(Received for publication, May 11, 1995)
From the
Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II.
(Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase.
The
phosphorylation of endogenous hepatocyte proteins was affected
oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating
that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The
inhibition of basal kinase activity was accompanied by enhanced DNA
replication, an effect which could be reproduced by microinjected
mutant cAMP-subresponsive RI. It is concluded that the basal
cAMP-kinase activity exerts a tonic inhibition of hepatocyte
replication. (Rp)-8-Br-cAMPS and microinjected RI also
desensitized hepatocytes toward inhibition of DNA synthesis by
interleukin-1. This indicates that basal cAMP-kinase activity can
have a permissive role for the action of another (interleukin-1
)
signaling pathway.
cAMP signaling via activation of cAMP-dependent protein kinase
(cAK) ()is a major pathway of cell regulation. Both cAK type
I and type II have catalytic (C) subunits whose activity is blocked
when complexed with the regulatory (RI, RII) subunits. cAMP binding to
R facilitates dissociative
activation(1, 2, 3, 4, 5) .
cAK can be specifically inhibited in intact cells by overexpression of
R subunits with deficient cAMP-binding sites (6) or by
introducing smaller peptides blocking the active site of C (for review
see (7) ). A simpler and more versatile method is to expose
cells to (Rp)-cAMPS(8, 9, 10) ,
which is a diastereoisomer of adenosine 3`,5`-phosphorothioate with
sulfur in the equatorial position. (Rp)-cAMPS is believed to
antagonize cAMP by binding to R without dissociating the kinase
holoenzyme(11) . Cases where (Rp)-cAMPS has been a
weak or incomplete cAMP antagonist have been ascribed to low affinity
for the cAMP-binding sites of R, low lipophilicity, and inhibition of
cyclic nucleotide phosphodiesterase, thereby increasing endogenous
cyclic nucleotide(12, 13) .
In the present study
improved cAMP antagonists were searched for by screening a number of (Rp)-cAMPS analogs with enhanced lipophilicity. The compounds
were tested for ability to antagonize cAMP activation of isolated cAKI
and cAKII under near physiological assay conditions and as cAMP
antagonists in intact cells with different cAK isozyme expression. The
cell systems were: 1) cAMP-dependent gene transcription and cell
rounding in 3T3 fibroblasts overexpressing either RI or
RII
(14, 15) , 2) cAKI-mediated apoptosis in
IPC-81 promyelocytic leukemia cells(16, 17) , 3)
inhibition of DNA replication mediated by cAK in primary rat
hepatocytes(18) , and 4) inhibition of glucagon-induced
modulation of protein phosphorylation in
hepatocytes(19, 20) .
So far, little is known about
the biological significance of the basal cAK activity in cells not
stimulated with agents acting to increase the cAMP level. Study of
fibroblasts microinjected with a peptide inhibitor of cAK suggests that
decrease of the basal cAK activity is essential for cells to enter
M-phase(21) . On the other hand, S-49 lymphoma cells which lack
functional C subunit (22, 23, 24) appear to
have a normal cell cycle transit. In the present study the basal cAK
activity in hepatocytes was decreased by microinjection of RI or by
treatment with (Rp)-8-Br-cAMPS. The basal phosphorylation was
assessed by two-dimensional gel electrophoresis and autoradiography of
proteins from P
-prelabeled hepatocytes.
Evidence will be presented 1) that basal cAK activity exerts a tonic
inhibition of DNA replication (G
/S transit) in primary rat
hepatocytes, and 2) that basal cAK activity has a permissive effect on
interleukin-1
(IL-1
) action on hepatocyte DNA replication.
This appears to be a new type of link between the cAMP and IL-1
signaling pathways, whose interdependence have been
debated(25, 26, 27, 28, 29) .
The relative antagonistic effectiveness of (Rp)-analogs was not explained by their affinity for RI and RII only, and it was tested whether they differed in ability to stabilize the holoenzyme relative to the dissociated forms of cAK. This was done by assaying the ability of (Rp)-analogs to activate diluted cAKI and cAKII. It turned out that some (Rp)-analogs could act as partial agonists, and it was tested whether they under certain conditions could cooperate with cAMP to partially activate cAKII in intact cells.
The phosphotransferase activity of isolated cAK was assayed (17) at 37 °C and pH 7.2 in 15 mM HEPES with 5
mM magnesium acetate, 70 µM phosphoacceptor
peptide (Kemptide), 0.1 mM [-
P]ATP, 0.1 mM EGTA, 1
mM dithioerythritol, 0.5 mg/ml bovine serum albumin, and 130
mM KCl. The concentration of C subunit was either 0.15 nM (incubation time 30 min) or 10 nM (incubation time 1
min). The cellular content of RI, RII, and C was determined as
described earlier (17, 31) and related to protein
content as determined by the Bio-Rad version of the Coomassie Blue dye
binding assay, using
-globulin as standard.
The IPC-81 promyelocytic leukemia
cells were cultured in Dulbecco's minimal essential medium with
7% heat-inactivated horse serum. Cells in logarithmic growth phase (0.3
10
/ml) were treated with apoptosis inducer
(PGE
, cholera toxin), which sometimes was added after 30
min of preloading with (Rp)-cAMPS analog. After 9 h of
incubation, the cells were fixed in 3.5% formaldehyde with 10 µg/ml
bisbenzimide H 33258 and scored for apoptosis based on fragmentation
and hypercondensation of chromatin(17) .
Hepatocytes were
isolated from male Wistar rats (150-250 g) by collagenase
perfusion and grown on collagen gel with synthetic hepatocyte-selective
medium(18) . The medium was supplemented with insulin (0.2
nM) and dexamethasone (100 nM) 2 h after seeding, and
with epidermal growth factor (9 nM) 20 h after seeding. In
some incubations (Rp)-cAMPS analog was added after 43.5 h and
glucagon (2 nM) or IL-1 (0.4 nM) after 44 h of
culture. Hepatocyte replication was determined by pulse labeling of DNA
with [
H]thymidine (0.7 µCi/ml) from 55 to 56
h after seeding. Hepatocytes to be injected were cultured on dishes
with grids. They received cytoplasmic injections 52 h after seeding (18) and were pulsed with [
H]thymidine
(0.7 µCi/ml) from 60-61 h in culture. Processing for
autoradiography and determination of pulse labeling index were as
detailed previously(18) .
Figure 1:
(Rp)-cAMPS analogs as
antagonists of forskolin-induced cAMP actions in fibroblasts with or
without overexpression of RI and RII. 3T3 fibroblasts were treated with
forskolin (6 µM for wild type cells and 10 µM for the RI and RII
expressors) and various
concentrations of the following phosphorothioate analogs of cAMP: (Rp)-cAMPS ((Rp)-cA;
), (Rp)-8-Cl-cAMPS
(Rp8Cl-cA;
), (Rp)-8-Br-cAMPS (Rp8Br-cA;
) or (Rp)-N
-phenyl-cAMPS (RpN
P-cA;
; only data with 0.3 mM analog shown). The dotted
lines in panel A show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for
half-maximal antagonism of forskolin. The percentage of rounded
fibroblasts was scored as described under ``Experimental
Procedures.'' A representative region from a dish with cells
treated with forskolin alone is shown in panel D, and after
treatment with forskolin and 300 µM (Rp)-8-Cl-cAMPS in panel E. The data represent
the average of three separate experiments, each run in triplicate, the
error bars indicating the S.E. Further details are given under
``Experimental Procedures.''
The fibroblast results suggested that (Rp)-8-Br/Cl-cAMPS should be better cAMP antagonists than (Rp)-cAMPS in cells expressing mainly cAKI. Apoptosis in
IPC-81 leukemia cells is mediated by activation of cAKI(16) ,
which is the dominant isozyme in such cells (Table 1). IPC cell
apoptosis was induced by cholera toxin or PGE, which acted
via cAK since they were inefficient in a cell subclone (Fig. 2)
with mutant cAMP-subresponsive cAK(17) . The apoptogenic
actions of cholera toxin and PGE
were completely blocked by (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS. (Rp)-cAMPS was about 10-fold less potent and provided only
partial protection (Fig. 2).
Figure 2:
(Rp)-cAMPS analog antagonism of
IPC leukemia cell apoptosis induced by cholera toxin or
PGE. More than 80% of IPC-81 leukemia cells (open
symbols) became apoptotic (panel C) after treatment for 9
h with 0.03 nM cholera toxin (CT; panel A) or 0.1
µM PGE
(panels B and C).
Nearly all the cells were protected against CT and PGE
by (Rp)-8-Cl-cAMPS (
) and (Rp)-8-Br-cAMPS (
; panel D), (Rp)-cAMPS (
) being less potent. The dotted lines show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for
half-maximal antagonism. The solid symbols (▾) represent
data on the RI-mutated (Ala
Asp),
cAMP-subresponsive IPC cell line, in which cholera toxin gave a slight
induction of apoptosis which was abolished by (Rp)-8-Br-cAMPS.
The data presentation and statistics were as explained in the legend to Fig. 1.
The relative antagonistic
potency of (Rp)-8-Br/Cl-cAMPS and (Rp)-cAMPS in
RI-dominated cells (Fig. 1A and 2) was mirrored in
studies of isolated cAKI. cAMP activation of type I kinase under near
physiological pH, ionic strength, and temperature was antagonized more
potently by (Rp)-8-Cl-cAMPS and (Rp)-8-Br-cAMPS than
by (Rp)-cAMPS, and the inverse was true for type II kinase. It
was also noted that (Rp)-N-phenyl-cAMPS
was a less potent cAKI antagonist than (Rp)-cAMPS (Fig. 3).
Figure 3:
Differential antagonism of cAMP activation
of isolated cAKI and II by (Rp)-cAMPS analogs. cAKI from
rabbit muscle (panel A) or cAKII from bovine heart (panel
B), each at 10 nM, was incubated for 1 min under
phosphotransferase conditions with 0.3 µM cAMP and various
concentrations (abscissa) of (Rp)-cAMPS (), (Rp)-8-Br-cAMPS (
), (Rp)-8-Cl-cAMPS (
),
or, in the case of cAKI, (Rp)-N
-phenyl-cAMPS (
). The dotted lines show the concentrations of (Rp)-8-Br/Cl-cAMPS and of (Rp)-cAMPS required for
half-maximal antagonism of cAMP. The data represent from 7 to 10
determinations from three separate experiments. The bars represent the
S.E. Further details are given in ``Experimental
Procedures.''
This prediction was tested experimentally at cAK concentrations low
enough (0.15 nM) to obtain significant dissociation of
subunits even in the absence of cyclic nucleotide. It appeared that (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS hardly
destabilized (dissociated) cAKI holoenzyme at all, and much less than (Rp)-N-phenyl-cAMPS. (Rp)-cAMPS
and (Rp)-8-chlorophenylthio-cAMPS had intermediate
destabilizing effects. For cAKII (Rp)-8-Br/Cl-cAMPS were about
as dissociative as (Rp)-cAMPS (Fig. 4). These data
provided a rational explanation for the high potency and selectivity of (Rp)-8-Br/Cl-cAMPS as antagonists of cAMP activation of cAKI,
but also raised the possibility that (Rp)-analogs might act as
weak partial agonists rather than as pure antagonists of cAMP.
Figure 4:
(Rp)-cAMPS analog activation of
dilute cAKI and II. cAKI from rabbit muscle (hatched bars) or
cAKII from bovine heart (open bars), each at 0.15 nM,
was incubated for 30 min under phosphotransferase assay conditions in
the absence of cyclic nucleotide (CONTR) or with 50 µM of (Rp)-8-Br-cAMPS (Rp8Br-cA), (Rp)-8-Cl-cAMPS (Rp8Cl-cA), (Rp)-8-chlorophenylthio-cAMPS (Rp8CPT-cA), (Rp)-cAMPS ((Rp-cA), (Rp)-N-phenyl-cAMPS (RpN
P-cA), or cAMP (cA). The
data (representing determinations from three to four separate
experiments) are given with standard error of the
mean.
To
know if (Rp)-analogs would be likely to act as partial
agonists in intact cells, they were first tested for ability to
activate isolated cAK at a concentration (50 nM) approaching
that in intact cells (Table 1). Neither cAKI nor cAKII was
activated more than 7% by any of the (Rp)-analogs (data not
shown). This suggests that they will be incapable of significantly
activating cAK intracellularly when acting alone. In the intact cell,
however, the analogs coexist with endogenous cAMP, which might
cooperate with (Rp)-analogs to activate cAK. The principle was
demonstrated for isolated cAKII that (Rp)-analog bound to one
site of R might cooperate with cAMP (not shown) or an agonistic cAMP
analog (Fig. 5) bound to the other site. It was tested if the
degradation-resistant site AI/AII-selective (Table 2) N-butyryl-cAMP could cooperate similarly with the
site BII-selective (Rp)-8-Cl-cAMPS in intact fibroblasts. This
turned out to be the case in RII
-transfected fibroblasts (Fig. 6A), whereas (Rp)-8-Cl-cAMPS
desensitized the RI
-transfected cells against N
-butyryl-cAMP (Fig. 6B). The
RII-transfected cells were activated synergistically also when the site
BI/BII selective 8-methylamino-cAMP was combined with the site AI/AII
preferring (Rp)-N
-phenyl-cAMPS (Fig. 7A). Very little synergism was noted when the
same analog pair was tested on the RI overexpressing fibroblasts (Fig. 7B). Since (Rp)-N
-phenyl-cAMPS selects site A of
both RI and RII (Table 2) its more modest synergism with
8-methylamino-cAMP in RI cells could not be explained by lack of
cAMP-binding site complementarity, indicating that cells chiefly
expressing the type I kinase may be less subject to the activating
effect of (Rp)-cAMPS analogs.
Figure 5:
(Rp)-8-Cl-cAMPS complementation
of cAKII activation by N-benzoyl-cAMP. cAK type I
(
) or type II (
) was incubated with, respectively, 0.2 and
0.1 µMN
-benzoyl-cAMP (N
B-cA) and various concentrations (abscissa) of (Rp)-8-Cl-cAMPS. The experimental
details were otherwise similar to those of the experiments shown in Fig. 3.
Figure 6:
(Rp)-8-Cl-cAMPS complements N-butyryl-cAMP action in fibroblasts predominantly
expressing type II kinase. Activation of 3T3 fibroblast cAK was
assessed by increased cAMP-responsive gene element-directed luciferase
activity (
,
,
) and cell rounding (
,
,
). The luciferase activity (left ordinate) is given as
percent of the activity (routinely about 2
10
counts/min/mg extract protein) in extracts from cells treated
with 10 µM forskolin and 300 µM
isobutylmethylxanthine. The data are given after subtraction of rounded
control cells (<7%) and luciferase activity in extracts from control
cells (<8%). The upper panel (A) represents H1.9
fibroblasts overexpressing the RII
subunit, and the lower
panel (B) represents RN1.25 fibroblasts overexpressing
the RI
subunit (for details of expression see Table 1). The dashed lines show the dose-response curve for N
-butyryl-cAMP when combined with 250 µM (Rp)-8-Cl-cAMPS (Rp8Cl-cA;
,
), the thin solid lines show the curve for N
-butyryl-cAMP combined with 40 µM 8-chlorophenylthio-cAMP (8CPT-cA;
,
), and
the thick solid lines show N
-butyryl-cAMP
alone (
,
). Due to the close correlation between
cAMP-responsive gene element-directed gene expression and cell rounding
both parameters are represented by common lines. The data shown are
from representative experiments. Similar results were obtained when the
concentration of 8-chlorophenylthio-cAMP was 30 or 60 µM rather than 40 µM (not shown). Further details of the
assays for luciferase expression and cell rounding are given under
``Experimental Procedures.'' Analog site affinities are
listed in Table 2.
Figure 7:
(Rp)-N-phenyl-cAMPS
complements 8-methylamino-cAMP action in fibroblasts predominantly
expressing type II kinase. The upper panel (A)
represents H1.9 fibroblasts overexpressing the RII
subunit, and
the lower panel (B) represents RN1.25 fibroblasts
overexpressing the RI
subunit. The experimental set up was as
described in the legend to Fig. 6, except that the cells were
exposed to various concentrations (abscissa) of the
site BI/BII-selective 8-methylamino-cAMP rather than the site AI/AII
selective N
-monobutyryl-cAMP. The dashed lines show the dose-response curve for 8-methylamino-cAMP when combined
with 250 µM (Rp)-N
-phenyl-cAMPS (Rp-N
P-cA;
), the thin solid
lines show the curve for 8-methylamino-cAMP combined with 80
µM 8-chlorophenylthio-cAMP (8-CPT-cA;
,
), and the thick solid lines show 8-methylamino-cAMP
alone (
,
).
The validity of the observed
(partially) agonistic actions of (Rp)-analogs (Fig. 4-7) depends crucially on their chemical purity.
Early reports that (Rp)-cAMPS was a partial agonist for kinase
activation (41, 42) were later dismissed as being due
to contamination by (Sp)-cAMPS and
cAMP(8, 43, 44) . High performance liquid
chromatography analysis of the presently used (Rp)-cAMPS and (Rp)-N-phenyl-cAMPS after incubation with
kinase for 1 h at 37 °C revealed single peaks comprising more than
99.1% of the UV-absorbing material applied. Comparison of the site AI
mapping data for N
-phenyl-cAMP and its (Rp)- and (Sp)-analogs reveals (Table 2) that
at most 0.9% of the (Rp)-N
-phenyl-cAMPS
preparation could be (Sp-N
-phenyl-cAMPS,
even if all the binding activity were due to contaminating (Sp)-N
-phenyl-cAMPS. Similarly, if all
the binding activity were due to N
-phenyl-cAMP
this potential contaminant could at most be present at 0.1% of the
concentration of (Rp)-N
-phenyl-cAMPS. To
know if a contamination of this extent could explain the activating
properties of (Rp)-N
-phenyl-cAMPS, it was
made 0.1% in N
-phenyl-cAMP and 1% in (Sp)-N
-phenyl-cAMPS and assayed for
kinase activation. Neither the concentration dependence nor the maximal
level of activation were affected by such admixture of N
-phenyl-cAMP and (Sp)-N
-phenyl-cAMPS. Furthermore, (Rp)-cAMPS treated with phosphodiesterase had the unaltered
ability to activate the kinases (data not shown). The results obtained
were therefore due to the (Rp)-analogs and not contaminants. (Rp)-cAMPS has been reported to induce dissociation of type I
isozyme only in the absence of MgATP(45) . Under the conditions
of the present study (Fig. 4), (Rp)-cAMPS was
activating whether ATP was 50 µM, 100 µM, or
1 mM (data not shown).
Figure 8:
(Rp)-cAMPS analog effects on
basal, interleukin-1-inhibited, and glucagon-inhibited hepatocyte
DNA replication. Primary rat hepatocytes treated with 0.4 nM interleukin-1
(IL-1
) or 2 nM glucagon
from 44 to 56 h after seeding had about half as many
[
H]thymidine-labeled nuclei (ordinate)
as control cells. The effects of IL-1
(panel A) or
glucagon (panel B) were completely overcome by (Rp)-8-Cl-cAMPS (panels A and B;
) and (Rp)-8-Br-cAMPS (panels A and B;
),
whereas the effect of glucagon was only partially overcome by (Rp)-cAMPS (panel B;
). Panel C illustrates the low percentage of
[
H]thymidine-labeled nuclei in hepatocytes
treated with 2 nM glucagon, and panel D shows the
much higher labeling when the cells had been cotreated with 150
µM (Rp)-8-Cl-cAMPS and 2 nM glucagon. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS not only
antagonized IL-1
and glucagon, but also increased the labeling
index above the level in control cells (panel A, solid
symbols). The data are given with standard error of the mean for
analog concentrations tested in three or four separate
experiments.
Figure 9:
Autoradiograms of two-dimensional PAGE of
extracts from P
-prelabeled cultured rat
hepatocytes: effects of (Rp)-8-Br-cAMPS, glucagon, and
interleukin-1
. Hepatocytes were labeled with
P
from 43 h after seeding and treated with 300 µM (Rp)-8-Br-cAMPS from 43.5 to 44.5 h (panels B and D), 2 nM glucagon from 44 to 44.5 h (panels C and D), 0.4 nM interleukin-1
(IL-1
) from 44 to 44.5 h (panel E), or left
untreated as control (panel A). The proteins were separated by
two-dimensional gel electrophoresis and autoradiographic spots
subjected to computer analysis as described under ``Experimental
Procedures.'' The arrows in panel A (spots
1-14) point to proteins whose phosphorylation state changed
after treatment with either glucagon, IL-1
, or (Rp)-8-Br-cAMPS. Treatment with (Rp)-8-Br-cAMPS (panel B) decreased the phosphorylation of three proteins (spots 1-3) and increased the phosphorylation of two
proteins (spots 4 and 5). Glucagon (panel C)
stimulated the phosphorylation of eight proteins (spots 1-3 and 10-14), whereas the phosphorylation of
two proteins (spots 4 and 5) was inhibited. (Rp)-8-Br-cAMPS appeared to abolish the phosphorylation
response to 2 nM glucagon (panel D). Four
phosphoproteins (spots 6-9) were uniquely affected by
IL-1
(panel E). The autoradiograms shown are
representative examples from four to nine separate
experiments.
Figure 10:
Microinjected RI
increased basal hepatocyte DNA replication and counteracts
interleukin-1
inhibition of DNA synthesis. Primary rat hepatocytes
cultured for 52 h were microinjected with mutant RI
or exposed to (Rp)-8-Br-cAMPS. 0.4 nM Interleukin-1
(IL-1
) was added to some of the
cultures 30 min later. After 60 h in culture the cells were pulsed (1
h) with [
H]thymidine and processed for
autoradiography. Both injection of RI
and addition
of (Rp)-8-Br-cAMPS counteracted the inhibitory effect of
IL-1
on DNA replication. The labeling index increased also in
control cells after injection of RI
or addition of (Rp)-8-Br-cAMPS. The standard error of the mean of three to
six experiments is indicated by error bars, each involving the
microinjection of 360-460 cells for every experimental condition.
Further details are given under ``Experimental
Procedures.''
An eventual involvement of cGMP-dependent protein kinase was addressed by testing whether (Rp)-8-Cl-cGMPS or (Rp)-8-Br-cGMPS could counteract the glucagon action or increase basal DNA replication. None of the (Rp)-cGMPS analogs were efficient in either respect (data not shown). This observation argues against the cGMP kinase being the target for (Rp)-8-Br/Cl-cAMPS. Furthermore, microinjected R (Fig. 10) is completely specific for cAK.
The novel compounds (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were superior to the parent compound (Rp)-cAMPS as cAMP antagonist for isolated cAKI (Fig. 3) and in cells predominantly expressing cAKI, like IPC
leukemia cells (Fig. 2), RI-transfected fibroblasts (Fig. 1), and primary hepatocytes (Fig. 8). The
available evidence indicates that (Rp)-8-Br/Cl-cAMPS in these
cells acted solely by decreasing the cAK activity: 1) in RI-transfected
fibroblasts the relative antagonistic potencies of (Rp)-8-Br-cAMPS, (Rp)-8-Cl-cAMPS, and (Rp)-cAMPS (Fig. 1A) were about as for
isolated cAKI (Fig. 3A). 2) IPC cells treated with (Rp)-8-Br/Cl-cAMPS were as resistant to cAMP elevating agents
as mutant IPC cells with cAMP-subresponsive cAK (Fig. 2). 3) The
effect of (Rp)-8-Br/Cl-cAMPS on hepatocyte DNA replication was
reproduced by microinjected R-subunit (Fig. 10), which is a
completely specific inhibitor of the C subunit of cAK. 4) Halogenated (Rp)-cGMPS analogs, which interact preferentially with the
cGMP-kinase(46) , could not mimic the effects of (Rp)-8-Br/Cl-cAMPS on hepatocyte DNA replication. 5) (Rp)-8-Br/Cl-cAMPS selectively counteracted the action of the
classical cAMP elevating agent glucagon with regard to protein
phosphorylation in hepatocytes (Fig. 9; Table 3and Table 4). It is therefore proposed that (Rp)-8-Br-cAMPS
and (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as
first line cAMP antagonist, particularly for studies in cells
expressing predominantly cAKI.
(Rp)-Analogs could decrease
even the basal cAK activity, as judged by depressed basal
phosphorylation of proteins whose phosphorylation increased in response
to elevated cAMP (Fig. 9; Table 3). That basal cAK
activity can be responsible for protein phosphorylation is supported by
studies of a subline (kin) of S-49 lymphoma cells
with genetically deficient expression of functioning C
subunit(47) , and a possible decrease of phosphorylation of
pyruvate kinase observed in hepatocytes in suspension treated with (Rp)-cAMPS(20) . (Rp)-8-Br-cAMPS enhanced the
phosphorylation of proteins whose phosphorylation was negatively
regulated by cAMP (Fig. 9; Table 3). This indicates that
the basal cAK activity is responsible also for negative regulation of
protein phosphorylation, which therefore must be a phenomenon sensitive
to very slight activation of cAK. The presumably indirect mechanisms
whereby cAK depresses protein phosphorylation can involve inhibition of
other kinases (48, 49) or stimulation of
phosphatases(49, 50, 51, 52) .
Since kin S-49 cells appear to have normal
proliferation rate and cell cycle traverse (22, 24, 47) the basal cAK activity cannot be
universally essential for cell cycle traverse. Evidence has been
presented, however, that down-regulation of cAK activity is required
for induction of fibroblasts mitosis(21) . The present study
pointed to two other proliferation-related actions of the basal cAK
activity in primary hepatocytes. First, their DNA replication was
enhanced when the basal cAK activity was decreased by either (Rp)-8-Br-cAMPS or by microinjection of R subunit ( Fig. 8and Fig. 10). This indicates that the basal cAK
activity exerted a tonic inhibition of G
/S transition rate
in epidermal growth factor-stimulated hepatocytes. Second, the
inhibitory action of IL-1
on hepatocyte DNA replication was nearly
abolished when the basal cAK activity was inhibited ( Fig. 8and
10). This indicates that the basal cAK activity permits the
IL-1
-induced inhibition of DNA replication. The links between cAMP
and IL-1
-signaling pathways have been debated, and obviously
differ between cell types(25, 26, 27) . There
is evidence for increased cAMP in response to IL-1
(28) and for IL-1
-induced activation of cAK without any
increase of cAMP(29) . The present study added the more subtle
concept of permissive action of cAK for IL-1
action to the list of
links between the two signaling pathways. Such permissive actions of
the basal cAK activity may be more common than hitherto realized, since
few studies have attempted to decrease the basal cAK activity.
Hopefully, the advent of the improved (Rp)-cAMPS analogs may
help elucidate more such cases. It may be noted that without the clear
evidence that cAK-dependent phosphorylation events were unaffected by
IL-1
(Fig. 9; Tables III and IV) the data of Fig. 8A would easily have been misinterpreted as
proving a mediator role of cAK activation for IL-1
action.
Obviously, that an effect is blocked by inhibitors of cAK is not
conclusive evidence that the observed effect was mediated by increased
activation of cAK.
A novel and unexpected observation was that adenine modifications affected the action of (Rp)-cAMPS analogs on the equilibrium between the dissociated and holoenzyme forms of cAK, the usefulness of (Rp)-8-Br/Cl-cAMPS as cAKI antagonists being related to the very low dissociation of cAKI isozyme in the presence of these (Rp)-analogs (Fig. 4). Unfortunately, all available (Rp)-analogs induced significant dissociation of cAKII (Fig. 4), and, under certain conditions, could contribute to partial activation of cAKII in intact cells (Fig. 6A and 7A). (Rp)-Analogs should therefore be used with caution in studies of cells predominantly expressing cAKII. Ongoing experiments are aimed at synthesizing novel (Rp)-analogs with improved agonist activity toward cAKII. These efforts will be guided by the observations of the present study (Fig. 4).