Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase
II. ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN
VIVO*
Fumio
Hayashi
§,
Isao
Matsuura¶,
Shu
Kachi
,
Tomoko
Maeda,
Maki
Yamamoto,
Yuka
Fujii,
Han
Liu,
Matsuyo
Yamazaki¶,
Jiro
Usukura, and
Akio
Yamazaki¶**
From the Department of Biology, Faculty of Science,
Kobe University, Kobe 657, Japan, the Departments of
¶ Ophthalmology and ** Pharmacology, the Kresge Eye
Institute, Wayne State University, School of Medicine, Detroit,
Michigan 48201, and the Department of Anatomy, School of
Medicine, Nagoya University, Nagoya 466, Japan
Received for publication, January 31, 2000, and in revised form, June 27, 2000
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ABSTRACT |
Retinal cGMP phosphodiesterase (PDE)
is regulated by P
, the regulatory subunit of PDE, and GTP/T
, the
GTP-bound subunit of transducin. In the accompanying paper
(Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki,
M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known
Ps contain a specific phosphorylation motif for
cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog
rod photoreceptor outer segments (ROS) isolated by a new method, we
show that Cdk5 is involved in light-dependent P
phosphorylation in vivo. Under dark conditions only
negligible amounts of P were phosphorylated. However, under
illumination that bleached less than 0.3% of the rhodopsin, ~4% of
the total P was phosphorylated in less than 10 s. P
dephosphorylation occurred in less than 1 s after the light was
turned off. Analysis of the phosphorylated amino acid, inhibition of
P phosphorylation by Cdk inhibitors in vivo and in
vitro, and two-dimensional peptide map analysis of P
phosphorylated in vivo and in vitro indicate
that Cdk5 phosphorylates a P threonine in the same manner in
vivo and in vitro. These observations, together with
immunological data showing the presence of Cdk5 in ROS, suggest that
Cdk5 is involved in light-dependent P phosphorylation in
ROS and that the phosphorylation is significant and reversible. In an
homogenate of frog ROS, PDE activated by light/guanosine
5'-O-(3-thiotriphosphate) (GTPS) was inhibited by P
alone, but not by P complexed with GDP/T or GTPS/T. Under
these conditions, P phosphorylated by Cdk5 inhibited the light/GTPS-activated PDE even in the presence of GTPS/T. These observations suggest that phosphorylated P interacts with and inhibits light/GTPS-activated PDE, but does not interact with GTPS/T in the homogenate. Together, our results strongly
suggest that after activation of PDE by light/GTP, P is
phosphorylated by Cdk5 and the phosphorylated P inhibits
GTP/T-activated PDE, even in the presence of GTP/T in
ROS.
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INTRODUCTION |
The hydrolysis of cGMP by
PDE1 in vertebrate ROS is
directly involved in visual signal transduction (1, 2). The inactive PDE is composed of P
, catalytic subunits, and two Ps,
regulatory subunits (3-6). In amphibian ROS, PDE is regulated
similarly to that in mammalian ROS (7, 8): PDE catalytic activity is
controlled by P and GTP/T. In frog ROS membranes, bleached rhodopsin stimulates GTP/GDP exchange on T (9), and the GTP/T formed is released from membrane-bound T (9, 10). The free GTP/T interacts with P, and P complexed with GTP/T
is released from P/membranes (10-12). PDE is thereby activated.
The release of the P complex is detected even in an isotonic buffer
containing Mg2+, and P complexed with GTPS/T can
be isolated using sequential column chromatography (10). During PDE
activation, P-less P binds tightly to
membranes.2 In the recovery
processes of frog ROS, after GTP hydrolysis by T, P remains in
the complex with GDP/T (10). When the GDP/T/P complex
interacts with membrane-bound T, P is released from the
complex and reassociates with P, resulting in the turnoff of PDE
(10). The P complex with GDP/T is very tight, and the GDP/T/P complex can be isolated by sequential column
chromatography (10).
It has been suggested that P phosphorylation is involved in the PDE
regulatory mechanism. P is phosphorylated by PI-stimulated kinase
(13), PKC (14), PKA (15), and P kinase (16, 17). In the
PI-dependent P phosphorylation (13), threonine 35 or serine 40 in P may be phosphorylated. PKC (14) and PKA (15) phosphorylates threonine 35 in P. The phosphorylated P has a higher inhibitory activity against GTP/T-activated PDE than that of
nonphosphorylated P. The important point in the P
phosphorylations by these protein kinases is that the P
phosphorylations appear not to occur when P binds to GTPS/T.
We have shown that P phosphorylations by PI-stimulated kinase (13)
and PKA (15) were inhibited by GTPS/T. In the case of P
phosphorylation by PKA (15), the inhibition is due to the
unavailability of the phosphorylation site in P, because a P
region, including threonine 35, is involved in its interaction with
GTP/T, and the region is masked when P is complexed with
GTP/T. The same kind of inhibition was also observed in the
ADP-ribosylation of P, because the P ADP-ribosylation site
(arginines 33 or 36) is masked when P is complexed with GTP/T
(18, 19). It is very likely that the phosphorylation of threonine 35 in
P occurs when P is complexed with P. We have shown that
arginine 33 or 36 in P is ADP-ribosylated when P is complexed
with P (18).
In contrast to these P phosphorylations, P phosphorylation by
P kinase appears to be light-dependent and thus can be
brought into agreement with in the current model of phototransduction (16, 17). In the phosphorylation, P complexed with GTP/T is the
best substrate for P kinase, and the P phosphorylation is
dependent upon GTP in ROS membranes. These results indicate that the
P phosphorylation occurs after PDE activation. Threonine 22 in P
is phosphorylated. The phosphorylated P loses its affinity to
GTP/T, but gains a 10~15 times higher ability to inhibit PDE activity than that of nonphosphorylated P. Thus, the phosphorylated P more effectively inhibits GTP/T-activated PDE than
nonphosphorylated P, and the inhibition occurs even in the presence
of GTP/T. These observations imply that 1) the P phosphorylation
is probably involved in the recovery phase of phototransduction to the
dark state, 2) after activation of PDE, GTP/T may interact with
another effector and the interaction may be associated with mechanisms for the recovery of phototransduction, and 3) the lifetime of GTP/T-activated PDE can be regulated by the P phosphorylation when the P phosphorylation functions.
In this series of experiments, we showed that Cdk5 phosphorylates P
complexed with GTP/T in vitro (in the accompanying paper (20)) and in vivo (in this paper). In the accompanying paper (20), we have shown that P preserves an amino acid sequence required
for the phosphorylation by Cdk5 and that the P kinase is Cdk5
complexed with p35, a Cdk5 activator. We have also demonstrated that
recombinant Cdk5/p35 phosphorylates P in a
GTPS-dependent manner in ROS membranes, suggesting that
Cdk5 is involved in the phosphorylation of P complexed with
GTP/T. In the present study, we link these observations with
light-dependent P phosphorylation in vivo
(21). Using frog photoreceptor outer segments isolated by a new method,
we show that Cdk5 is involved in the light-dependent P
phosphorylation in vivo, that the P phosphorylation is
significant and reversible, and that the P phosphorylation and
dephosphorylation are rapid enough to be involved in the recovery phase
of phototransduction. Moreover, in an homogenate of photoreceptor outer
segments, the phosphorylated P inhibits light/GTPS-activated PDE,
even in the presence of GTPS/T. These observations suggest that
the P phosphorylation verified in the in vitro system
(16, 17, 20) functions similarly in functional, isolated photoreceptor outer segments.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemical reagents were purchased from the
following sources: [-33P]ATP,
[-32P]ATP, [3H]cGMP and
[33P]phosphorus from NEN Life Science
Products; ATP, cGMP, GTP, and GTPS from Roche Molecular
Biochemicals; phosphocreatine, creatine phosphokinase, PMSF, leupeptin,
pepstatin A, aprotinin, olomoucine, roscovitine, iso-olomoucine, and PI
from Sigma; okadaic acid from LC Services; molecular sieve (4A 1/16)
from Wako Pure Chemicals; Immobiline DryStrip gels, a
chemiluminescence detection kit, and Pharmalyte (pH 8-10.5) from
Amersham Pharmacia Biotech; nitrocellulose membranes (0.2 µ) and
Bio-Lytes (pH 5-7 and pH 6-8) from Bio-Rad; and
L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin (sequencing grade) from Promega. Cdk5 and
p35 antibodies used (Santa Cruz Biotechnology) were the same as those
used in a previous study described in the accompanying paper (20).
Characterization of these antibodies was also described in the
accompanying paper (20). The MAP kinase antibody used (New England
Biolabs) was prepared against peptides corresponding 350-360 amino
acids of human p44 MAP kinase. A P antibody was prepared using a
peptide corresponding to bovine P
Arg24-Gly46. This antibody recognizes
bovine and frog P. All experiments described were carried out using
frogs (Rana catesbiana).
Protein Preparation--
Recombinant bovine Cdk5/p35 was
prepared by a Mono S column (22). Cdk5 and p35 cloned from bovine
retina have the same cDNA sequences as those of bovine brain
proteins (20). We note that recombinant p35 is expressed as a ~25-kDa
protein, as described previously (22-24). The ~25-kDa protein (p25)
is a truncated form of p35, but p25 has been shown to activate Cdk5.
P kinase was isolated from a soluble fraction of frog ROS using a
Mono Q column (16). Frog P (10) and its phosphorylated form (16)
were isolated as described. Recombinant bovine P was prepared as
described previously (18). Frog GDP/T was purified as
described previously (9, 10). GTPS/T was prepared from
GDP/T by using GTPS, urea-treated ROS membranes, and T, as
described previously (9). To prepare P complexed with
GTPS/T or GDP/T, these Ts were incubated with equimolar
concentration of P at 4 °C overnight, and these complexes were
isolated by gel-filtration columns.
Isolation of Frog Photoreceptor Outer Segments and Determination
of the Level of P Phosphorylation in the Outer Segments--
Frogs
were fully dark-adapted (>12 h) at room temperature. Before bleaching
samples, all manipulations were done under infrared light.
Photoreceptor outer segments were isolated as described previously (19). Briefly, the frontal hemisphere of an eyeball was removed by a razor blade, and the resulting eye-cup was vertically cut in half. The half-eye-cup was put on two layers of curved filter
paper (Toyo Filter Paper 5B, 3 × 6 cm). After its vitreous body
was soaked by the filter papers, the retina layer adhered to the filter
paper. The retina could be peeled off from the eye-cup when the curved
filter paper was flattened. The pigment epithelium layer stayed on the
eye-cup. The retina was covered with 100 µl of Ringer's solution
(105 mM NaCl, 2.5 mM KCl, 1.2 mM
MgCl2, 10 mM HEPES (pH 7.5), 2 mM
taurine, and 5 mM glucose) containing [33P]phosphorus (~1 mCi/ml) and incubated for 30 min
under O2/CO2 (95:5) atmosphere.
[33P]Phosphorus was used to avoid illumination of
rhodopsin by the Cerenkov effect of [32P]phosphorus. The
pH value of the incubation medium was exactly adjusted to 7.50 by the
addition of 0.1 N NaOH. After rinsing with Ringer's
solution, the retina was placed on a flat end of a plastic plunger, and
the excess Ringer's solution was removed by dry filter paper. The
retina on the plastic plunger was exposed to white light (2% rhodopsin
bleached/min) for the indicated periods and quickly frozen by attaching
its photoreceptor outer segment layer to the surface of a copper block
pretreated with liquid N2. The retina was moved into cold
acetone (
20 °C, 100 ml) containing 5 g of molecular sieve (4A
1/16) and incubated (20 °C, 3 days) with acetone. The acetone was
replaced each day. Acetone was removed by decantation, and dehydrated
retinas were dried under vacuum. A piece of an adhesive tape (Scotch
3M) was attached to the outer segment surface of the retina layer.
After the backing filter paper was removed, another piece of
tape was attached to the neural retina layer. By pulling these
two tapes apart, the outer segment layer and the neural retina layer,
including the photoreceptor inner segments, were separated. The outer
segment layer attached to the tape is observed by light microscopy (× 500) (Fig. 1A). The purity of the outer segments will be
described later. The outer segment layer was solubilized with 100 µl
of buffer A (1% Triton X-100, 1% deoxycholic acid, 150 mM
NaCl, 50 mM Tris/HCl (pH 7.5), 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 50 nM okadaic acid) containing 1% SDS, heated at 95 °C for
5 min, and diluted with 900 µl of buffer A. Insoluble materials were
spun down by ultracentrifugation. The protein concentration was
determined by the bicinchoninic acid protein assay method (25) after
proteins were precipitated with deoxycholic acid/trichloroacetic acid.
P and phosphorylated P in each sample (1.0 mg of protein) were
immunoprecipitated using a P-specific antibody and further purified
by SDS-PAGE. Radioactive bands corresponding to phosphorylated P
were detected by using an image analyzer and were cut out. Phosphoamino
acid analysis of the P was carried out as described
previously (16). 33P-Labeled spots were detected on
an image analyzer. Radioactive spots were recovered, and their
radioactivities were measured.
The purity of photoreceptor outer segments isolated was investigated
using antibodies against MAP kinase, which are believed not to be
present in outer segments (20). Photoreceptor outer segments and the
neural retinal layer were solubilized in 200 µl of SDS-sample buffer
containing 70 mM DTT and heated at 95 °C for 5 min.
Proteins (20 µg) in these samples were isolated by SDS-PAGE, and MAP
kinase was detected by Western blot. The protein concentration was
measured as described previously (25).
Determination of Cdk5 Localization in Frog Retinas--
Fresh
frog retina was immediately fixed with 4% paraformaldehyde in 0.13 M phosphate buffer (pH 7.4) for 2 h at 4 °C. After washing with the same buffer (×3), the retina was equilibrated with
30% sucrose solution. Cryosections (14 µm thick) were prepared with
a Cryostat (Leica CM3050 Bensheim, Germany), and mounted on
glass slides. These specimens were blocked with the phosphate-buffered saline buffer containing 1% (w/v) bovine serum albumin and 0.5% Triton X-100 for 30 min at room temperature. Sections were incubated with a Cdk5 antibody at the IgG concentration of 1 ng/ml for 2 h.
For controls, the same concentration of the antibody was mixed with the
peptide antigen (20 ng/ml) prior to application. Specimens were
incubated with a secondary antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG (15 mg/ml), for 1 h. These specimens were washed with the phosphate buffer (×3). Finally, antibody binding sites
were visualized with 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt and nitro blue tetrazolium chloride.
Immunological Detection of Phosphorylated P--
Before
illumination of samples, all manipulations were done under infrared
light. Retinas were isolated from frog eye-cups as described above.
These retinas were incubated in Ringer's solution (20 min). After
exposure to white light (2% rhodopsin bleached/min) for indicated
times, these retinas were quickly frozen as described above. As a
control, all procedures were carried out without light. The
photoreceptor outer segment layer was isolated from dried retina as
described above and then solubilized with 50 µl of 1% SDS containing
100 mM DTT. Solubilized outer segment sample was diluted by
10-fold with buffer B (2% Triton X-100, 1% Pharmalyte (pH 8-10.5),
0.5% Bio-Lyte (pH 5-7), 0.5% Bio-Lyte (pH 6-8), 1 mM
PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 100 nM okadaic acid, and 9.2 M urea in final
concentrations). P and phosphorylated P in the solubilized outer
segments (50 µg protein) were isolated by two-dimensional gel
electrophoresis using Immobiline DryStrip gels, consisting of
isoelectric focusing (pH 6-11) in the first dimension and SDS-gradient
gel (10-20%) electrophoresis in the second dimension. P and
phosphorylated P were blotted on nitrocellulose membranes, and
membranes were blocked by 5% milk in Tris-buffered saline containing
0.1% Tween 20. A P-specific antibody diluted in the same blocking
buffer was used to identify P, and the bound antibody was detected
by a chemiluminescence detection kit. After development of x-ray films,
P spots were scanned by Paragon 1200A3 ProScanner and relative
density (mm2 × OD) was calculated by Molecular Analyst
software (Bio-Rad). The location of phosphorylated P in gels was
confirmed using 33P-phosphorylated P.
Detection of Effects of Cdk Inhibitors on P Phosphorylation in
Vivo and in Vitro--
In the in vivo system, Cdk
inhibitor, olomoucine (100 µM), or roscovitine (50 µM) were applied to retinas incubated in Ringer's solution containing [33P]phosphorus. Other experimental
conditions were the same as those described above. An inactive analogue
of olomoucine, iso-olomoucine (100 µM), was used as a
control. Isolation of P and phosphoamino acid analysis of
phosphorylated P were carried out as described above. Effects of
these inhibitors on P kinase and recombinant Cdk5/p35 were also
measured using P phosphorylation in vitro (16, 20).
Two-dimensional Peptide Map Analysis of P Phosphorylated in
Vitro and in Vivo--
Phosphopeptide maps of P phosphorylated
in vitro and in vivo were compared. As P
phosphorylated in vitro, frog P (10 µg) was
phosphorylated by using ~10 µCi of [-33P]ATP and
P kinase, as described previously (16, 20). As P
phosphorylated in vivo, after incubation of retinas in
Ringer's solution containing [33P]phosphorus, P was
phosphorylated by 10-min light exposure (20% rhodopsin was bleached),
as described above. These phosphorylated Ps were isolated by
immunoprecipitation using a P-specific antibody and SDS-PAGE. P
radioactive spots were identified by autoradiography and cut out.
Extracted Ps were digested with trypsin, and the resulting peptides
were analyzed using two-dimensional peptide map analysis as described
previously (26). Radioactive spots were detected by an image
analyzer (BAS2000, Fuji Film).
Measurement of Effects of Nonphosphorylated and Phosphorylated
Ps on Light/GTPS-activated PDE--
Dark-adapted intact frog ROS
was prepared from six frogs by Percoll density gradient centrifugation
(13). The intact ROS was suspended in buffer C (65 mM KCl,
35 mM NaCl, 10 mM HEPES (pH 7.8), 2 mM MgCl2, 0.2 mM PMSF, 5 µg/ml
aprotinin), and its aliquot (10 µl) was divided into siliconized
plastic tubes. Each tube was tightly sealed with an aluminum foil and
stored at 90 °C. Each frozen ROS was thawed just before use and
sheared by passing through a siliconized fine pipette tip. The ROS
homogenate (final concentration of rhodopsin, 2.5 µM) was
added to 180 µl of buffer C containing 0.45 mM GTPS
and 1 mM BAPTA in a lucent glass chamber with magnetic
stirrer. PDE activity was assayed by a pH electrode (Beckman S506A) in
the presence or absence of 300 nM P, GDP/T/P,
GTPS/T/P, and GTPS/T and phosphorylated P. Reaction
was started by adding 4 mM cGMP. To stimulate PDE activity,
a light flash that bleached 0.003% rhodopsin was given at time 0. After each measurement, continuous white light was applied to see a
maximum reaction rate.
Analytical Methods--
P phosphorylation by P kinase (16)
and recombinant Cdk5/p35 (20) was performed as described. Protein
concentration was assayed with bovine serum albumin as a standard (27).
Concentration of frog P (10, 20) and recombinant P (20) was
measured as described. It should be emphasized that all experiments
were carried out more than two times, and the results were similar. Data shown are representative of these experiments.
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RESULTS |
Localization of Cdk5 in Frog Retinas--
In pervious studies (16,
17, 20), we showed that Cdk5 is in a soluble fraction of frog ROS, that
P complexed with GTPS/T is the best substrate for Cdk5, and
that P phosphorylation by Cdk5 is dependent upon GTPS in frog ROS
membranes. We also showed that all known Ps have a special
phosphorylation motif for proline-directed kinase, including Cdk5 (20).
These biochemical observations strongly suggest that Cdk5 is in
photoreceptor outer segments. In this study, we carried out two
experiments to confirm the localization of Cdk5 in photoreceptor outer
segments: immunodetection of Cdk5 in outer segments isolated by a new
method and an immunohistochemical search for Cdk5 in retina. In the
first experiment, photoreceptor outer segments were separated from
other neural retinal layers, including photoreceptor inner segments
(Fig. 1). Light microscopy (×500) showed
that outer segment layer contains yellow rod-shape cells (Fig.
1A). The yellow color detected appears to indicate the
presence of bleached rhodopsin. The neural retinal layer also contained
similar rod-shape cells; however, the color of these cells was white
(data not shown). We note that pigment epithelium cells were not
contaminated in this preparation, because no black cells were detected
in the preparation. To check the purity of the outer segment layer
isolated, first we compared the protein profile in the outer segments
with that in neural retinal layers. We found that protein profiles are
different on SDS gels (Fig. 1B, lanes 1). In
addition, the purity of the outer segment layer was also examined by
comparing of the contents of MAP kinase in the outer segment layer with
that in the other neural retinal layers, because, using an
immunohistochemical method, we have already suggested that MAP kinase
is present in the neural retinal layers, but not in the outer segment
layer (20). We found that the immunological signal of MAP kinase was
clearly observed in the neural retinal layers, but not in the outer
segment layer (Fig. 1B, lanes 3), indicating that
MAP kinase contents in the outer segment preparation are not enough to
be detected by Western blotting. These observations suggest that the
outer segment layer is reasonably separated from the other neural
retinal layers. Under these conditions, Cdk5 was clearly observed in
both outer segment and other neural retinal layers (Fig. 1B,
lanes 2). This observation strongly suggests that Cdk5 is
present in both the photoreceptor outer segment layer and the other
neural retinal layers in frog retina. This observation also shows that
Cdk5 contents in the outer segment layer appears to be less than that
in the neural retinal layers. We note that the specificity and
sensitivity of the Cdk5 antibody have already been shown in the
accompanying paper (20).
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Fig. 1.
Isolation and purity of photoreceptor outer
segment layer from frog retinas. A, isolation. Frog
retinas were isolated and incubated in Ringer's solution as described.
Then retinas were quickly frozen by attaching their photoreceptor outer
segment layer to the surface of a copper block pretreated with liquid
N2. Retinas were then dehydrated and dried as described. A
piece of a Scotch 3M tape was attached to the outer segment surface of
the retina layer, and another piece of tape was attached to the
neural retina layer. By pulling these two tapes apart, the outer
segment layer was isolated from the neural retina layers including the
photoreceptor inner segments. The outer segment layer was observed by
light microscopy (× 500). The picture shown was taken on a dark
background. The cleft shown was formed when dehydrated retina was dried
in vacuo. B, purity of the outer segments
prepared by the method. In addition to Cdk5, the presence of MAP kinase
in the outer segment layer (a) and other neural retinal
layers (b) was investigated. After solubilization of these
preparations in 100 µl of SDS-sample buffer containing 70 mM DTT, proteins (20 µg) were separated by SDS-PAGE and
detected by Coomassie Blue staining or Western blotting. Lanes
1, proteins stained by Coomassie Blue; lanes 2, Cdk5
detected by Cdk5 antibody; and lanes 3, MAP kinase detected
by MAP kinase antibody.
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In the immunohistochemical search for Cdk5 in frog retina, the
strongest immunological signal was observed in the inner plexiform layer (Fig. 2B). Other strong
signals were in the outer plexiform layer and at the interface between
the inner segments and the outer nuclear layer. These observations are
consistent with previous data showing the location of Cdk5 in rat
retina (28). The immunological signal in the outer segment layer, on
the contrary, was less intense than that of those layers. However, we
believe that this signal is significant for the following reasons. 1)
In the same photoreceptor cell, the signal in the outer segment layer
is stronger than the signal in the outer nuclear layer; 2) in retinal
cells, the signal in the outer segment layer is clearly stronger than
that in the inner nuclear layer; and 3) the background signal seen
between outer segments is similar to the signal in the control retina, and the signal in the outer segments is much stronger than the background signal. The similar immunological signals were also observed
in bovine retinal cells (data not shown). Based on three different
observations (biochemical observations in previous studies (16, 17,
20), detection of Cdk5 in an homogenate of highly purified outer
segments (Fig. 1), and this immunohistochemical observation), it is
reasonable to conclude that Cdk5 is present in photoreceptor outer
segments.
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Fig. 2.
Localization of Cdk5 in frog retina.
Cdk5 in fresh frog retina was localized using a Cdk5 antibody and
alkaline phosphatase-conjugated anti-rabbit IgG. For a control, the
antibody treated with the peptide antigen prior to application was
used. The cell layers seen are: OS, outer segments;
IS, inner segments; ONL, outer nuclear layer;
OPL, outer plexiform layer; INL, inner nuclear
layer; and IPL, inner plexiform layer. As shown in the
accompanying paper (20), the specificity of the antibody was enough to
distinguish Cdk5 in an homogenate of retina, and the antibody was
sensitive to frog Cdk5.
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Light-dependent P Phosphorylation in Vivo--
In
previous studies (16, 17, 20), we have shown that P complexed with
GTPS/T is the best substrate for P kinase (Cdk5) and that
GTPS is required for P phosphorylation by Cdk5 in photoreceptor
outer segment membranes. These observations imply that P
phosphorylation is light-dependent in photoreceptors, because binding of GTPS to T is completely dependent upon
illuminated rhodopsin (1, 7). Indeed, the light-dependent
P phosphorylation was detected in vivo (21). We combined
and extended these observations. After incubation with
[33P]phosphorus, retinas were illuminated under various
light conditions, quickly frozen, and dehydrated. The photoreceptor
outer segment layer was then isolated from these retinas as described
above. P was isolated from these photoreceptor outer segments by a
P-specific antibody and SDS-PAGE, and subsequently phosphorylated
amino acids in the P were identified and their radioactivities were
measured. Under dark conditions the phosphorylated amino acid was
barely detected (Fig. 3A), and
only less than 0.5% of the total P was found to be phosphorylated
(Fig. 3C). However, a threonine residue in P was clearly
phosphorylated in illuminated photoreceptors (Fig. 3A). Two
phases of phosphorylation were observed. After an initial rapid
phosphorylation, the phosphorylation increased gradually during
illumination (Fig. 3B). In the rapid phosphorylation, the
P phosphorylation was detected when 0.03% of rhodopsin was bleached
(1 s). During the slow phosphorylation, 10 ± 4% of the total
P (n = 5) was phosphorylated after bleaching of 10%
of rhodopsin (Fig. 3C), and 16 ± 6% of the total P
(n = 5) was phosphorylated after bleaching of 20% of
rhodopsin (data not shown). Thus, as the rapid phosphorylation, ~4%
of the total P was estimated to be phosphorylated after less than
0.3% of the rhodopsin was bleached in less than 10 s (Fig.
3A). These observations indicate that P phosphorylation
in photoreceptor outer segments is light-dependent and
significant and that the P phosphorylation is fast enough to be
involved in the recovery phase of phototransduction. When the light was
turned off, the P was dephosphorylated rapidly (Fig. 3, A
and B). The P dephosphorylation could be observed in less
than 1 s. Together, these results indicate that the P phosphorylation is reversible and that the dephosphorylation is also
fast.
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Fig. 3.
Light-dependent
P phosphorylation in
vivo. A, light dependence. Frog retinas were
incubated in a Ringer's solution containing
[33P]phosphorus under dark conditions. Under various
light conditions, retinas were illuminated and rapidly frozen. Then
photoreceptor outer segment layers were isolated from lyophilized
retinas. For the illumination, light was adjusted to bleach 2%
rhodopsin/min. Outer segment layers obtained from lyophilized retinas
were solubilized with 100 µl of buffer A containing 1% SDS
(95 °C) and diluted with 900 µl of buffer A. P in each sample
(1.5 mg) was immunoprecipitated using a P-specific antibody. P in
the precipitate was further purified by SDS-PAGE, and phosphoamino acid
analysis of the P was carried out. P-Ser, phosphoserine;
P-Thr, phosphothreonine; P-Tyr, phosphotyrosine;
D, dark; L10", light 10 s;
L60", light 60 s; L60"D10", light
60 s and then dark 10 s; L60"D60", light
60 s and then dark 60 s. B, radioactivity of the
phosphorylated threonine residue isolated in A. The
radioactivity was measured after scratching radioactive spots of the
threonine residue. C, immunological detection of
light-dependent P phosphorylation. Retinas were
illuminated for 5 min under the same conditions described in
A. As a control, retinas were not illuminated. Then
photoreceptor outer segments were isolated from these retinas. Ps in
these photoreceptor outer segments (50 mg) were isolated by
two-dimensional gel electrophoresis (IEF, isoelectric
focusing (pH 6-11), in the first dimension, and SDS-PAGE,
polyacrylamide gradient 10-20%, in the second dimension) and detected
using the P-specific antibody with a chemiluminescence detection
kit. After development of x-ray films, P spots were scanned, and
relative densities were calculated. The location of phosphorylated P
in gels, indicated by a open arrowhead, was identified using
P phosphorylated by recombinant Cdk5/p35 in vitro.
a, P in dark outer segments; b, P in
bleached outer segments.
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We note that incorporation of the radioactivity into a P serine
residue was sometimes detected in the in vivo system,
especially when the level of the P threonine phosphorylation was
increased by high bleaching (Fig. 3A). The incorporation was
weak because only less than 10% of the radioactivity incorporated into
the threonine residue was observed in the serine residue. As described below, we will show that Cdk5 is involved in the
light-dependent phosphorylation of the P threonine.
However, the serine phosphorylation seems not to be due to Cdk5,
because only threonine 22 in P (frog and bovine) is phosphorylated
by Cdk5 in vitro (16, 20). Since we do not know the full
amino acid sequence of frog P, it is also possible that the P
contains another Cdk5 phosphorylation site, including a serine residue,
and the phosphorylation of the serine was detected in vivo,
but not in vitro, because of structural hindrance. However,
this possibility is very slim, because P appears not to have a rigid
conformation, and such structural hindrance seems not to be present in
P in vitro (20). We also note that the involvement of PKC
and PKA in the serine phosphorylation is unlikely, because these
protein kinases have been shown to phosphorylate only threonine 35 in
P in vitro (14, 15). It is also possible that the frog
P contains a serine phosphorylation site for these protein kinases,
and the site could not be phosphorylated by structural hindrance under
the in vitro conditions. However, the possibility was small,
because P seems not to have a rigid conformation, as described
above. In the case of PI-stimulated kinase (13), we suggested that
threonine 35 or serine 40 in frog P was phosphorylated in
vitro. However, the possibility of serine 40 phosphorylation is
also small, because under bleached conditions the site appears to be
masked by GTP/T in vivo, as described in the
Introduction. At present, the mechanism and function of the serine
phosphorylation are unknown.
Effects of Cdk Inhibitors on P Phosphorylation in Vitro and in
Vivo--
We used Cdk inhibitors, olomoucine, and roscovitine to
determine whether P phosphorylation in vivo is due to a
Cdk. These Cdk inhibitors have been used in vitro (0.2-10
µM) and in vivo (10-100 µM)
(29-31). We found that under the in vitro conditions both
olomoucine and roscovitine inhibited P phosphorylation by frog P
kinase or by recombinant bovine Cdk5/p35 with 50% inhibition ~2 and
~7 µM, respectively (Fig.
4A). Iso-olomoucine, an
inactive analog of olomoucine, did not inhibit the P
phosphorylation. We also found that 100 µM olomoucine
completely inhibited the light-dependent phosphorylation of
P threonine in vivo, but iso-olomoucine did not (Fig.
4B). Moreover, 50 µM roscovitine drastically
reduced the P threonine phosphorylation. These observations strongly suggest that a Cdk is involved in the light-dependent P
phosphorylation.
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Fig. 4.
Effect of Cdk inhibitors on
P phosphorylation in vitro
and in vivo. A, in
vitro. P (2 µg) was phosphorylated with 200 ng P kinase
( ,  ,  ) or 10 ng of recombinant Cdk5/p35 ( ,  ,  ) in 30 ml of the reaction mixture in the presence of various concentrations of
olomoucine (, ) and roscovitine (, ). As a control for
olomoucine, iso-olomoucine was used (, ). P kinase and
recombinant Cdk5/p35 were prepared by using a Mono Q column and a Mono
S column, respectively. One-hundred percent indicates the level of P
phosphorylation without an inhibitor. B, in vivo.
After incubation of frog retinas in Ringer's solution containing
[33P]phosphorus in the presence or absence of
olomoucine (100 µM), iso-olomoucine (100 µM), and roscovitine (50 µM). P
phosphorylation was stimulated by illumination (1 min). Phosphoamino
acid analysis of the phosphorylated P was carried out as described.
P-Ser, phosphoserine; P-Thr, phosphothreonine;
P-Tyr, phosphotyrosine.
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PI-stimulated kinase, PKC or PKA, have been shown to phosphorylate P
in vitro (13-15). However, it should be emphasized that involvement of these protein kinases in the light-dependent
phosphorylation of P threonine (Figs. 3 and 4) is very unlikely for
the following reasons. 1) As summarized in the Introduction, these
protein kinases appear not to function in the
light-dependent P phosphorylation in vivo,
because the amino acid(s) phosphorylated by these kinases seem to be
masked by GTP/T when P interacts with GTP/T (15, 18, 19). 2)
It is possible that frog P could have another threonine, other than
threonine 35, as a phosphorylation site for these protein kinases, and
the site would be available in vivo, but not in
vitro, because of structural hindrance. However, this possibility
is small, because P appears not to have a ridged conformation, as
suggested (20). 3) The Cdk inhibitors used are very specific to Cdks
(31). Very high concentration of these inhibitors is required to
inhibit PKC and PKA in vitro; IC50 values for
various isozymes of PKC are >100 µM roscovitine and
>800 µM olomoucine, and IC50 values for PKA
(bovine heart) are >1,000 µM roscovitine and >2,000
µM olomoucine (31). In addition, similar high
concentration of these inhibitors is also required for the inhibition
of other protein kinases such as cGMP-dependent protein kinase (bovine tracheal smooth muscle, IC50 >1,000) and
casein kinase 2 (rat liver, IC50 >2,000) (31). Although
the effect of these inhibitors on frog kinases is unknown, it is
unlikely that frog PI-stimulated kinase, PKC, PKA, or unknown protein
kinases, other than Cdks, could be inhibited by less than 100 µM amounts of these Cdk inhibitors in
vivo. Especially, it is very unlikely that any frog protein kinase
was completely inhibited by 100 µM olomoucine in
vivo (Fig. 4B).
Evidence for the Involvement of Cdk5 in P Phosphorylation in
Vivo--
As described above, a Cdk is involved in the P
phosphorylation in vivo. To identify the Cdk as Cdk5,
two-dimensional phosphopeptide maps of both P phosphorylated by P
kinase (Cdk5) in vitro and P phosphorylated in
illuminated retina (10-min illumination) were compared (Fig.
5). We found that only one kind of the
radioactive peptide was produced from these Ps and that the peptide
derived from P phosphorylated in vivo was found in the
same location as that of P phosphorylated in vitro.
Together with data showing that only threonine 22 is phosphorylated by
Cdk5 in vitro (16, 20), these observations indicate that the
same threonine residue in P is phosphorylated in vivo and
in vitro. These observations imply that the Cdk involved in
the light-dependent P phosphorylation in vivo
is Cdk5. Since under our conditions, photoreceptors were illuminated
for 10 min (~20% rhodopsin illumination), these results also
indicate that the same threonine residue is phosphorylated in these two
phases of P phosphorylation. These observations also strongly
support our previous conclusion that protein kinases known to
phosphorylate P threonine 35 are not involved in the light-dependent phosphorylation of P.
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Fig. 5.
Two-dimensional peptide map analysis of
P phosphorylated in vitro and
in vivo. As P phosphorylated in
vitro, frog P was phosphorylated by P kinase with
[-33P]ATP. As P phosphorylated in vivo,
frog retinas were incubated in Ringer's solution containing
[33P]phosphorus, and P in these retinas was
phosphorylated by illumination (10 min). These phosphorylated Ps
were isolated by a P-specific antibody and SDS-PAGE. These
phosphorylated Ps were digested with trypsin, and resulting peptides
were analyzed using two-dimensional peptide map analysis. a,
peptides from P phosphorylated in vivo; b,
peptides from P phosphorylated in vitro; c, a
mixture of peptides from P phosphorylated in vivo and
P phosphorylated in vitro. * indicates the starting point
of the two-dimensional peptide map.
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Effects of Phosphorylated P on Light/GTPS-activated
PDE--
In previous studies (16, 17), using systems reconstituted
with isolated proteins, we showed that P phosphorylated by P
kinase (Cdk5) more effectively inhibits GTPS/T-activated PDE than
nonphosphorylated P. This is because the phosphorylated P loses
its affinity to GTPS/T, but gains a 10~15 times higher ability
to inhibit GTPS/T-activated PDE than that of nonphosphorylated P. In this study, we investigated whether these phenomena were also
observed in an homogenate of frog photoreceptor outer segments, a
system containing all proteins of outer segments. We checked the
effects of nonphosphorylated and phosphorylated Ps, alone or
complexed with T, on the time course of
light/GTPS-dependent activation of PDE (Fig.
6). We found that P complexed with
GDP/T could not inhibit the PDE activity; however, the same
concentration of P alone inhibited the PDE activity (Fig.
6A). This implies that GTP hydrolysis is not sufficient for
the deactivation of light/GTP-activated PDE in a frog ROS homogenate
and that P must be released from GDP/T for the PDE deactivation.
This is consistent with previous results obtained in systems
reconstituted by isolated proteins (10, 32). We also found that P
complexed with GTPS/T did not inhibit the light/GTPS-activated
PDE; however, P phosphorylated by P kinase (Cdk5) inhibited the
light/GTPS-activated PDE, even in the presence of GTPS/T in
the homogenate (Fig. 6B). Moreover, we found that in the
homogenate, lesser amounts of phosphorylated P were required to
inhibit light/GTPS-activated PDE than that of nonphosphorylated P
(data not shown). These results indicate that the nonphosphorylated
P retains its complex with GTPS/T in the homogenate; however,
after the P is phosphorylated by Cdk5, the phosphorylated P
cannot keep its complex with GTPS/T and inhibits effectively
GTPS/T-activated PDE. We note that the effect of P
phosphorylation on the time course of
light/GTPS-dependent PDE activation could not be
measured by adding ATP to the homogenate, because rhodopsin in the
homogenate is also phosphorylated, and the phosphorylated rhodopsin may
affect the light/GTPS-activated PDE activity (33, 34). We also note
that 300 nM amounts of these Ps, free or
complexed with GDP/T or GTPS/T, were used in these
experiments, because by using the high concentration of P, we tried
to clearly show that even a small portion of nonphosphorylated P was
not released from its complexes with GDP/T or GTPS/T. Under
similar conditions, previous studies used 35-200 nM P
to inhibit light/GTP-activated PDE activity measured with a pH
electrode (35, 36).
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Fig. 6.
Effects of P and
phosphorylated P on
light/GTPS-activated PDE. Using an
homogenate of intact frog photoreceptor outer segments (2.5 µM rhodopsin), PDE activity was assayed by a pH electrode
in 180 µl of buffer C containing 1 mM BAPTA, 50 nM okadaic acid, 0.45 mM GTPS, and 4 mM cGMP in the presence or absence of 300 nM of
P, GDP/T/P, GTPS/T/P, or GTPS/T and
phosphorylated P. As a photic stimulus, a light flash that bleached
0.003% rhodopsin was given at time 0. Under light conditions that
illuminated 0.3% of rhodopsin/s, the PDE activity was 30 µM/s. A, effects of P and GDP/T/P on
the time course of light/GTPS-dependent PDE activation.
a, buffer only; b, GDP/T/P; c,
P. B, effects of GTPS/T/P and GTPS/T and
phosphorylated P on the time course. a, buffer only;
d, GTPS/T/P; e, GTPS/T and
phosphorylated P.
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DISCUSSION |
In previous studies (16, 17), we showed that P is
phosphorylated by P kinase in a GTP-dependent manner and
that the phosphorylated P loses its affinity to GTP/T, but gains
high affinity to P to inhibit cGMP hydrolysis by P. In the
study described in the accompanying paper (20), we have demonstrated that P has a special phosphorylation motif for Cdk5 and that P
kinase is Cdk5/p35. In the present study, using an in vivo system, we have shown the following results: 1) P is phosphorylated in a light-dependent manner, 2) Cdk5 is involved in the
P phosphorylation, 3) the P phosphorylation is significant and
rapid enough to be involved in the recovery phase of phototransduction,
4) dephosphorylation of P is also rapid, indicating that the P
phosphorylation is reversible. In addition, using immunodetection in
highly purified photoreceptor outer segments and an immunohistochemical
search of the retina, we have detected significant signals of Cdk5 in photoreceptor outer segments. Moreover, using an homogenate of photoreceptor outer segments, we have demonstrated that the
phosphorylated P inhibits light/GTPS-activated PDE, even in the
presence of GTPS/T. Although factors that regulate the P
phosphorylation are not identified yet, these observations clearly
indicate that the P phosphorylation by Cdk5 can play important roles
in PDE regulation.
In this study, we used frog outer segments isolated by a new method. We
have shown that the purity of the outer segments isolated by the method
is high enough to show the localization of Cdk5 in the outer segments.
Nishizawa et al. (37) also recently reported a novel method
to isolate bovine photoreceptor cells containing outer segments and the
majority of the inner segments. The concept of their method is similar
to that of ours, because they used nitrocellulose membranes to attach
the photoreceptor cell monolayer. Then, the photoreceptor cell layer
was separated from other retinal layers attached to a filter paper. It
is unknown why photoreceptor cells isolated by their method contain
both outer and inner segments. We, however, speculate that there are
several reasons for the difference as follows. 1) The species used to
obtain photoreceptors may be critical because the form and size of
photoreceptors, especially the structural strength of the ciliary
connection, may be different in different species. We used frog retina
and they used bovine retina. We have never tried to isolate bovine
outer segments by our method. Thus, we do not know whether our method
is fitted to isolate inner segment-free outer segments from bovine
retina. 2) Dehydration of retina may be important to weaken the
structure of ciliary connection. We dehydrate retinas before
separation of outer segments from inner segments, but they did not. In
any case, both methods will be useful to isolate photoreceptors and their segments and can be improved by attention to the
differences in methods and results.
It should be emphasized that this series of studies does not exclude
the turnoff mechanism of GTP/T-activated PDE by hydrolysis of GTP.
We anticipate that both P phosphorylation and GTP hydrolysis are
involved in the deactivation of GTP/T-activated PDE in
phototransduction and that the P phosphorylation functions under
some special conditions, although the relationship between PDE turnoff
by P phosphorylation and by GTP hydrolysis is unknown now. However,
it is clear that GTP hydrolysis is not enough to turnoff
GTP/T-activated PDE in frog ROS membranes. After hydrolysis of GTP
by T, P is still complexed with GDP/T, and the P complex
cannot inhibit GTP/T-activated PDE (Ref. 10; Fig. 6A).
The GDP/T/P complex is easily extracted and isolated from frog
ROS membranes (10), indicating that the interaction between GDP/T
and P is very tight and specific. Indeed, the GDP/T/P complex
is easily prepared by mixing GDP/T with P (32). We have shown
that T
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ABSTRACT
INTRODUCTION
