Investigative Ophthalmology & Visual Science, Vol. 29, No. 12, December 1988
Copyright © Association for Research in Vision and Ophthalmology
Protein Phosphorylation in Cultured Rat RPE
Effects of Pro rein Kinose C Acrivorion
Cynrhia A. Heth and Susan Y. Schmidt
Incubation of confluent cultures of rat retinal pigment epithelium (RPE) with 32P-orthophosphate
resulted in the incorporation of 32 P into proteins, RNA and the nucleoside phosphates ADP, GDP,
ATP and GTP. RPE cultures incubated with phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, did not significantly change the incorporation of 32 P into total protein, RNA
or the nucleoside phosphates ADP, GDP, ATP and GTP. However, PMA exposure specifically
increased phosphorylation of five proteins with molecular weights of 80 kilodaltons (K), 56K, 35K,
33K, and 29K having isoelectric points between 4.3 and 6.5. PMA treated cultures also showed
dephosphorylation of two proteins having molecular weights of about 33K. The observed increase in
80K phosphorylation suggests that protein kinase C is present and activated by PMA in the RPE.
Invest Ophthalmol Vis Sci 29:1794-1799,1988
Phosphorylation and dephosphorylation of proteins occurs following extracellular stimulation of cell
surface receptors by effector substances such as
growth factors, hormones, and neurotransmitters,
and is one mechanism by which the functional states
of proteins are altered and cellular responses are regulated. Phosphorylation of proteins is catalyzed within
cells by several different protein kinases whose functions are regulated by receptor-mediated increases in
a variety of intracellular second messengers such as
calcium, cyclic nucleotides, and diacylglycerol
(DAG).1"5 For example, an increase in the intracellular concentration of DAG in the presence of calcium
directly activates protein kinase C,6"8 which in turn
phosphorylates specific proteins. 910 Tumor promoters such as phorbol-12-myristrate-13-acetate
(PMA) are synthetic analogs of DAG which activate
protein kinase C" and have been widely used to
identify the endogenous substrates of protein kinase
C within cells.12
The current studies were done to define the baseline levels of protein phosphorylation in cultured rat
retinal pigment epithelium incubated with 32P and to
examine changes in protein phosphorylation upon
exposure to PMA.
Materials and Methods
Cell Culture
Cultures of retinal pigment epithelium (RPE) were
prepared from 5-day-old Long-Evans rats as previously described.13 The use of animals in this investigation conformed to the ARVO Resolution on the
Use of Animals in Research. Cells were plated at
equal density in 24 well plates and maintained in
BME medium containing 20% NuSerum (Collaborative Research, Lexington, MA) for 14 days. Cultures
reached confluency in 9 days.
Incubations With 32 P and PMA
Confluent cultures were incubated at 37°C with
Earle's medium, pH 7.4, containing 1 mM CaCl2,0.1
raM PO4, 0.2% bovine serum albumin, and 3.04 /iCi
32
P orthophosphoric acid (8500-9120 Ci/mM, carrier
free, New England Nuclear, Boston, MA). To determine the time course of 32P incorporation into proteins and RNA incubation times of 1, 5, 15, 30, 60
and 90 min were examined. To test the effects of
PMA on protein phosphorylation, following 1 hr of
incubation with 32P, some cultures were exposed to
PMA (final concentration 1.62 /xM, effective concentration for 3T3-L1 cells,14) in 2% BSA for 15 min.
Control cultures received 2% BSA alone for the 15
min incubation.
From the Berman-Gund Laboratory for the Study of Retinal
Degenerations, Harvard Medical School, Massachusetts Eye and
Ear Infirmary, Boston, Massachusetts.
Supported by NIH Grants EY-05790 (CAH) and EY-03815
(SYS), Bethesda, Maryland, and a grant from the National Society
to Prevent Blindness to CAH and the National Retinitis Pigmentosa Foundation, Baltimore, Maryland.
Submitted for publication: March 2, 1988; accepted June 29,
1988.
Reprint requests: Cynthia A. Heth, PhD, Berman-Gund Laboratory for the Study of Retinal Degenerations, Massachusetts Eye and
Ear Infirmary, 243 Charles Street, Boston, MA 02114.
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No. 12
Fig. 1. Representative
two-dimensional
silver
stained gels (A) and corresponding autoradiograms
(B) of control RPE cultures
(left) and cultures treated
with FMA (right). Molecular weight markers (MW)
are shown at top center. The
acidic range (pi 4.0) of each
gel is located at the left and
the basic end (pi 8.5) is located at the right. Protein
patterns appear unchanged
by PMA treatment (A),
however 32P incorporation
is altered in several proteins
(B) and is shown in more
detail in Figure 2.
PHO5PHORYLATION OF RPE PROTEINS / Herh ond Schmidr
1795
A
CONTROL
B
I
Sample Processing
Incubations were terminated by removing the 32P
containing media from the cultures. Each well was
washed three times with Tris buffered saline, pH 7.4
at 4°C. Cells were extracted with 160 fi\ of 2% SDS,
sonicated on ice, centrifuged (140,000 g, 30 min) at
4°C, and divided into aliquots for protein analysis,
trichloroacetic acid (TCA) precipitation, and two-dimensional gel electrophoresis. Samples not immediately processed were stored at -70°C. Protein concentration was determined according to Lowry et al15
using bovine serum albumin as a standard. Total 32P
incorporation into protein and RNA was determined
by TCA precipitation at 4°C; 32P incorporation into
protein alone was determined after TCA preparations
were heated to 90°C for 15 min as previously described.16 TCA precipitated samples were drawn
through filters under vacuum and radioactivity in the
dried niters was determined by liquid scintillation
counting. Values for 32P radioactivity incorporated
into protein or RNA in RPE cell extracts were quantitated as cpm/mg protein and were normalized with
respect to the radioactivity in 10 ^1 of medium (con-
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sidered to be 100%). Samples containing 15 ng of
protein were analyzed by two-dimensional gel electrophoresis on 10% gels17 using an ampholyte mixture to produce a pH range of 4.0 to 10. Gels were
stained with Coomassie blue (0.25%) or silver,18 dried
using a slab gel dryer (Bio-Rad, Richmond, CA,
model 224) and exposed to Kodak-Xomat film to
produce autoradiograms.
High pressure liquid chromatography (HPLC) was
used to analyze the nucleoside phosphates from control cultures and cultures treated with PMA. For
HPLC analysis, cultures were scraped and sonicated
in 200 nl of 3.6% perchloric acid. The samples were
centrifuged (4000 g, 10 min) and the supernatants
were neutralized with 0.6 mM tri-N-octylamine in
chloroform. The resulting aqueous phase (150 n\) was
applied to a radial-compression anion-exchange column (Partisil 10-SAX resin, Baxter Scientific, Bedford, MA) at 45 °C.19 The buffer gradient was from 1
mM NaH2PO4 (pH 3.30) to 0.2 M NaH2PO4; 0.5 M
NaCl (pH 4.60) over a time course of 50 min. Eluted
peaks were detected by UV absorbance at 280 nm
and radioactivity was measured using a Berthold flow
through radioactivity monitor. The area under each
1796
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1988
Vol. 29
CONTROL
m
A
CONTROL
20-35 K
20-35 K +PMA'
• * >
B
Fig. 2. Two regions of the autoradiograms shown in Figure IB are enlarged to more clearly show the localization of yiP incorporation into
proteins having isoelectnc points between 4.0 (left) and 7.5 (right). In the high molecular weight range (A) specific PMA-induced increases in
J2
P are associated with two proteins: 80K, pi 4.3 and 56K, pi 4.8, shown at the black arrows. In the lower molecular weight range (B), exposure
to PMA induced an increase in 32P incorporation into three proteins having molecular weights of 35K, 33K and 29K (black arrows, pis of 4,8,
4.6, and 4.7, respectively) white causing a decrease in 32P incorporated into two proteins having molecular weights of 34K and 33K (open
arrows, pis of 5.3 and 7.5 respectively). Phosphorylation of most other proteins from cultures treated with PMA remained at control levels.
nucleotide peak was integrated using point-to-point
baselines and Waters Multimethod analysis. Nucleotides in RPE extracts were identified by comparison
with the elution profiles of nucleoside standards.
Cultures were also incubated with 3H adenine or 3H
guanosine and processed as above for HPLC to verify
the positions of ADP, ATP, GDP and GTP.
A. 1 . 2 x 1 0 J
B
40
60
80
- 1.7 x 1 0 6
100
Incubation Time
Fig. 3. The time course of 32P incorporation into TCA-precipitable RPE proteins is plotted as the percent of medium radioactivity.
Three cultures were analyzed at each time point for two media:
Medium A (D) contained 1.2 X 105 cpm/10 nl Medium B (O)
contained 1.7 X 106 cpm/10 pi. Incorporation of 32P into protein
over the incubation times examined is linear, directly related to the
32
P content of the labeling medium, and does not plateau within 90
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Results
Incubation of RPE cultures with PMA did not result in any apparent changes in protein patterns as
analyzed by silver stained two-dimensional gels (Fig.
1A). Autoradiograms of two-dimensional gels
showed that more than 100 RPE proteins have incorporated 32P (Fig. IB). While 32P incorporation into
most of these proteins appeared unaffected by PMA,
several proteins showed specific increases or decreases in 32P radioactivity. The most notable PMAinduced increases in phosphorylation were associated
with proteins having molecular weights of 80K and
56K (Fig. 2A), and 35K, 33K and 29K (Fig. 2B) all of
1797
PHOSPHORYLATION OF RPE PROTEINS / Herh and Schmidr
No. 12
which migrate to isoelectric points in the acidic range
of the gel (between 4.0 and 6.5). Concurrently, PMAtreated cultures showed specific dephosphorylation
of two proteins having molecular weights of approximately 33K and isoelectric points between 5.5 and
6.5 (Fig. 2B). Following each experiment, the incubation media was examined for nucleotides or proteins
which may have been released from broken cells. No
nucleotides or proteins could be detected in the
media, which suggests that the RPE cells were intact
and the large number of phosphorylated proteins
seen in the two-dimensional autoradiograms were
not the result of cell lysis during the incubations,
which could lead to nonspecific phosphate incorporation.
The incorporation of 32P into TCA precipitable
proteins by the RPE cells occurred in a linear fashion
between 1 and 90 min and remained proportional to
the amount of radioactivity in the medium (Fig. 3).
Incorporation of 32P into protein and RNA in control
cultures (25.8 ± 5.4 and 24.0 ± 3.7 cpm/mg protein,
mean ± SEM, n = 8) appeared lower than those in
PMA treated cultures (42.3 ± 9.9 and 38.3 ± 14.1
cpm/mg protein, respectively, n = 6); these values,
however, were not significantly different from control
(P > 0.05).
The concentration and specific activity of the nucleoside diphosphates and triphosphates, ADP, GDP,
ATP and GTP, in control cultures and cultures
treated with PMA were determined by HPLC (Fig. 4).
Concentrations of ADP, GDP, ATP and GTP were
similar in control cultures and cultures exposed to
PMA; the nucleoside triphosphate concentrations
were three to four times higher than the concentration of the nucleoside diphosphates (Table 1). The 32P
labeling of diphosphates was comparable to that of
triphosphates in both control and PMA-treated cultures (Fig. 4B, C). Exposure of RPE cultures to PMA
did not effect the specific activity of either ATP or
GTP (P > 0.05). The 32P-specific activities of ATP
and GTP in duplicate control cultures from three
experiments were 166 ± 25.6 and 118 ± 35.0, respectively (mean ± SEM). The specific activities of ATP
and GTP in cultures exposed to PMA in all three
experiments were comparable to control values; 172
± 20.4 and 105 ± 26.0, respectively (mean ± SEM).
HPLC PROFILES
LATP
Minutes
C.
ADP
* ATP
GDP
GTP
/y K
40
Minutes
Fig. 4. Representative trace of absorbance at 280 nm used to
quantitate nucleoside phosphates ADP, GDP, ATP and GTP as
nmoles/mg protein from RPE control cultures (A). Simultaneously, 32P incorporation into each nucleotide in control cultures
(B) was measured as the radioactive samples travel through a
Berthold detector, which produces a 5 min delay in the elution
profile. Treatment of RPE cultures with PMA did not alter either
the radioactive elution profile or the specific activities of nucleotides (C), or the UV absorbance profile (not shown).
Discussion
This study provides evidence that protein kinase C
is present in cultured rat RPE based on the observation that protein phosphorylation was enhanced in
the presence of PMA. In particular, phosphorylation
offiveproteins having apparent molecular weights of
80K, 56K, 35K, 33K and 29K was increased by ex-
Table 1. Concentrations of nucleoside diphosphates and triphosphates
Control
+PMA
ADP
GDP
ATP
GTP
ATP/ADP
GTP/GDP
1.2 ±0.1
1.0 ±0.1
0.5 ±0.1
0.5 ±0.1
4.1 ±0.4
4.3 ± 0.5
2.3 ± 0.3
2.4 ±0.1
3.4
4.1
4.6
4.8
Values are given as the mean nmoles/mg protein ± SEM for duplicate
control and PMA-treated cultures from three experiments. Analysis of these
values by t-test showed there was no significant difference in nucleoside
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concentrations between control cultures and cultures exposed to PMA (P
> 0.05). High ratios of triphosphate to diphosphate indicate the cells are
unperturbed by PMA or the experimental conditions.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1988
posure to PMA compared with baseline phosphorylation in the absence of PMA. An increase in 80K
phosphorylation in response to activation of protein
kinase C by PMA has been reported in many cells
and is widely used as a marker for protein kinase C
activity.1214 The five RPE proteins showing an increase in phosphorylation represent the major substrates of protein kinase C in the rat RPE, however we
can not rule out the possibility that PMA or protein
kinase C may have activated other kinases in the RPE
which phosphorylated these proteins. In addition,
two proteins having an apparent molecular weight of
33K were dephosphorylated in the presence of PMA.
The dephosphorylation of these two proteins may reflect either decreased activity of another kinase or
increased phosphatase activity, as it has been shown
that phosphatases are also substrates of protein kinase
C.20 The present study provides a baseline for phosphorylation of proteins in RPE and shows the
changes in the phosphorylation of specific substrates
which occur under conditions which mimic increased
intracellular DAG and subsequent protein kinase C
activation.
The pattern of RPE proteins resolved in this study
by two-dimensional gel electrophoresis and silver
staining are very similar in molecular weight and relative isoelectric points to the RPE proteins cataloged
previously.21"23 Although RPE phosphoproteins have
been found in the molecular weight ranges corresponding to RPE cytoskeletal proteins,24 surface proteins25 and glycoproteins,26 further studies are needed
to identify the RPE phosphoproteins and clarify their
functions.
The exposure of RPE to PMA resulted in specific
changes in protein phosphorylation and did not produce detectable metabolic disturbance or disruption
of RPE cells. We believe the observed increases in
phosphorylation following exposure to PMA were
not a result of protein synthesis, as it has been shown
that brief (15 min) exposures to PMA alters only the
state of protein phosphorylation and not the rate of
protein synthesis.14 ATP and GTP levels remained
comparable to control levels and changes in nucleoside phosphates were not detected in these experiments. The ATP: ADP ratios of RPE cells are indicative of the active maintenance of high energy metabolites and are close to the ratio found in rat brain
(3.5:1).27 These data suggest that when the demand
for ATP or GTP as 32P donors is high, which presumably occurs during PMA-induced protein phosphorylation, production of ATP and GTP is increased to
keep the concentrations of these nucleotide triphosphates near equilibrium.
The phosphorylation of the 80K protein suggests
that protein kinase C-dependent phosphorylation of
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Vol. 29
specific substrates occurs in the RPE. Protein kinase
C or other protein kinases may play a regulatory role
in the pigment epithelium through phosphorylation
of substrates which are involved in cell growth, differentiation and maintenance of cell shape, regulation of ionic fluxes, release of lysosomal enzymes,
endocytosis and transcytosis. Direct measurement of
protein kinase C activity in the RPE should support
the hypothesis that there are receptors on the RPE
which are linked, by phospholipase C and the inositol
phosphate/diacylglycerol second messenger system,
to the regulation of protein kinase C. RPE receptors
have previously been demonstrated for transferrin28
and recently phosphorylation by protein kinase C has
been implicated in transferrin receptor cycling2930 in
other cell types. RPE cells also have receptors for
retinol-binding protein31 and insulin-like growth factor.28 RPE response to agonists suggests these cells
may also have specific receptors for neurotransmitters,32"35 peptides,36"38 rod outer segments,3940 mannose-containing ligands41'42 and other factors.4344
Whether the interaction of specific ligands with these
receptors generates DAG and activates protein kinase
C remains to be determined.
Key words: retinal pigment epithelium, protein kinase C,
phosphorylation, two-dimensional gel electrophoresis, high
pressure liquid chromatography, nucleoside phosphates
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