparations with compromised
membrane structures, intense bleaches without proper recycling of the
visual chromophore, and long phosphorylation time courses.
At
intense bleaches in vivo, less than half of the Rho* is
phosphorylated (Ohguro et al., 1995), suggesting that some
steric hindrance, such as the binding of arrestins, prevents
phosphorylation of each Rho molecule. This would be consistent with a
report that only 25% of Rho* is accessible to transducin (Fung,
1983). This mechanism could limit phosphorylation of Rho* at high
levels of illumination but allow each Rho* to be phosphorylated, at low
levels of illumination; however, as ROS membranes are fragmented and
arrestins are removed or diluted in vitro, this accessibility
could be enhanced.
Analysis of phosphorylation of
C-terminally truncated forms of Rho (Ohguro et al., 1993,
1994b, 1995) or corresponding peptides (Fig. 7) (Pullen and
Akhtar, 1994) revealed that the four terminal amino acids (VAPA) are
critical in the selectivity of phosphorylation. Multiple
phosphorylation appears to be sequential (as described previously by
Ohguro et al.(1993)) with a neighboring hydroxyl-containing
amino acid being next to the primary site of phosphorylation.
Interestingly, Sung et al.(1994) discovered that truncation of
Rho at Gln, as found in retinitis pigmentosa, prevented
phosphorylation at Ser
(Fig. 7), and produced 15%
longer time-to-peak in the electrophysiological responses. The complete
truncation of the C terminus at Gly
leads to more
prolonged responses, suggesting that Rho phosphorylation is needed for
normal inactivation of the phototransduction cascade (Chen et
al., 1995b). Phosphorylation at different sites may have important
physiological consequences in serving different inactivation pathways
of Rho*.
Figure 7:
Preferential phosphorylation of the
C-terminal region of Rho. Sites of phosphorylation of Rho were
identified by Ohguro et al. (1993, 1994b, 1995, and present
work); sites of phosphorylation of the peptides were taken from Pullen
and Akhtar(1994). The symbols represent observed pattern of
phosphorylation of the underlined residues for
monophosphorylated and multiply phosphorylated species. ↕ and
&cjs3707; represent frequent mutations or truncations at the C terminus
that are associated with retinitis pigmentosa, respectively (Macke et al., 1995).
These studies emphasize the importance of correlating in vitro and in vivo studies. From a combination of both approaches, we interpret Rho phosphorylation as a marker of conformational change in Rho* and RK, and we suggest that heterogeneity of phosphorylation may serve many different physiological functions during light responses in rod photoreceptors.