From the
Upon illumination, rhodopsin kinase (RK) phosphorylates the
visual pigment rhodopsin, which is thought to partially terminate the
biochemical events that follow photon absorption. RK enzymology was
explored by mutagenesis of the residues Ser
Rhodopsin kinase (RK)
RK is
activated by Rho* and phosphorylates multiple sites on the C terminus
of Rho*. Neither opsin nor Rho is a substrate for RK, although the
sites of phosphorylation on Rho are accessible. Thus, the binding of RK
to the cytosolic surface of Rho* (but not Rho) is required for the
subsequent phosphorylation of the C terminus of Rho* (Fowles et
al., 1988; Palczewski et al., 1991).
Analysis of the
primary sequence reveals that the kinase catalytic domain is located in
the midregion of the RK sequence (Lorenz et al., 1991), which
has an overall structure related to the
The goal of the present study was to explore
the mechanism of RK regulation by autophosphorylation. Several
mutations in the major autophosphorylation sites allowed us to identify
changes produced by phosphorylation of these residues with regard to
the enzymatic mechanism of RK action.
Peptide C (DDEASTTVSKTETSQVARRR) and an acidic
peptide (RRRDDEEEEESAAA) were synthesized using a fully automated batch
instrument (430A, Applied Biosystems, Inc., Foster City, CA) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on 0.1 mmol scale.
After deprotection, the peptide was purified by preparative reverse
phase HPLC (>95% pure).
In a centrifugation assay (Buczyet
al., 1991), dephosphorylated WT RK bound to phosphorylated Rho*.
In our experimental conditions,
The K
We especially thank Dr. Robert J. Lefkowitz (Duke
University) in whose laboratory numerous experiments reported here were
performed. We thank J. P. Van Hooser, H. Kendall, C. Brown, and G.
Irons for excellent technical assistance. We thank Dr. Janina
Buczyfor help with RK assays during the course of this work,
Dr. Ann A. DePaoli-Roach for the generous gift of PrP 2A, and Dr. Ann
Milam for comments on the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Thr
(major autophosphorylation sites), and Lys
(a
distal residue). We found the following to be true. (i) Double
mutations at residues Ser
and Thr
to Ala or
Asp decrease autophosphorylation to substoichiometrical levels, while
single mutations at either residue independently reduce
autophosphorylation by half. (ii) Phosphorylation of residue
Ser
influences the affinity of RK for heparin-Sepharose
only moderately, whereas Thr
and Lys
are
important for this interaction. RK K491A does not phosphorylate acidic
peptides, suggesting that this residue participates in substrate
binding. (iii) Mutations in the autophosphorylation region affect the K
for ATP, suggesting that this region is
involved in binding of ATP to the catalytic site. (iv) RK mutants S488A
or S488D and RK S488A and T489A have an increased ability to
phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation
region change the initial site of phosphorylation on photolyzed
rhodopsin (Rho*), implying that this region may regulate selectivity of
the site of phosphorylation.
(
)specifically
phosphorylates photoactivated rhodopsin (Rho*) in retinal photoreceptor
cells. The phosphorylation of Rho* leads to inhibition of G protein
binding (G
, transducin) through increased binding of the
regulatory protein arrestin (Wilden et al., 1986). Bound
arrestin uncouples G
from further activation by Rho* and
thus terminates the activation pathway of phototransduction (Wilden et al., 1986; Bennett and Sitaramayya, 1988). Arrestin binds
to Rho* only when one or two of the several sites at the C terminus of
Rho* have been phosphorylated (Ohguro et al., 1994). RK is
therefore an important component regulating the level of activation of
the G
pool on a disc membrane (reviewed by Lagnado and
Baylor(1992)). Rho, G
, RK, and arrestin are functional
archetypes for related G protein-coupled transduction systems that
involve different types of receptors, G proteins, G protein-coupled
receptor kinases (GRKs), and arrestin isoforms (reviewed by Inglese et al.(1993) and Palczewski and Benovic(1991)).
-adrenergic receptor
kinase (Inglese et al., 1993). The N terminus of RK contains a
domain implicated in the recognition of Rho* (Palczewski et al., 1993). RK is post-translationally processed at the C terminus by
farnesylation, proteolysis, and
-carboxyl methylation (Inglese et al., 1992a), modifications that appear to be important for
the light-dependent association of the kinase with Rho* (Inglese et
al., 1992b). Additionally, RK is modified by multiple
intramolecular phosphorylations at the C and N termini of the enzyme
(Lee et al., 1982; Kelleher and Johnson, 1990; Palczewski et al., 1992). Recently, the sites of autophosphorylation in
the primary structure of RK were identified as Ser
(minor)
near the N terminus, and Ser
and Thr
(major) near the C terminus (Palczewski et al., 1992).
Autophosphorylation changes the affinity of RK for different forms of
Rho*, with the weakest interaction occurring between phosphorylated RK
and phosphorylated Rho*. It was proposed that autophosphorylation of RK
facilitates its dissociation from Rho* (Buczyet al., 1991) by changes in the rate of dissociation (k
) from phosphorylated Rho* (Pulvermüller et al., 1993).
Proteins and Reagents
Several forms of Rho were
prepared from ROS according to published procedures as follows: urea
washed Rho (Shichi and Somers, 1978); phosphorylated Rho by
regeneration of phosphorylated opsin with 11-cis-retinal
(Palczewski et al., 1989; Hofmann et al., 1992); and
G-Rho lacking the C-terminal 19 amino acids by digestion
with endoproteinase Asp-N (Palczewski et al., 1991). The
catalytic subunit of protein phosphatase 2A (PrP 2A) was purified from
rabbit skeletal muscle (Shenolikar and Ingebritsen, 1984) or was a
generous gift from Ann A. DePaoli-Roach (Indiana University,
Indianapolis). GRKs (2, 3, and 5) were prepared from Sf9 cells using a
baculovirus expression system as recently described (Söhlemann et al., 1993; Premont et al., 1994). G
was purified from bovine brain using the method of Casey et
al.(1989).
Mutagenesis
Site mutations were prepared using
standard polymerase chain reaction cassette mutagenesis procedure
(Innis and Gelfand, 1990) employing the NcoI and EcoRI sites of RK in the pBlueScript II vector (Stratagene).
Following verification of the sequence by dideoxynucleotide sequencing,
the RK coding region was subcloned into the eukaryotic expression
vector pCMV5 (Andersson et al., 1989).
Purification of RK on a Heparin-Sepharose
Column
Bovine RK was purified on a heparin-Sepharose column as
described elsewhere (Palczewski, 1993). Bovine RK or mutants were
expressed in COS-7 cells transfected with the appropriate plasmid
constructs using the diethylaminoethyl dextran method (Inglese et
al., 1992a, 1992b). RK or mutants from COS cells were purified as
follows. Cells from two 150-mm dishes were harvested by scraping with
75 mM Tris/HCl buffer, pH 7.5, containing 1 mM MgCl, 1 mM dithiothreitol, 20% adonitol, 10
µg/ml leupeptin, and 20 µg/ml aprotinin. The cell suspension
was diluted 1:1 with 10 mM BTP buffer, pH 7.5, containing 0.4%
Tween 80, homogenized (Tenbroeck Tissue Grinder, Wheaton), and
centrifuged at 125,000
g for 7 min (Beckman
Optima
TLX). The supernatant containing RK was treated
with PrP 2A (0.2 µg) or ATP (0.4 mM) for 1 h at room
temperature and loaded onto a heparin-Sepharose column (5
50
mm, Pharmacia Biotech Inc.) that had been equilibrated with 10 mM BTP buffer, pH 7.5, containing 0.3% Tween 80. RK was eluted with a
NaCl gradient (0-750 mM) in BTP buffer, pH 7.5,
containing 0.3% Tween 80 at a flow rate of 30 ml/h using a quaternary
HPLC pump system (Hewlett-Packard, model 1050). One-ml fractions were
collected, and a protein elution profile was monitored by absorption at
280 nm. Aliquots from all fractions were tested for the presence of RK
activity.
Assay for RK Activity
RK activity was measured
using [-
P]ATP (100-500 cpm/pmol,
DuPont NEN) and urea-washed ROS membranes in 20 mM BTP buffer,
pH 7.5, at 30 °C (Palczewski, 1993). RK concentration was
determined by direct ELISA using recently described anti-RK antibodies
(Palczewski et al., 1993) and a known amount of purified RK as
a standard.
Identification of the Initial Phosphorylation Sites in
Rhodopsin Phosphorylated by GRKs
The initial sites of
phosphorylation (monophosphorylated) species of Rho* by mutants of RK
and GRKs were evaluated using urea-washed ROS as described for WT RK
(Ohguro et al., 1994).
Mutagenesis, Expression, and Purification of
RK
Mutations in the RK autophosphorylation domain were as
follows. 1) Ala was substituted for Ser and Thr
to confirm the sites of autophosphorylation in RK and to
determine the contribution of autophosphorylation to RK activity. In
addition, we examined the mutation of Lys
, distal to the
RK autophosphorylation region (see Fig. 1), to determine the
influence of this residue on the enzymatic properties of RK. Ala has
been used in scanning mutagenesis techniques to remove side chain atoms
beyond the
-carbon while allowing minimum perturbations in
secondary structure and tertiary conformation (Cunningham and Wells,
1989; Gibbs and Zoller, 1991). 2) Asp was employed to mimic the
phosphorylated state of the kinase (see Kurland et al. (1992)).
Figure 1:
Illustration of RK mutants and GRK
family alignment of autophosphorylation region. A, mutations
of the autophosphorylation sites examined in this study. B,
alignment of the autophosphorylation regions of GRKs 1-6. The
dendrogram on the left displays the relative percent homology
between the kinases (based on the catalytic domains); this homology is
mirrored in the autophosphorylation region of the respective kinases.
The boldfaceletters are identical residues among all
GRKs, and the underline indicates experimentally determined
autophosphorylation sites (Palczewski et al., 1992; Premont et al., 1994). The GRK designation is followed by the common
name.
RK expressed in COS cells was purified on
DEAE-Sepharose (Palczewski et al., 1988), heparin-Sepharose
(Buczyet al., 1991), or a combination of both and
yielded 5-25 µg of RK (from two 150-mm plates), which
constituted
10-30% of the total protein obtained from these
steps. The enzyme was identified by immunoblotting and ELISA using an
RK-specific antibody (Palczewski et al., 1993). The RK
activity correlated well with the relative amount of RK protein as
determined from the ELISA and was specifically inhibited by the RK
antibody (data not shown).
Effects of Mutations at the Autophosphorylation Region of
RK on Its Interaction with Heparin-Sepharose
Autophosphorylation
of RK significantly changed its affinity for heparin (Fig. 2).
Using a heparin-Sepharose column, the fully autophosphorylated form of
RK was eluted at 90 mM NaCl (fractions 6-7),
partially phosphorylated RK was eluted at
200 mM NaCl
(fractions 8-9), and dephosphorylated RK was eluted at
290
mM NaCl (fractions 10-12) (Fig. 2A)
(Buczyet al., 1991). RK extracts from either ROS,
retina (Buczyet al., 1991), or COS cells (data not
shown) have similar amounts of unphosphorylated and phosphorylated
forms of the enzyme. Thus, RK extracts were dephosphorylated with PrP
2A, and the phosphatase, which binds weakly to heparin (Fowles et
al., 1989), was removed during the heparin-Sepharose
chromatography step. Employing these steps, dephosphorylated RK was
separated from phosphorylated forms before experiments related to the
autophosphorylation reaction were performed. This procedure also
allowed us to eliminate all interfering phosphorylated proteins and
protein kinases present in COS cell extracts. In fact, no protein
kinase activity was detected with generic substrates such as casein,
histone H2, and Kemptide, a peptide substrate for cyclic-AMP-dependent
protein kinase (data not shown). As shown in Fig. 2A, an
extract of COS cells that had been transfected with a carrier DNA did
not yield any significant phosphorylation of Rho*. All mutants were
stable for at least 1 h at 30 °C or 2 days when stored on ice.
Figure 2:
Chromatography of RK on a
heparin-Sepharose column. The extract from COS cell transfected with RK
plasmid was loaded on a heparin-Sepharose column (5 50 mm,
Pharmacia), and RK was eluted with a NaCl gradient (0-750
mM) in BTP buffer, pH 7.5, containing 0.3% Tween 80. Aliquots
from all fractions were tested for the presence of RK activity. A, RK extract that was preincubated with ATP (opencircles), RK extract that was preincubated with PrP 2A (closedcircles), or COS extract from cells
transfected with empty plasmid (opentriangles); B, RK S488A pretreated with ATP (opencircles) or PrP 2A (closedcircles); C, RK S488D,T489D (closedcircles) and RK
S488D (opentriangles) pretreated with PrP 2A; D, RK T489A treated with ATP (opencircles)
or PrP 2A (closedcircles); E, RK
S488A,T489A pretreated with ATP (opencircles) or PrP
2A (closedcircles); F, RK K491A pretreated
with ATP (opencircles) or PrP 2A (closedcircles). Dottedlines in all panels corresponded to elution positions of phosphorylated and
dephosphorylated WT RK.
The elution profiles of the autophosphorylated or dephosphorylated
forms of RK S488A from heparin-Sepharose were shifted by one fraction
toward higher salt as compared with the native enzyme.
Dephosphorylation of RK S488A by PrP 2A, using conditions that
dephosphorylated WT RK, led to only partial dephosphorylation (Fig. 2B), suggesting that RK S488A was a poor substrate
for PrP 2A compared with WT RK. Dephosphorylated RK S488D was eluted
from the heparin-Sepharose column similarly to WT RK, while the double
mutant RK S488D T489D had lower affinity for heparin (Fig. 2C). The elution profile of RK T489A from
heparin-Sepharose was significantly different (Fig. 2D).
The phosphorylated and dephosphorylated forms of this mutant were
eluted close together in a position typical for dephosphorylated WT RK.
RK S488A and T489A (Fig. 2E) was insensitive to ATP or
PrP 2A treatment. These results suggest that phosphorylation of residue
Ser influences the affinity of RK for heparin-Sepharose
only moderately, whereas Thr
residue was more critical
for this interaction. Because RK K491A had lower affinity for heparin (Fig. 2F), residue Lys
may comprise part
of the RK heparin binding site. This effect was reflected in the lower
IC
of heparin for WT RK (
0.3 mM) versus RK K491A (>10 mM) (data not shown).
Effects of Mutations at the Autophosphorylation Region of
RK on the Stoichiometry and Rate of
Autophosphorylation
Autophosphorylation of WT RK and RK K491A
(data not shown) had similar stoichiometries and rates, while single
mutations at residues 488 or 489 to Ala or Asp yielded a kinase with
autophosphorylation stoichiometries reduced by half, as shown, for
example, for RK S488D (Fig. 3). Double mutations at residues
Ser and Thr
to Ala or Asp almost eliminated
autophosphorylation (Fig. 3). Each of these residues, Ser
or Thr
, could be phosphorylated independent of the
other, indicating that there was no hierarchical phosphorylation. The
RK/GRK2 chimera did not undergo phosphorylation (data not shown). The
overall stoichiometry of autophosphorylation was lower than
anticipated, which may be explained by a fraction of the RK being
denatured or the RK concentration being consistently overestimated.
Figure 3:
Time course and stoichiometry of RK
autophosphorylation. A, autoradiogram of RK and RK S488D,T489D
autophosphorylation. B, time course and stoichiometry of RK
autophosphorylation. From the top: WT RK (closedcircles); RK S488D (opentriangles);
and RK S488D,T489D (opencircles). Known amounts of
purified, dephosphorylated RK (1-5 µg) were incubated with 10
µM [-
P]ATP (3,000-6,000
cpm/pmol) in BTP buffer containing 1 mM MgCl
, 100
mM NaCl at 30 °C. At selected time points, aliquots of the
reaction mixture were removed, mixed with an equal volume of 10% SDS,
and analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970).
Radioactive bands were visualized by autoradiography, cut out,
dissolved in 30% H
O
, and mixed with
scintillation counting solution. Radioactivity was measured with a
scintillation counter.
Kinetic Parameters of RK Mutants
The Kfor Rho* (
2-4
µM) was similar for WT RK and all mutants with changes in
the autophosphorylation region (). RK S488A and RK
S488A,T489A had a 20-fold higher K
for
ATP, while RK S488D and RK S488D,T489D and the chimeric mutant had a K
similar to WT RK, suggesting that Asp
can be utilized for the hydrogen bond network with the phosphates of
ATP. While Thr
mutants had no effect on K
for ATP, double mutant RK S488A,T489A
had the highest V
, suggesting that Ser
and Thr
residues may also influence the ATP
phosphotransfer.
70% of RK is associated with
membranes, while we did not detect a stable complex between RK
S488D,T489D and P-Rho* (data not shown), supporting an idea that
autophosphorylation may influence the rate of RK dissociation from
P-Rho*.
Phosphorylation of a Model Peptide
Following
endopeptidase Asp-N cleavage, all phosphorylation sites in the Rho
molecule are removed; however, the resulting truncated Rho*
(G-Rho*) can still bind RK and stimulate phosphorylation
of an exogenous acidic peptide (Palczewski et al., 1991).
Although the mechanism of peptide phosphorylation by RK is probably
different from that involving receptor phosphorylation as reflected by
large differences in V
/K
(Palczewski et al., 1989), the assay of peptide
phosphorylation in the presence of G
-Rho* might reveal
differences between mutated RKs (). Phosphorylation of the
model peptide by the majority of RK mutants occurs at similar rates (4
÷ 5 nmol of P
/(min
mg of RK)) and was not
appreciably enhanced by G-Rho*, in contrast to the 4-fold enhancement
seen with WT RK. RK K491A, which interacted poorly with heparin, did
not phosphorylate acidic peptides, suggesting that these residues
participate in binding or catalysis of peptide phosphorylation. These
results indicated that the ability of Rho* to stimulate peptide
phosphorylation is altered when a mutation at the autophosphorylation
domain is present.
Ser
RK binds to Rho*, presumably to the metarhodopsin I
and II conformers (Pulvermüller et al., 1993; Paulsen and
Bentrop, 1984). However, several lines of evidence suggest that the
C-terminal region of Rho, the site of multiple phosphorylations
catalyzed by RK, is already exposed and available for interaction with
enzymes before Rho assumes the activated Rho* conformation (discussed
in Palczewski et al.(1991)). Here we found that a mutant with
SerMutants of RK Are Active in the
Dark
converted to Ala has 7 ± 1% of activity toward
Rho (in dark) compared with phosphorylation of Rho* (in light). Mutant
RK S488D and double mutant RK S488A,T489A had the capability to
phosphorylate Rho in the dark 5 ± 1 and 11 ± 1%,
respectively. WT RK and other mutants were inactive in phosphorylating
Rho in the dark. Also, RK S488A, RK S488D, and RK S488A,T489A had
higher affinity for Rho than WT RK. In the centrifugation assay,
20-30% of the RK mutants were associated with the membranes,
compared with less than 5% for the WT RK (data not shown).
Sites of Rho* Phosphorylation by RK Mutants and
GRKs
The initial sites of Rho* phosphorylation were studied
employing conditions recently described (Ohguro et al., 1994)
with some modifications. Urea-washed bovine ROS were phosphorylated by
RK or its mutants in conditions that favored a phosphorylation
stoichiometry of 1 mol of P/mol of Rho*. The C-terminal
19-residue peptide of Rho, containing all sites phosphorylated by RK,
was analyzed by proteolytic subdigestion of
P-labeled
peptides on HPLC. Standard phosphopeptide sequences were verified by
mass spectroscopy as described recently (Ohguro et al., 1994).
Similar to the phosphorylation by RK isolated from ROS, the
COS-expressed WT enzyme phosphorylated mainly Ser
and to
a lesser extent Ser
on Rho* (Fig. 4). Mutations of
RK Ser
or Thr
to Ala made these mutants
almost completely selective for Rho* Ser
. Mutation of
Ser
to Asp changed this specificity back to that of WT
RK, as the primary sites of phosphorylation were observed to be at Rho*
Ser
and Rho* Ser
. To explore this important
observation further, three additional GRKs and a RK/GRK2 chimera were
tested in this assay. Initial sites of phosphorylation by GRK2, GRK3,
and RK/GRK2, which have the highly charged amino acid sequence DEED in
the autophosphorylation region (Fig. 1), were different from that
obtained for RK S488D,T489D mutants, suggesting that other regions than
the autophosphorylation domain also are involved in the substrate
specificity. G
subunits, which are known to
accelerate receptor phosphorylation by GRK2 and -3 (reviewed by Inglese et al. (1993)), had no effect on the initial sites of
phosphorylation, suggesting that the action of these subunits is to
bring these GRKs to membranes rather than to change the mechanism of
phosphorylation. GRK5, which has similar sequence to RK and undergoes
autophosphorylation in sites equivalent to these RK sites (Premont et al., 1994), yielded similar phosphorylation to WT RK. These
results suggest that the autophosphorylation region may influence the
stability of the activated form of RK, thereby leading to modification
of the receptor at specific sites.
Figure 4:
Initial sites of phosphorylation of Rho*
by WT RK, RK mutants, and other GRKs. The bargraph shows the relative degree of phosphorylation for individual Ser
residues in the C terminus of Rho* by each kinase studied for
monophospho-Rho*. The C-terminal sequence of Rho* in which these
initial sites of phosphorylation occur is shown below. The differences
between experiments were below 5%.
Autophosphorylation of RK
exhibits clearly distinct features from that of GRK5 (Premont et
al., 1994; Kunapuli et al., 1994). (i) In contrast to
GRK5 the autophosphorylation of RK is not influenced by ROS membranes
or Rho* (Kelleher and Johnson, 1990). (ii) Differently
autophosphorylated RK and autophosphorylation mutants display strong
effects on the affinity to heparin, whereas a GRK5 mutant was not
affected by heparin (Kunapuli et al., 1994). Interestingly,
heparin only modestly inhibits RK (K0.3 mM) and is a very potent inhibitor of GRK5 (150
nM). Thus, it is conceivable that both enzymes utilize
different mechanisms of heparin binding. (iii) Mutants in the
autophosphorylation domain have similar kinetic parameters for RK,
while GRK5 mutants phosphorylate poorly agonist-occupied receptors,
possibly due to a decrease in K
for ATP.
The functional consequences of autophosphorylation of GRK5 and GRK6
remain to be established.
Table: Effects of mutations in the autophosphorylation
region of RK on the kinetics of Rho* and peptide phosphorylation
for Rho* was determined by varying the
Rho* concentration from 0.2 to 20 µM at an ATP
concentration of 100 µM. The reaction time (with
illumination) was 10 min. The K
for ATP
and V
were determined by varying the ATP
concentration from 0.2 to 20 times the estimated K
at a Rho* concentration of 20 µM. The reaction
time (with illumination) was 10 min. The amount of RK used in the
reactions was chosen so that no more than 5% of Rho* was phosphorylated
and less than 1% of ATP was used during this assay. The K
values were calculated by
Lineweaver-Burk analysis as described by Segel (1975). Rho
concentration was determined spectrophotometrically at 498 nm assuming
a molar absorption coefficient of 40,600 (Wald and Brown, 1953) and a
molecular mass of 40 kDa. ATP concentration was determined
spectrophotometrically.
, G-protein of the rod cell transducin; Rho*, photolyzed
rhodopsin; WT RK, wild-type rhodopsin kinase; ROS, rod outer
segment(s); GRK, G protein-coupled receptor kinase; PrP, protein
phosphatase; HPLC, high pressure liquid chromatography; ELISA,
enzyme-linked immunosorbent assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.