(Received for publication, November 7, 1996)
From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750
The cAMP-dependent protein kinase
holoenzyme is assembled from regulatory (R) and catalytic (C) subunits
that are expressed in tissue-specific patterns. Despite the dispersion
of the R and C subunit genes to different chromosomal loci, mechanisms
exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved.
We have created null mutations in the RI and RII
regulatory
subunit genes in mice, and find that both result in an increase in the level of RI
protein in tissues that normally express the
isoforms. Examination of RI
mRNA levels and the rates of RI
protein synthesis in wild type and RII
mutant mice reveals that the
mechanism of this biochemical compensation by RI
does not involve
transcriptional or translational control. These in vivo
findings are consistent with observations made in cell culture, where
we demonstrate that the overexpression of C
in NIH 3T3 cells results
in increased RI
protein without increases in the rate of RI
synthesis or the level of RI
mRNA. Pulse-chase experiments
reveal a 4-5-fold increase in the half-life of RI
protein as it
becomes incorporated into the holoenzyme. Compensation by RI
stabilization may represent an important biological mechanism that
safeguards cells from unregulated catalytic subunit activity.
The cAMP-dependent protein kinase (PKA)1 is a key regulatory enzyme responsible for the intracellular transduction of a variety of extracellular signals and for the maintenance of numerous aspects of cellular homeostasis (1). The holoenzyme is composed of a regulatory (R) subunit dimer complexed with two catalytic (C) subunits. Two molecules of cAMP bind to each R subunit causing release of enzymatically active C subunits, which then modify the activity of target proteins by reversible phosphorylation of serine or threonine residues located within an appropriate consensus sequence (2).
Four R subunit isoforms and two C subunit isoforms of PKA have been
characterized in the mouse (3). They are highly conserved among
mammals, encoded by unique genes located on separate chromosomes, and
show unique patterns of gene expression. The -isoforms are expressed
ubiquitously while
isoforms show more restricted patterns of
expression. RI
is induced relatively late in development and is
highly expressed in neural tissues (4-6). RII
is expressed during
embryogenesis in mouse brain, spinal cord, and liver (7). In adult mice
RII
protein is most abundant in brain and brown and white adipose
tissue, with lower expression in testis and ovary (8). C
is most
abundant in the brain, but lower levels of C
mRNA are found in
all tissues examined (9).
PKA holoenzymes can be separated by ion-exchange chromatography and analysis of a variety of mammalian tissues has revealed significant differences in the ratio of type I (RI-containing) to type II (RII-containing) holoenzyme (10). In rats and mice, brain and adipose tissue contain principally the type II holoenzyme, while heart and liver contain mainly type I. The ratio of type I to type II holoenzyme in individual tissues also varies across species. While mouse and rat hearts possess mainly the type I holoenzyme, beef and guinea pig hearts have principally the type II holoenzyme, with human and rabbit hearts showing equivalent amounts of both holoenzymes (11).
The type I to type II holoenzyme ratios can also change dramatically
during cell development. Differentiation of Friend erythroleukemic cells results in a large increase in total PKA activity and a shift
from equimolar amounts of type I and type II holoenzyme to a majority
of RII-containing holoenzyme (12). A similar selective increase in
the RII
regulatory subunit occurs in differentiating ovarian
follicles treated with estradiol and follicle-stimulating hormone (13).
Selective increases in the RI
regulatory subunit and the type I
holoenzyme occur during the differentiation of L6 myoblasts, which also
show increases in total PKA activity (14). A similar phenomenon has
been observed during the differentiation of 3T3-L1 cells (15).
Although the ratio of type I to type II holoenzyme varies in different
cell types and stages of differentiation, total R and C subunit levels
are thought to be equivalent in a variety of tissues (16). How this
extremely tight coordination of R and C subunits is achieved in all
tissues remains to be determined; however, experiments performed in
cell cultures have revealed one potential mechanism (17, 18). The
ubiquitous RI subunit has been shown to be unstable when not
associated with the C subunit in the type I holoenzyme. In
Kin
cells that lack detectable C subunit, RI
subunits
are rapidly degraded and the steady-state level of RI
is reduced
(17, 19). In contrast, overexpression of the C subunit in NIH 3T3 cells elicits a coordinate increase in RI
protein (18).
In this report we show that loss of RI or RII
in gene-disrupted
mice results in biochemical compensation by RI
with no change in
RI
mRNA levels. We demonstrate in cell culture that this
compensation is due to a decrease in the turnover rate of RI
protein
when it associates with the C subunit. The capacity of RI
to
compensate for changes in C subunit expression provides a mechanism to
protect cells from unregulated C subunit activity during developmental
and hormonally induced changes in PKA subunits.
Generation of RI and RII
mutant mice has been
described (8, 20). Both mutant and wild type mice used in the
experiments were age-matched and maintained on the same mixed
C57BL/6 × 129Sv/J genetic background.
Wild type mouse NIH 3T3 fibroblasts and
C3T3 cells (NIH 3T3 cells stably transfected with a plasmid
containing the zinc-inducible metallothionein promoter driving
expression of the mouse C
subunit) were maintained in Dulbecco's
modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).
Exponentially growing cells in 10 cm plates were treated for 24 h
with 90 µM zinc sulfate in DMEM containing 10% FBS and
then harvested as described previously (18).
Brain and white adipose tissue were
isolated from RI and RII
mutant and wild type animals,
immediately placed in liquid nitrogen, and stored at
70 °C.
Samples were thawed into homogenization buffer (250 mM
sucrose, 100 mM NaPO4, pH 7.0, 150 mM NaCl, 1 mM EDTA, 4 mM EGTA, 4 mM dithiothreitol, 0.5% Triton X-100, 2 µg/ml leupeptin,
3 µg/ml aprotinin, 0.2 mg/ml soybean trypsin inhibitor, 1 mM AEBSF), sonicated, and centrifuged at 16,000 × g, and the supernatant was collected and assayed for protein
concentration using a Bradford assay (Bio-Rad). Total protein (40 µg)
was run on 10% polyacrylamide gels and transferred to nitrocellulose
membranes. Blots were then blocked overnight and probed with
affinity-purified polyclonal antibodies to RI
, C
, or RII
.
Blots were then washed and incubated with horseradish
peroxidase-conjugated secondary antibodies and visualized using the
Amersham ECLTM system.
Wild type NIH 3T3 cells and
C3T3 cells were treated with 90 µM zinc sulfate for
24 h. Cells were then washed twice in labeling media (Hanks'
balanced salt solution, 5% NaHCO3, 1% bovine serum albumin, 25 mM Hepes, pH 7.2, 100 units/ml penicillin, 100 µg/ml streptomycin) and then incubated for 1 h at 37 °C with
200 µCi/ml EXPRE35S35S protein-labeling mix
(Dupont NEN). After 1 h, cells were harvested by washing twice in
cold phosphate-buffered saline (20 mM NaPO4, pH
7.0, 150 mM NaCl) followed by addition of lysis buffer (250 mM sucrose, 25 mM Tris, pH 7.2, 25 mM NaCl, 5 mM MgCl2, 1 mM AEBSF, 1% Triton X-100, 1% sodium deoxycholate).
Plates were then scraped, transferred to Eppendorf tubes, sonicated,
and spun for 1 h at 100,000 × g. Supernatants
were recovered and stored at
70 °C. To determine
35S-incorporation into total protein, 2 µl from each
sample was spotted onto Whatman GF/C filters, and protein was
precipitated in 10% trichloroacetic acid, followed by three washes in
3% trichloroacetic acid/1% sodium pyrophosphate. Filters were then
dried and counted in liquid scintillation fluid. Samples containing
equivalent total radioactivity were brought to a final volume of 100 µl in a lysis buffer containing 100 mM NaCl and 40 µM cAMP and incubated for 2.5 h with
affinity-purified polyclonal anti-RI
antibodies followed by 30 min
with 3 µl of a 10% suspension of Protein A-Insoluble (Sigma). Reactions were then overlaid on a cushion of
lysis buffer containing 1 M sucrose and centrifuged to
pellet the immunoprecipitates, which were stored at
70 °C. Pellets
were resuspended and run on 10% SDS-polyacrylamide gel electrophoresis
(PAGE) gels. Gels were fixed for 30 min in 10% methanol, 5% acetic
acid, followed by a 30-min incubation in AmplifyTM. Gels
were then dried and exposed to XARTM Kodak film for 24 h. For determination of RI
translation rates in adipocytes, wild
type and RII
mutant mice were sacrificed, and white adipose tissue
from uterine fat pads was weighed and immediately minced using fine
razor blades, and then placed in scintillation vials containing 1 ml of
adipocyte media (100 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1 mM
NaH2PO4, 12 mM Hepes, pH 7.2, 2.5 mM CaCl2, 1 mg/ml glucose, 1% bovine serum
albumin, 33.6 mg/liter NaHCO3) in a 37 °C rotating water
bath at 40 rpm. After placing all of the fat pads in culture, 1 mg/ml
collagenase was added to each vial and incubated for 1 h to
dissociate the cells. At the end of 1 h, cells were washed 4 times
in 5 volumes of adipocyte media to remove the collagenase and
resuspended in 1 ml of adipocyte media containing 200 µCi/ml
EXPRE35S35S protein-labeling mix and placed in
a 40 rpm rotating water bath at 37 °C. At the end of 1 h, cells
were washed 4 times in 5 volumes of adipocyte media without bovine
serum albumin, and the pellets were immediately frozen at
80 °C.
Immunoprecipitation and analysis of RI
protein were performed as
described above.
After labeling of NIH 3T3 and
C3T3 cells for 1 h with 200 µCi/ml
EXPRE35S35S protein-labeling mix, duplicate 10 cm plates were washed twice in DMEM, 10% fetal bovine serum and then
incubated in DMEM, 10% fetal bovine serum plus 90 µM
zinc sulfate containing 4 mM L-methionine. At
each time point the cells were harvested and processed as described above. For the immunoprecipitation reactions, equivalent amounts of
total protein were loaded rather than equivalent counts.
HPLC analysis was
performed as described (6). Wild type and RII mutant mice were
sacrificed, and uterine fat pads were immediately isolated, weighed,
and stored at
70 °C. Fat pads were homogenized in buffer (20 mM Tris, pH 7.6, 0.1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 10 mM
dithiothreitol, 5 mM magnesium acetate, 250 mM
sucrose, 1 µg/ml leupeptin, 3 µg/ml aprotinin, 100 µg/ml soybean
trypsin inhibitor, 0.5 mM AEBSF, 100 µM ATP) and centrifuged for 30 min at 16,000 × g, and the
supernatants were assayed for protein concentration using a Bradford
assay (Bio-Rad). Samples diluted with homogenization buffer to a final concentration of 1-2 mg/ml were loaded onto a DEAE/HPLC column and
eluted using a linear NaCl gradient from 0 mM to 250 mM. Fractions were collected and assayed for kinase
activity in the presence and absence of 5 µM cAMP with
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) as a substrate (21).
The method used for measuring total
amounts of RI and C
mRNA has been described (9). Briefly,
total nucleic acid samples isolated by proteinase K digestion and
phenol/chloroform extraction were incubated with a single-stranded
[32P]CTP-labeled RNA probe at 70 °C for 16 h.
Following hybridization, samples were digested with RNase A and T1,
precipitated in 10% trichloroacetic acid, and filtered onto Whatman
GF/C filters. The amount of RNase-resistant probe was determined by
liquid scintillation counting. RI
- and C
-specific mRNA in
each sample was determined by comparison to a standard curve
constructed with known amounts of M13 DNA containing the sense strand
of the RI
and C
cDNAs. The results, calculated as picograms
of RNA hybridized per µg of total nucleic acid, were converted to
molecules/cell by assuming 6 pg of DNA/cell.
We have previously reported that
targeted disruption of the neural-specific RI gene in mice results
in deficiencies in hippocampal long term potentiation and long term
depression (20, 22). Western blots using protein extracts from the
cerebral cortex and hippocampus of RI
mutant mice were compared with
age-matched controls to quantitate changes in RI isoforms. This
analysis demonstrated a compensatory increase in RI
protein in both
tissues (Fig. 1), whereas no changes were observed in C
or RII isoforms (data not shown). RI
protein levels were determined
by densitometry of Western blots from wild type and RI
mutant
protein extracts. Densitometry analysis revealed an approximate 40%
increase in RI
protein in both the cerebral cortex and hippocampus
of RI
mutant mice (Table I). In order to address
whether the increase in RI
protein was due to an elevation in
transcription from the RI
gene, solution hybridization experiments
were performed using total nucleic acid isolated from cortex and
hippocampus of wild type and RI
mutant mice. This analysis revealed
no change in RI
mRNA levels in mutant tissues (Table I).
|
The RII regulatory subunit is highly expressed
in both white adipose tissue (WAT) and brown adipose tissue in mice. A
targeted disruption of the RII
gene has been created that displays
marked alterations in both WAT and brown adipose tissue metabolism (8). In order to address potential compensation by other regulatory subunits
in mice carrying a null mutation in the RII
gene, Western blots were
performed on WAT from wild type and RII
mutant mice. RII
mutant
mice showed a complete loss of the RII
protein (Fig. 2A). Separate Western blots examining the levels of
RI
and C
revealed a 3-4-fold increase in RI
protein in RII
mutant WAT, while C
protein was reduced by approximately 43%
(Fig. 2B). RI
mRNA levels in total nucleic acid
samples from WAT of wild type and RII
mutant mice were
identical (Table II).
|
The
large increase in RI protein observed in WAT from RII
mutant mice
suggests that the RI
subunit has replaced RII
and formed a type I
holoenzyme. HPLC analysis of WAT obtained from wild type mice revealed
that the majority of PKA activity was associated with the type II
holoenzyme together with a small free C subunit peak (Fig.
3). In contrast, WAT from RII
mutant mice contained
only type I holoenzyme. Western blots using protein from HPLC fractions
containing the type I holoenzyme peak confirmed the presence of RI
and C
in these fractions (data not shown). Peak activity fractions
were also assayed in the presence of the heat-stable PKA inhibitor,
PKI, which confirmed that all the kinase activity was
PKA-dependent.
The Rate of Translation of RI
In order to address
the mechanism of RI compensation in RII
mutant mice,
pulse-labeling experiments were performed in primary cultures of white
adipocytes from wild type and RII
mutant mice. No significant
difference was observed in the rate of translation of RI
protein
between wild type and RII
mutant mice after a 1-h pulse (Fig.
4B). Western blots from the same extracts
used to perform the pulse-labeling experiments confirmed that RI
protein was substantially increased in RII
mutant mice (Fig.
4A). This implies that the increased RI
protein must be
due to stabilization of the protein.
Altered RI
Loss of either RI or RII
would result in an
excess of C subunit over R unless a compensatory mechanism exists to
maintain the R/C balance. The observed increase in RI
appears to be
a response to this imbalance. We used a cell line stably transfected with the C subunit to characterize further the mechanism of RI
compensation under conditions where the C subunit is expressed in
excess of R. We have previously shown that when C is overexpressed in
NIH 3T3 cells there is a specific increase in RI
with no change in
RII subunits (18). This increase in RI
resulted in the appearance of
new type I holoenzyme, analogous to the results shown in Fig. 3 for
RII
mutant WAT (18, 23). We therefore used these C
-overexpressing 3T3 cells for metabolic labeling studies to determine the mechanism of
RI
compensation. Wild type NIH 3T3 fibroblasts and C
3T3 cells stably expressing a zinc-inducible expression vector for the mouse C
catalytic subunit (18) were treated with 90 µM zinc
sulfate for 24 h and subsequently analyzed by Western blotting for
RI
and C
. Zinc treatment of C
3T3 fibroblasts resulted in a
27-fold increase in C
protein compared with values obtained from
wild type 3T3 cells (Table III). As previously observed
(18), an elevation in RI
protein was also seen upon overexpression
of C
(Fig. 5A). Western blot analysis of a
range of protein dilutions from C
3T3 cells and wild type 3T3 cells
showed a 4-fold increase in RI
protein (Table III).
|
Solution hybridization experiments demonstrated that mRNA levels
for RI remained constant despite the elevation in RI
protein (Table III). The increase in RI
protein could be due to either an
elevated rate of translation or a stabilization of RI
protein. In
order to determine the mechanism, zinc-treated NIH 3T3 and C
3T3
cells were labeled for 1 h with [35S]methionine
followed by immunoprecipitation of RI
protein. The rate of synthesis
of RI
protein in wild type NIH 3T3 and C
3T3 cells was equivalent
(Fig. 5B). Western blot analysis from the same extracts confirmed that
C subunit protein was indeed overexpressed in the zinc-treated C
3T3
cells as expected (data not shown).
Pulse-chase experiments were performed to determine the half-life of
RI protein in wild type NIH 3T3 cells and C
3T3 cells. The
half-life of RI
in wild type NIH 3T3 cells was approximately 3.5 h as measured by immunoprecipitation of
35S-labeled RI
protein from cell extracts after a cold
chase with unlabeled methionine. In contrast, the half-life of RI
protein in zinc-treated C
3T3 cells was 13.5 h (Fig. 5C). This
represents a 4-fold increase in the half-life of the RI
protein upon
overexpression of C
and is in good agreement with the 4-fold
increase in RI
protein observed in this experiment.
The ability of mammalian cells to assemble and regulate multisubunit protein complexes usually relies on some type of autoregulatory loop. Whereas bacteria frequently solve the problem of coordinate regulation by utilizing multigene operons controlled by a single promoter, in higher animals the genes encoding interacting subunits are dispersed, frequently onto different chromosomes. Nevertheless, expression from these genes generally results in stoichiometric levels of protein subunits. The problem of coordinate regulation becomes crucial when an excess of one of the subunits might lead to undesirable biological effects in the cell. The PKA holoenzyme system is an example of such a case in which an excess of catalytic subunit could result in unwanted biological effects and reduce the ability of the cell to regulate activity by cAMP. Four separate regulatory and two catalytic subunits are produced, sometimes within the same cell, and yet most tissues are able to maintain an equimolar ratio of R and C subunits (16).
In this report we have artificially perturbed the expression of RI
and RII
subunits using targeted gene disruption in mice and examined
the compensatory mechanisms that regulate R/C subunit balance in
neurons and adipose tissue. In neurons of RI
mutant mice, levels of
RI
increase and at least partially replace the lost RI
subunit.
In adipose tissue from RII
mutant mice, we find a dramatic
compensation by RI
and only a modest loss in total C subunit. In
both cases, the increase in RI
protein is due to stabilization by
incorporation into holoenzyme. Since it was not possible to quantitate
the changes in RI
half-life by pulse-chase experiments in whole
animals, we have used a cell culture model system in which the
overexpression of exogenous C subunit elicits an increase in RI
very
similar in magnitude to that observed in WAT from RII
mutants.
Pulse-chase experiments in the cell culture system demonstrate a 4-fold
increase in RI
half-life when it is incorporated into holoenzyme and
stabilized by interaction with C subunit.
Previous studies have shown that the R and C subunits are stabilized
against proteolysis when assembled as a holoenzyme. Stabilization of
RI through binding to the C subunit has been demonstrated in S49
mouse lymphoma cells (17). Kin
cells, which lack
detectable C subunit, show a 10-fold increase in the turnover rate of
RI
protein and a significant decrease in steady-state RI
levels
when compared with wild type S49 cells (17, 19). However, when wild
type S49 cells are treated with agents that raise cAMP and separate the
R and C subunits, the RI
protein is destabilized to the same extent
observed in Kin
cells. The C subunit is also exposed to
degradative pathways when released from the holoenzyme complex. Chronic
activation of LLC-PK cells with cAMP can lead to the loss of more than
75% of the cell's complement of C subunit within 12.5 h
(24).
What are the rules governing the assembly of type I and type II holoenzymes in vivo? Experiments in cell culture have shown that C subunits preferentially assemble with RII subunits rather than RI subunits (25, 26). NIH 3T3 cells and wild type WAT express both RI and RII subunits. However, when holoenzymes are separated by ion-exchange chromatography only the type II holoenzyme is observed (8, 23). When NIH 3T3 cells are programmed to overexpress exogenous C subunit, the formation of new type I holoenzyme occurs (18, 23), suggesting that there is an ordered assembly of first type II and then type I holoenzyme. In contrast, overexpression of RI subunits does not alter the amount of type II holoenzyme nor does it result in increased formation of type I holoenzyme. This suggests that the total amount of free C subunit is rate-limiting with respect to formation of first type II and then type I holoenzyme (6, 25).
Emerging from these studies is an appreciation of the cell's capacity
to maintain cAMP-mediated control of C subunit activity and the
important role played in this process by RI. A simple model
describing the dynamic assembly of R and C subunits is depicted in Fig.
6 using the example of WAT from wild type and RII
mutant mice. In adipocytes, the RII
subunits preferentially
associate with C, leaving a pool of free RI
that is rapidly
degraded. Type I holoenzyme is only formed when the level of C subunits
exceeds the level of RII subunits (in this case caused by the loss of RII
). In this situation RI
can successfully compete for binding to the pool of free C subunits and is therefore stabilized in a
holoenzyme complex. Preferential binding of RII subunits to C probably
does not arise because of intrinsic differences between RI and RII
subunits in their affinity for C, as these affinities have been shown
to be quite similar (27). We propose that the phenomenon occurs as a
result of a lower Ka for cAMP-dependent activation of the RI holoenzyme compared with the RII holoenzyme. Free
RI subunits have been shown by numerous investigators to have a higher
affinity for cAMP than do RII subunits. Published values for the
Kd of RI-cAMP binding range from 0.1 (28) to 1 nM (29). In contrast, higher Kd values
for RII-cAMP binding are consistently reported, ranging from 4 (30) to
6 nM (31). We have shown that the apparent
Ka for cAMP activation of RI
holoenzyme is about
4-fold lower than that for RII
holoenzyme when measured in cell
extracts (8). Given the enhanced sensitivity to activation of
RI-containing holoenzyme, we predict that C subunits would shift
preferentially to the RII-containing holoenzyme complex until the RII
binding capacity of the cell is saturated.
When the concentration of free C subunit increases due to the loss of
RI or RII
, RI
rapidly responds to this perturbation via
protein stabilization in a holoenzyme complex, thus protecting the cell
from unregulated C subunit activity and rescuing the C subunit from
rapid proteolysis. This biochemical adaptation provides a very
effective mechanism for regulating the ratio of type II to type I
holoenzyme formed in a given tissue and for maintaining regulation when
C subunit levels change.
Modulation of RI turnover rate may represent an important biological
mechanism for maintaining equivalent amounts of R and C subunits. Loss
of this ability to maintain cAMP-dependent regulation of C
subunit activity during the process of cellular differentiation could
have catastrophic consequences, a phenomenon that we have recently
observed in mutant mice lacking RI
altogether.2 RI
null mutants display
early embryonic lethality with severe developmental abnormalities.
We thank Charles Rubin for the RII
antibody, Brian Hemmings for the C
antibody, Randy Matthews for
carefully reviewing the manuscript, Thong Su for help with the HPLC
analyses, and Bjørn Skälhegg for comments on the cell culture
experiments.