Compensatory Regulation of RIalpha Protein Levels in Protein Kinase A Mutant Mice*

(Received for publication, November 7, 1996)

Paul S. Amieux , David E. Cummings , Kouros Motamed , Eugene P. Brandon , Lauren A. Wailes , Kim Le , Rejean L. Idzerda and G. Stanley McKnight Dagger

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 RIbeta and RIIbeta regulatory subunit genes in mice, and find that both result in an increase in the level of RIalpha protein in tissues that normally express the beta  isoforms. Examination of RIalpha mRNA levels and the rates of RIalpha protein synthesis in wild type and RIIbeta mutant mice reveals that the mechanism of this biochemical compensation by RIalpha 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 Calpha in NIH 3T3 cells results in increased RIalpha protein without increases in the rate of RIalpha synthesis or the level of RIalpha mRNA. Pulse-chase experiments reveal a 4-5-fold increase in the half-life of RIalpha protein as it becomes incorporated into the holoenzyme. Compensation by RIalpha stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.


INTRODUCTION

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 alpha -isoforms are expressed ubiquitously while beta  isoforms show more restricted patterns of expression. RIbeta is induced relatively late in development and is highly expressed in neural tissues (4-6). RIIbeta is expressed during embryogenesis in mouse brain, spinal cord, and liver (7). In adult mice RIIbeta protein is most abundant in brain and brown and white adipose tissue, with lower expression in testis and ovary (8). Cbeta is most abundant in the brain, but lower levels of Cbeta 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 RIIbeta -containing holoenzyme (12). A similar selective increase in the RIIbeta regulatory subunit occurs in differentiating ovarian follicles treated with estradiol and follicle-stimulating hormone (13). Selective increases in the RIalpha 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 RIalpha 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, RIalpha subunits are rapidly degraded and the steady-state level of RIalpha is reduced (17, 19). In contrast, overexpression of the C subunit in NIH 3T3 cells elicits a coordinate increase in RIalpha protein (18).

In this report we show that loss of RIbeta or RIIbeta in gene-disrupted mice results in biochemical compensation by RIalpha with no change in RIalpha mRNA levels. We demonstrate in cell culture that this compensation is due to a decrease in the turnover rate of RIalpha protein when it associates with the C subunit. The capacity of RIalpha 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.


EXPERIMENTAL PROCEDURES

Mice

Generation of RIbeta and RIIbeta 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.

Cell Culture

Wild type mouse NIH 3T3 fibroblasts and Calpha 3T3 cells (NIH 3T3 cells stably transfected with a plasmid containing the zinc-inducible metallothionein promoter driving expression of the mouse Calpha 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).

Western Blot Analysis

Brain and white adipose tissue were isolated from RIbeta and RIIbeta 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 RIalpha , Calpha , or RIIbeta . Blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using the Amersham ECLTM system.

Translation Rate Determination

Wild type NIH 3T3 cells and Calpha 3T3 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-RIalpha 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 RIalpha translation rates in adipocytes, wild type and RIIbeta 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 RIalpha protein were performed as described above.

Pulse-chase Experiments

After labeling of NIH 3T3 and Calpha 3T3 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 and Protein Kinase Activity

HPLC analysis was performed as described (6). Wild type and RIIbeta 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).

Solution Hybridization

The method used for measuring total amounts of RIalpha and Calpha 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. RIalpha - and Calpha -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 RIalpha and Calpha 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.


RESULTS

Compensatory Increase in RIalpha in Cerebral Cortex and Hippocampus of RIbeta Null Mutant Mice

We have previously reported that targeted disruption of the neural-specific RIbeta 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 RIbeta mutant mice were compared with age-matched controls to quantitate changes in RI isoforms. This analysis demonstrated a compensatory increase in RIalpha protein in both tissues (Fig. 1), whereas no changes were observed in C or RII isoforms (data not shown). RIalpha protein levels were determined by densitometry of Western blots from wild type and RIbeta mutant protein extracts. Densitometry analysis revealed an approximate 40% increase in RIalpha protein in both the cerebral cortex and hippocampus of RIbeta mutant mice (Table I). In order to address whether the increase in RIalpha protein was due to an elevation in transcription from the RIalpha gene, solution hybridization experiments were performed using total nucleic acid isolated from cortex and hippocampus of wild type and RIbeta mutant mice. This analysis revealed no change in RIalpha mRNA levels in mutant tissues (Table I).


Fig. 1. Increases in RIalpha protein in cerebral cortex and hippocampus from RIbeta null mutant mice. Western blot comparing wild type (+/+, n = 4) and RIbeta null mutant (-/-, n = 4) cerebral cortex and hippocampus using an affinity-purified polyclonal antibody that recognizes both RIalpha and RIbeta . 40 µg of total protein from homogenates of cerebral cortex and hippocampus were run in each lane.
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Table I.

RIalpha mRNA and protein levels in cerebral cortex and hippocampus


mRNA levela
Protein levelb
Cortex Hippocampus Cortex Hippocampus

molecules/cell densitometry units
Wild type 29  ± 2.5 29  ± 2.2 45  ± 4.0 44  ± 1.0
RIbeta mutant 28  ± 2.2 30  ± 2.7 63  ± 7.5 62  ± 7.4
% change  -3.4c +3.4c +40d +41d

a  Solution hybridization results represent the averages ± S.D. of four wild type and four knockout animals.
b  Protein levels shown are averages ± S.D. of laser scanning densitometry units from Western blots using four wild type and four RIbeta mutant animals for cortex and three wild type and three RIbeta mutant animals in hippocampus.
c  Not significant.
d  p < 0.05.

Disruption of RIIbeta Leads to Increased Levels of RIalpha in White Adipose Tissue

The RIIbeta regulatory subunit is highly expressed in both white adipose tissue (WAT) and brown adipose tissue in mice. A targeted disruption of the RIIbeta 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 RIIbeta gene, Western blots were performed on WAT from wild type and RIIbeta mutant mice. RIIbeta mutant mice showed a complete loss of the RIIbeta protein (Fig. 2A). Separate Western blots examining the levels of RIalpha and Calpha revealed a 3-4-fold increase in RIalpha protein in RIIbeta mutant WAT, while Calpha protein was reduced by approximately 43% (Fig. 2B). RIalpha mRNA levels in total nucleic acid samples from WAT of wild type and RIIbeta mutant mice were identical (Table II).


Fig. 2. Increases in RIalpha protein in WAT from RIIbeta null mutant mice. A, Western blot comparing wild type (+/+, n = 3) and RIIbeta mutant (-/-, n = 3) WAT using an antibody to RIIbeta . B, Western blot comparing wild type (+/+, n = 4) and RIIbeta mutant (-/-, n = 4) WAT using antibodies to RIalpha and Calpha . 40 µg of total protein from WAT homogenates were run in each lane.
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Table II.

mRNA and protein levels in wild type and RIIbeta mutant WAT


mRNA levela
Protein levelb
RIalpha Calpha RIalpha Calpha

molecules/cell densitometry units
Wild type 32  ± 3.6 165  ± 28 45  ± 40 101  ± 47
RIIbeta mutant 32  ± 6.4 185  ± 5.3 149  ± 26 58  ± 8
% change 0 +12c +231d  -43e

a  Solution hybridization results represent the averages ± S.D. of four knockout and three wild type animals.
b  Protein levels represent the averages ± SD of three wild type and three knockout animals analyzed by making dilutions of the mutant samples and comparing them by Western blot with wild type samples using laser scanning densitometry.
c  Not significant.
d  p < 0.05.
e  p < 0.1.

Assembly of Type I Holoenzyme in RIIbeta Null Mutants

The large increase in RIalpha protein observed in WAT from RIIbeta mutant mice suggests that the RIalpha subunit has replaced RIIbeta 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 RIIbeta mutant mice contained only type I holoenzyme. Western blots using protein from HPLC fractions containing the type I holoenzyme peak confirmed the presence of RIalpha and Calpha 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.


Fig. 3. HPLC profile of PKA from wild type and RIIbeta null mutant WAT. 2 mg of total protein from WAT homogenates from wild type (top) and RIIbeta mutant (bottom ) mice was resolved by HPLC/ion-exchange chromatography, and proteins were eluted with a linear salt gradient. Individual fractions were assayed for PKA activity using Kemptide as the substrate (closed circles). Fractions containing peak kinase activity were also assayed in the presence of 5 µM PKI peptide to demonstrate that the kinase activity was PKA-specific (open circles). Both panels show HPLC profiles from one wild type and one RIIbeta mutant mouse and are representative of three independent experiments run on different mice all with similar results.
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The Rate of Translation of RIalpha Protein

In order to address the mechanism of RIalpha compensation in RIIbeta mutant mice, pulse-labeling experiments were performed in primary cultures of white adipocytes from wild type and RIIbeta mutant mice. No significant difference was observed in the rate of translation of RIalpha protein between wild type and RIIbeta mutant mice after a 1-h pulse (Fig. 4B). Western blots from the same extracts used to perform the pulse-labeling experiments confirmed that RIalpha protein was substantially increased in RIIbeta mutant mice (Fig. 4A). This implies that the increased RIalpha protein must be due to stabilization of the protein.


Fig. 4. Pulse-labeling analysis of RIalpha synthesis in adipocytes. Adipocytes from wild type (+/+, n = 2) and RIIbeta mutant (-/-, n = 2) WAT were isolated and pulse-labeled for 1 h as described under "Experimental Procedures." A, Western blot analysis of the cell homogenates used for immunoprecipitation of RIalpha in panel B. B, each cell pellet was homogenized, and samples containing equivalent total trichloroacetic acid-precipitable counts were used to immunoprecipitate RIalpha protein with a polyclonal affinity-purified RIalpha antibody. Immunoprecipitates were run on SDS-PAGE gels and analyzed by autoradiography to assess the level of newly synthesized RIalpha .
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Altered RIalpha Stability in a Cell Culture Model of RIalpha Compensation

Loss of either RIbeta or RIIbeta 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 RIalpha 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 RIalpha 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 RIalpha with no change in RII subunits (18). This increase in RIalpha resulted in the appearance of new type I holoenzyme, analogous to the results shown in Fig. 3 for RIIbeta mutant WAT (18, 23). We therefore used these Calpha -overexpressing 3T3 cells for metabolic labeling studies to determine the mechanism of RIalpha compensation. Wild type NIH 3T3 fibroblasts and Calpha 3T3 cells stably expressing a zinc-inducible expression vector for the mouse Calpha catalytic subunit (18) were treated with 90 µM zinc sulfate for 24 h and subsequently analyzed by Western blotting for RIalpha and Calpha . Zinc treatment of Calpha 3T3 fibroblasts resulted in a 27-fold increase in Calpha protein compared with values obtained from wild type 3T3 cells (Table III). As previously observed (18), an elevation in RIalpha protein was also seen upon overexpression of Calpha (Fig. 5A). Western blot analysis of a range of protein dilutions from Calpha 3T3 cells and wild type 3T3 cells showed a 4-fold increase in RIalpha protein (Table III).

Table III.

mRNA and protein levels in zinc-treated NIH 3T3 and Calpha 3T3 cells


mRNA levela
Protein levelb
RIalpha Calpha RIalpha Calpha

molecules/cell densitometry units
NIH 3T3 105  ± 2 60  ± 3 28 36
Calpha 3T3 107  ± 2 12,100  ± 100 118 960
Fold increase 1 200 4 27

a  Solution hybridization results represent the averages of duplicate plates plus the range.
b  Protein levels were determined by making serial dilutions of the Calpha 3T3 extracts and comparing them by Western blotting with duplicates of NIH 3T3 cells using laser scanning densitometry.


Fig. 5. Stabilization of RIalpha protein in Calpha overexpressing NIH 3T3 cells. Wild type NIH 3T3 and Calpha 3T3 cells were treated for 24 h with 90 µM zinc sulfate. A, Western blot analysis of the extracts used for metabolic labeling studies in panel B. B, metabolic labeling of RIalpha . Duplicate plates were treated with zinc sulfate and then labeled with 35S as described under "Experimental Procedures." Lysates containing equivalent total trichloroacetic acid-precipitable counts were incubated with a polyclonal affinity-purified RIalpha antibody and immunoprecipitated with Protein A-Insoluble. Immunoprecipitated RIalpha was run on SDS-PAGE and analyzed by autoradiography. Control lysates were incubated with a polyclonal affinity-purified conalbumin antibody. C, pulse-chase analysis of RIalpha in wild type (left) and Calpha 3T3 (right) cells. Five sets of duplicate 10-cm plates of wild type and Calpha 3T3 cells were treated with zinc sulfate and labeled as above. Plates were then chased for a total of 24 h in DMEM plus 10% FBS containing 4 mM L-methionine and 90 µM zinc sulfate. Lysates were made at 0, 3, 6, 12, and 24 h, and RIalpha was immunoprecipitated and analyzed as above with the exception that lysates containing equivalent amounts of total protein rather than equivalent trichloroacetic acid-precipitable counts were used for the immunoprecipitations.
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Solution hybridization experiments demonstrated that mRNA levels for RIalpha remained constant despite the elevation in RIalpha protein (Table III). The increase in RIalpha protein could be due to either an elevated rate of translation or a stabilization of RIalpha protein. In order to determine the mechanism, zinc-treated NIH 3T3 and Calpha 3T3 cells were labeled for 1 h with [35S]methionine followed by immunoprecipitation of RIalpha protein. The rate of synthesis of RIalpha protein in wild type NIH 3T3 and Calpha 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 Calpha 3T3 cells as expected (data not shown).

Pulse-chase experiments were performed to determine the half-life of RIalpha protein in wild type NIH 3T3 cells and Calpha 3T3 cells. The half-life of RIalpha in wild type NIH 3T3 cells was approximately 3.5 h as measured by immunoprecipitation of 35S-labeled RIalpha protein from cell extracts after a cold chase with unlabeled methionine. In contrast, the half-life of RIalpha protein in zinc-treated Calpha 3T3 cells was 13.5 h (Fig. 5C). This represents a 4-fold increase in the half-life of the RIalpha protein upon overexpression of Calpha and is in good agreement with the 4-fold increase in RIalpha protein observed in this experiment.


DISCUSSION

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 RIbeta and RIIbeta 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 RIbeta mutant mice, levels of RIalpha increase and at least partially replace the lost RIbeta subunit. In adipose tissue from RIIbeta mutant mice, we find a dramatic compensation by RIalpha and only a modest loss in total C subunit. In both cases, the increase in RIalpha protein is due to stabilization by incorporation into holoenzyme. Since it was not possible to quantitate the changes in RIalpha 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 RIalpha very similar in magnitude to that observed in WAT from RIIbeta mutants. Pulse-chase experiments in the cell culture system demonstrate a 4-fold increase in RIalpha 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 RIalpha 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 RIalpha protein and a significant decrease in steady-state RIalpha 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 RIalpha 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 RIalpha . 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 RIIbeta mutant mice. In adipocytes, the RIIbeta subunits preferentially associate with C, leaving a pool of free RIalpha 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 RIIbeta ). In this situation RIalpha 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 RIalpha holoenzyme is about 4-fold lower than that for RIIbeta 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.


Fig. 6. Model for RIalpha compensation in RIIbeta mutant mice. In wild type WAT, RIalpha protein is synthesized but is unable to compete with the RIIbeta subunit for catalytic subunits and is thus rapidly degraded with a half-life of approximately 3 h. In RIIbeta mutant WAT, the absence of the RIIbeta protein results in a large increase in free catalytic subunits that associate with RIalpha to form new type I holoenzyme. The RIalpha protein is stabilized approximately 4-fold with a half-life of approximately 14 h. The loss in total catalytic subunit results from the lower affinity interaction between RIalpha and Calpha at physiological concentrations of cAMP resulting in increased degradation of the free catalytic subunit.
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When the concentration of free C subunit increases due to the loss of RIbeta or RIIbeta , RIalpha 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 RIalpha 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 RIalpha altogether.2 RIalpha null mutants display early embryonic lethality with severe developmental abnormalities.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM32875 and the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pharmacology, Box 357750, University of Washington, Seattle, WA 981957750. Tel.: 206-616-4237; Fax: 206-616-4230; E-mail: mcknight{at}u.washington.edu.
1    The abbreviations used are: PKA, protein kinase A; R, regulatory; C, catalytic; Kin-, mutant S49 mouse lymphoma cells; Calpha 3T3 cells, NIH 3T3 cells stably transfected with a zinc-inducible expression vector for Calpha ; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; WAT, white adipose tissue; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; high performance liquid chromatography.
2    P. S. Amieux and G. S. McKnight, unpublished results.

Acknowledgments

We thank Charles Rubin for the RIIbeta antibody, Brian Hemmings for the Calpha 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.


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