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Molecular and Cellular Biology, May 2000, p. 3442-3448, Vol. 20, No. 10
Department of Medicine, Division of
Metabolism, Endocrinology and Nutrition,1 and
Department of Pharmacology,3 University
of Washington, Seattle, Washington 98195; Mental Health
Research Institute, University of Michigan, Ann Arbor, Michigan
481094; and V.A. Puget Sound Health Care
System, Seattle, Washington 981082
Received 7 January 2000/Returned for modification 13 January
2000/Accepted 14 February 2000
Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of
the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by
binding the free catalytic (C) subunit with a high affinity and is also
known to export nuclear C subunit to the cytoplasm. The significance of
these actions with respect to PKI's physiological role is not well
understood. To address this, we have generated by homologous
recombination mutant mice that are deficient in PKI A variety of hormones,
neurotransmitters, and other molecules exert their actions on target
cells by means of the cyclic AMP (cAMP)-mediated signaling cascade. The
generation of intracellular cAMP by stimulating G protein-coupled
receptors linked to adenylyl cyclase leads to the activation of the
cAMP-dependent protein kinase (PKA). This signaling cascade, one of the
most versatile and multifunctional systems studied to date, is
responsible for the modulation of numerous processes, such as
secretion, enzyme activation, and transcription. It is also an
extraordinarily well-conserved mechanism of signal transduction, since
it is seen in a wide variety of organisms.
PKA is a holoenzyme, consisting of two regulatory (R) subunits and two
catalytic (C) subunits. Molecules of cAMP generated within the cell
bind to the R subunits, decreasing their affinity for the C subunits.
This releases the C subunits to diffuse throughout the cell and
phosphorylate target molecules. Apart from the R subunits, another
endogenous modulator of the C subunit is also present in most tissues:
protein kinase inhibitor (PKI). The PKIs are heat-stable proteins, 70 to 75 amino acids in length, that are high-affinity, specific
inhibitors of PKA (24). The N-terminal region of PKI
contains the sequence RRNAI, which acts as a pseudosubstrate site for
PKA and is required for PKI's inhibitory activity. In addition, other
amino acid residues in the N-terminal region of the protein also
contribute to the interaction between PKI and the C subunit (2,
18, 19). The synthetic peptide encompassing amino acids 5 to 24 retains PKI's inhibitory activity and has been used extensively as a
biochemical tool to probe the PKI signaling pathway (23).
Immunocytochemical localization studies have demonstrated that the C
subunit and PKI have access to both the cytoplasm and the nucleus
(11, 32). By injecting synthetic C subunit and PKI, Wen et
al. (32) have shown that PKI acts as a chaperone for nuclear
export of the C subunit by means of a distinct leucine-rich motif
within PKI. By enhancing the rate of export of the C subunit from the
nucleus, PKI is thought to affect the kinetics and/or extent of PKA
activity in the nucleus.
In all, there are three known isoforms of PKI, Transfection studies in cell culture have led to the speculation that
PKI, by virtue of its localization and affinity for the C subunit,
serves to reset the basal activity of PKA once it is activated, in
preparation for the next round of activation (33). Clearly,
transfection of excess PKI relative to the C subunit reduces the
transcriptional activity of PKA-regulated genes (14).
Despite these extensive in vitro studies, there are no clear
indications as to the physiological role of PKI. The presence of three
isoforms of PKI suggests that each may serve an important role in the
modulation of the cAMP-PKA signaling cascade. Their distinct patterns
of tissue expression may indicate specific roles in different tissues.
We addressed this question by generating targeted deletions of the PKI
genes in mice. This paper describes the generation and phenotype of
mouse mutants deficient in PKI Construction of the PKI
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Deficient Gene Expression in Protein Kinase Inhibitor
Null Mutant Mice
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, one of the
three isoforms of PKI. The mice completely lack PKI activity in
skeletal muscle and, surprisingly, show decreased basal and
isoproterenol-induced gene expression in muscle. Further examination
revealed reduced levels of the phosphorylated (active) form of the
transcription factor CREB (cAMP response element binding protein) in
the knockouts. This phenomenon stems, at least in part, from lower
basal PKA activity levels in the mutants, arising from a compensatory
increase in the level of the RI subunit of PKA. The deficit in gene
induction, however, is not easily explained by current models of PKI
function and suggests that PKI may play an as yet undescribed role in
PKA signaling.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, 
, and 
,
encoded by distinct genes (3, 10, 31). Each of these
isoforms has a unique tissue expression pattern while sharing a
nanomolar affinity for the C subunit (10, 29). PKI mRNA
is most abundant in skeletal muscle, with modest expression in the
heart and brain. In contrast, PKI is expressed very highly in the
testis, with little to no expression elsewhere. PKI mRNA is found at
low levels in most tissues, with somewhat higher levels in the testis
and heart. In some tissues that express multiple isoforms, for example, the brain or the testis, the pattern of expression is quite cell specific (25, 28).
, the PKA inhibitor abundant in
skeletal muscle.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
targeting vector and generation of
mutant mice.
A PKI genomic clone was isolated from a 129SV/J
mouse genomic library (21). A 7.5-kb genomic fragment
containing both exons of the PKI gene was used to construct a
targeting vector, PKI-Rec 1. An approximately 2.5-kb
EcoRI-HindIII fragment of the gene encompassing exon 1 was replaced with a neomycin phosphotransferase cassette to facilitate positive selection. This strategy deleted the
N-terminal two-thirds of PKI, including the inhibitory and nuclear
export domains.
-Rec 1 DNA was linearized with BamHI and then electroporated into REK3 ES cells
derived from 129SV/J mice (5). Recombinant cells were
selected with 200 µg of active G418 (Gibco)/ml. Resistant colonies
were picked, isolated, and screened by genomic Southern blot analysis.
Five clones were identified as having undergone specific homologous recombination and were microinjected into 3.5-day C57BL/6 blastocysts. These blastocysts were subsequently transferred to pseudopregnant foster mothers to yield six chimeric male mice. The chimeras were bred
to C57BL/6 females, and several PKI heterozygous offspring were
obtained. All experiments comparing wild-type and mutant mice used age-
and sex-matched animals on the C57BL/6 × 129SV/J hybrid background.
PKA kinase and PKI inhibitor assays. For kinase assays, hind leg skeletal muscle samples were homogenized in buffer (20 mM Tris, 0.1 mM EDTA, 0.5 mM EGTA, 5 mM magnesium acetate, 250 mM sucrose, 1% Triton X-100, 10 mM dithiothreitol, 0.1 mM ATP, pH 7.5) with protease inhibitors, followed by sonication and centrifugation for 10 min at 12,000 × g at 4°C. Protease inhibitors included 1 µg of pepstatin/ml, 2 µg of aprotinin/ml, 2 µg of leupeptin/ml, 125 µg of 4-(2-aminoethyl)-benzenesulfonyl fluoride/ml, and 78.5 µg of benzamidine/ml. Supernatant proteins (0.4 mg/ml) were assayed as described previously using kemptide as a substrate in the presence (total kinase) or absence (basal kinase) of 5 µM cAMP (9). Six to eight mice of each genotype were assayed separately in triplicate to obtain individual values for each mouse, which were then averaged for each genotype.
For inhibitor assays, hind leg skeletal muscle homogenates were made and assayed essentially as described previously (10). The homogenates were heated for 10 min at 95°C to inactivate endogenous kinases and then centrifuged as described above. Increasing amounts of supernatant proteins were added to a kinase assay mixture containing 1 nM purified bovine heart C subunit (Sigma). Each concentration was assayed in triplicate for two mice of each genotype, and the results are reported as percent of control C subunit activity in the absence of any added tissue extract. The experiment was repeated with similar results on a separate group of mice.Northern blots. For the fasting experiments, food was withdrawn from the mice in the evening, 3 h prior to lights out. They had access to water ad libitum. The mice fasted overnight for a period of 16 h before tissues were collected. For refeeding, the fasting mice were given access to mouse chow ad libitum for 6 h before tissues were collected. Isoproterenol treatment (0.5 mg/kg of body weight in 10 mM ascorbic acid-saline) was administered intraperitoneally, and tissues were harvested after 6 h. Total RNA was isolated from hind leg skeletal muscle, and Northern blots were run with 10 µg of RNA per lane as described previously (6) and subjected to phosphorimager analysis.
Skeletal-muscle cultures.
Mice were euthanized by
CO2 administration, and intact gastrocnemius muscles were
isolated from both legs. The muscles were rinsed in phosphate-buffered
saline and placed individually in separate wells of a 6-well tissue
culture plate along with 5 ml of Ham's F-10 medium. The plate was then
incubated at 37°C in a tissue culture incubator for 30 min. One
muscle of each pair was treated with 50 µM forskolin (diluted in
dimethyl sulfoxide; Sigma), and the contralateral muscle was treated
with the vehicle alone. The muscles were then returned to the incubator
for a defined period of time, after which they were rinsed in
phosphate-buffered saline and homogenized in 1.5 ml of boiling sodium
dodecyl sulfate lysis buffer (100 mM Tris [pH 6.8], 2% sodium
dodecyl sulfate, 10% glycerol). The homogenates were boiled for 10 min
before being aliquoted and frozen at 
80°C. Protein concentrations
were determined by the bicinchoninic acid protein assay (Pierce), and
the protein samples were supplemented to 10% -mercaptoethanol and
0.1% bromophenol blue before being subjected to polyacrylamide gel
electrophoresis analysis and Western blotting for total and
phosphorylated (phospho)-CREB (cAMP response element binding protein).
Western blots.
For PKA subunit analysis, hind leg skeletal
muscle was isolated from wild-type and knockout mice, immediately
frozen on dry ice, and stored at 80°C. Samples were thawed directly
in kinase homogenization buffer (10 ml/g of tissue), homogenized,
sonicated, and centrifuged for 10 min at 12,000 × g at
4°C. The supernatants were aliquoted and frozen at 80°C for
future use. Samples were then assayed for protein concentration by the
Bradford method (Bio-Rad). Thirty-five micrograms of total protein from
each animal was run in individual lanes of 10% polyacrylamide gels and
transferred to a nitrocellulose membrane. The blots were then blocked
for a minimum of 2 h in blocking buffer (10 mM Tris HCl [pH 8],
150 mM NaCl, 5% nonfat powdered milk, 0.05% Tween 20) and probed with anti-RI monoclonal antibody (Signal Transduction) or anti-RII or
anti-C (a kind gift from S. S. Taylor, University of
California
San Diego) polyclonal antibody in blocking buffer. The
blots were then washed and incubated with horseradish
peroxidase-conjugated secondary antibody and visualized using the
Amersham ECL system. Autoradiograms were scanned in a scanning
densitometer and analyzed with ImageQuant software (Molecular Dynamics).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of PKI homozygous mutant mice.
The targeting
vector used to target the PKI gene in embryonic stem cells is shown
in Fig. 1a. Homologous recombination
replaced the first exon of PKI, which encodes most of the protein,
with the neomycin resistance cassette. The mutant cells were used to generate heterozygous mice, which when bred gave rise to
homozygous mice at the expected Mendelian ratio of approximately 25%.
Genotyping of the offspring was performed by Southern blotting (Fig.
1b), and the deletion of the PKI gene was confirmed by Northern
blotting of RNA from various tissues from the mice. The homozygous
(knockout) mice showed a complete absence of the PKI transcript that
is readily apparent in the wild-type mice (Fig. 1c).
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transcript in the Sertoli
cells (but not in the germ cells) of the testis led to the hypothesis
that this protein may modulate the action of follicle-stimulating
hormone (FSH), which acts via the cAMP-PKA pathway to regulate
spermatogenesis (28). However, the knockout mice showed
normal litter sizes relative to their wild-type counterparts (7.9 ± 0.6 versus 7.3 ± 0.8, respectively; n = 13).
Extensive characterization of testicular function revealed no defects
in testis weights, sperm number, or sperm motility (data not shown). This suggests that PKI does not have a significant role in FSH signaling in the testis.
PKI activity in mutant mice.
Loss of PKI would be expected
to most drastically affect skeletal muscle, the tissue that contains
the greatest abundance of this protein. In addition, this tissue shows
negligible levels of PKI and PKI (10, 29). We
undertook a measurement of total PKI activity in this tissue from
wild-type and knockout animals. The results of the assay, depicted in
Fig. 2, show that addition of increasing
amounts of heat-inactivated extract from wild-type muscle to an
assay mix containing exogenous C subunit causes kinase activity
to drop nearly to zero. The addition of a similar amount of PKI
knockout muscle extract, however, does not cause a significant decline
of kinase activity. This indicates the functional absence of all PKI
activity in skeletal muscle, further validating the success of the
targeting strategy shown in Fig. 1. It also demonstrates the lack of
functional compensation by the other two isoforms of PKI in skeletal
muscle. This was further confirmed by probing a Northern blot
containing skeletal muscle RNA from four wild-type and four knockout
mice. PKI was nearly undetectable in both genotypes, while PKI
was expressed at low levels that were unchanged in the knockouts (data
not shown).
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deletion would be manifested by an increase in transcription of PKA-regulated genes in skeletal muscle. This tissue was chosen for analysis owing to the high level of
PKI activity in the wild types and the complete absence of PKI activity
in the knockouts.
Gene expression in skeletal muscle.
Few genes are known to be
transcriptionally regulated by PKA in skeletal muscle.
Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme involved in the
gluconeogenesis pathway (17, 22) that is primarily expressed
in liver but is also expressed at lower levels in skeletal muscle
(16, 34). The PEPCK gene is inducible by the cAMP-PKA
pathway, which can be stimulated either by fasting or by treatment with
-adrenergic agonists like isoproterenol (4, 17, 26).
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CREB phosphorylation. The 43-kDa nuclear protein CREB was originally identified as a factor that binds the conserved cAMP response element and is a target for phosphorylation by PKA (20). Phosphorylation on Ser-133 enhances the transcriptional activity of CREB and can be detected by antibodies raised specifically against phospho-CREB (12, 13). Mutagenesis of the single Ser residue renders the protein inactive as a transcription factor, suggesting that phosphorylation at this site is critical for PKA-dependent gene induction (13).
To evaluate whether the decreased gene expression was a consequence of lower levels of CREB activation, we examined phospho-CREB levels in skeletal muscle stimulated with a PKA activator. Since the time course of CREB phosphorylation and dephosphorylation is extremely short (under 1 h) (15), this experiment was better suited to in vitro skeletal muscle cultures. Organ cultures of gastrocnemius muscle were treated with forskolin, and preliminary experiments revealed that the highest expression of phospho-CREB occurred at about 8 min after stimulation, dropping substantially by 18 min. As shown in Fig. 4, phospho-CREB was present at lower levels in unstimulated (control) knockout muscle than in wild-type muscle at both time points, 8 (Fig. 4a) and 18 (Fig. 4b) min. Densitometric analysis, normalized for total CREB levels (Fig. 4a, bottom), revealed that knockouts contained about half as much phospho-CREB as wild types at the 8-min time point. Upon activation by forskolin, phospho-CREB levels rose 50% in the wild-type muscle (Fig. 4a). The fold increase in phospho-CREB levels was similar in the knockouts, resulting in stimulated phospho-CREB levels about half those of the wild types. The variability among mice in phospho-CREB levels mirrors the discrepancy of PEPCK expression among animals fed ad libitum and reflects true differences in phosphorylation, as there were equal levels of total CREB in all the lanes (Fig. 4a). Fig. 4b demonstrates an even greater difference in phosphorylation between wild types and knockouts at 18 min poststimulation, when induction by forskolin is no longer obvious. Again, levels of total CREB were unchanged (data not shown). These lower levels of phospho-CREB in the knockouts under both basal and induced conditions match the profile of gene activation that is seen in skeletal muscle (Fig. 3).
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PKA activity.
To determine whether changes in PKA activity may
underlie the changes in CREB phosphorylation, a study of PKA activity
was conducted with tissue extracts from wild-type and knockout skeletal muscle under two conditions. The absence of exogenous cAMP in the assay
mix represents conditions of basal PKA activation, while the presence
of exogenous cAMP represents total (inducible) levels of PKA. In the
former instance, the knockout tissue was seen to demonstrate a
significantly lower kinase activity than the wild type (Fig.
5), a result entirely consistent with the
lower basal levels of gene expression and CREB phosphorylation seen in
this tissue. Basal PKA activity is shown on the left in Fig. 5a and with an expanded axis in Fig. 5b. Upon addition of exogenous cAMP, total induced kinase activity levels (Fig. 5a, right) increased approximately 30-fold over basal levels. Interestingly, wild-type and
knockout tissues showed similar total induced kinase activities. Note
that the small increase in the knockout is not statistically significant (n = 6 knockouts and 8 wild types). Since
the levels of C subunit are the same in wild-type and mutant skeletal
muscle (see below), these results indicate that endogenous PKI, which is present in the wild types but not the mutants, has little or no
effect in this in vitro assay of total PKA activity. Perhaps under
these assay conditions, endogenous PKI, which may be only 20% as
abundant as the C subunit (30), is not sufficiently
concentrated to remain associated with the C subunit, despite our
efforts to stabilize PKI-C subunit interactions by preparing tissue
extracts in the presence of Mg and ATP. We have also determined that
the cAMP concentrations that give half-maximal PKA activation in wild types and knockouts are identical (data not shown).
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raises a
mechanistic question as to how this might occur. Since PKI interacts directly with the C subunit of PKA, one possibility is that this might
occur via a downregulation of C subunit in a compensatory mechanism.
Alternatively, there might be compensatory changes in the R subunits.
PKA subunit levels.
Western blots for the C subunit of PKA
in skeletal muscle showed no difference in C subunit between the
wild-type and the knockout mice (Fig. 6),
nor was there a difference in the RII regulatory subunit isoform.
However, the regulatory subunit RI showed an upregulation in the
knockouts. The level of RI in the knockouts was determined to be
1.6-fold that of the wild type by scanning densitometry. The
compensation by only one of the two regulatory subunits present in this
tissue indicates that the compensation is specific. Compensatory
changes in RI have been described earlier in RI and RII
knockout mice and have been shown to result from an increase in the
stability of RI (1). It is likely that a similar
mechanism is at play here, as there is no change in RI mRNA levels
in the knockout mice (data not shown). The increase in RI serves to
bring more of the existing C subunits under regulatory control,
lowering the basal kinase activity in the knockouts.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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When PKI was initially discovered, it was thought to act as a
substoichiometric inhibitor of PKA whose level might be regulated to
control both basal and cAMP-stimulated PKA activity (14, 31). However, more recent studies have uncovered a potentially more dynamic role for PKI in the cAMP-regulated phosphorylation of
nuclear proteins. A leucine-rich nuclear export signal was identified
on PKI that allows it to act as a chaperone for the C subunit of PKA,
facilitating export of the C-PKI complex from the nucleus
(32). The activities of PKI as both a direct inhibitor of
the C subunit and a regulator of C subunit nuclear localization suggested that it might play an important role in cAMP-mediated gene
regulation. In order to examine this potential physiological role, we
disrupted the gene for PKI in mice. Knockout mice are healthy and
fertile, with normal weight gain and motor behavior.
Potential compensatory mechanisms were investigated to determine
whether changes in other PKI or PKA signaling components might have
occurred in the PKI knockout mice. For example, since mice have
three genes encoding distinct PKI isoforms, a compensatory up-regulation in expression of PKI or PKI could partially
substitute for the loss of PKI. However, this clearly did not occur,
as there was no detectable PKI activity in knockout skeletal muscle. Compensatory changes have been observed within the PKA system in mice
carrying specific PKA subunit knockouts. Increases in the RI
regulatory subunit occur when there is a loss of another R isoform,
e.g., in RII, RI, and RII knockout mice, and the compensation
results from an increased association of RI with the C subunit to
replace the missing R isoform, with a consequent increase in RI
stability (1, 8). In the PKI knockout skeletal muscle, we
observed a significant up-regulation of RI protein, and as there was
no change in the RI mRNA level, we suggest that the same RI
protein stabilization mechanism is responsible. No change in the amount
of C subunit was observed in the knockout mice, as assessed both by
Western blot analysis and by an assay of total (cAMP-stimulated)
kinase. We conclude that the C subunit that would normally be
associated with PKI in the wild-type mice was instead associated
with the up-regulated RI. We suggest that this change underlies the
observed decrease in basal PKA activity in the knockouts. Because the
interaction with RI has a higher affinity than the interaction with
PKI (17), the C subunit associated with RI is
"locked" in a more inactive configuration.
A significant distinguishing feature of PKI and the R subunit is their
subcellular localization. The preponderance of data demonstrate that R
subunits are restricted to the cytoplasm, while PKI can clearly enter
the nucleus. In fact, recent literature has suggested that PKI is
predominantly nuclear until it binds the C subunit and chaperones it
out of the nucleus (11, 33). Previous studies, however,
demonstrated a substantial cytoplasmic pool of PKI associated with
microtubules (27). The observed compensatory increase in
RI in the PKI knockouts suggests that PKI is somewhat
interchangeable with R and that a significant fraction of the
cytoplasmic C subunit is normally associated with PKI. We conclude,
therefore, that there is a significant cytoplasmic pool of PKI that
is replaced by RI when PKI is lost by targeted deletion.
The most striking defects in the PKI mutant skeletal muscle are in
gene expression and transcription factor phosphorylation, and these
would not have been predicted to occur based on the known properties of
PKI. The loss of PKI activity in skeletal muscle would be expected to
lead to enhanced activity of the C subunit and loss of the rapid
nuclear export of C subunit that is thought to help terminate the PKA
signal. The expected result would be an increase in both basal and
induced CREB phosphorylation and an increase in PKA-regulated gene
expression. However, under basal conditions in fed animals, the
phosphorylation of CREB and the level of PEPCK mRNA are significantly
reduced in PKI knockout mice. This result might appear consistent
with the lower levels of basal PKA activity measured in the in vitro
kinase assay, but this defect cannot be overcome by conditions that
elevate cAMP and activate PKA. Fasting mice normally show an induction
of PEPCK mRNA, and this response can be mimicked by administration of a nonspecific -adrenergic receptor agonist like isoproterenol. However, in PKI knockout mice, neither fasting nor isoproterenol is
able to achieve full activation of PEPCK gene expression despite the
presence of equivalent levels of total (cAMP-stimulated) PKA activity
in knockout and wild-type skeletal muscle. CREB phosphorylation is also
not stimulated to as high a level in PKI-deficient mice as in wild-type
mice, suggesting that the defect in gene expression is in a step prior
to phosphorylation of transcription factors.
One interpretation is that the cell operates at the low end of the cAMP
concentration curve and that the cAMP-stimulated kinase activity levels
attained in vivo are much lower than the maximal levels measured in
vitro. In this scenario, the knockouts never reach the same levels of
PKA activation in vivo as do the wild types but are still able to
regulate their phospho-CREB and PEPCK levels, albeit around a lower set
point. However, it is notable that cytoplasmic PKA signaling appears to
be unaffected, since no difference was observed in skeletal muscle
glycogen levels between wild types and knockouts (data not shown). An
alternate interpretation of our data is that PKI actually facilitates
phosphorylation of transcription factors and subsequent gene
transcription in an unknown manner. Perhaps C-PKI represents a mobile
pool of C subunit that could be very important, since much of the PKA
holoenzyme is not only restricted to the cytoplasm but is bound to
subcellular organelles and signaling complexes by association with
AKAPs (A kinase anchoring proteins). It is possible that PKI regulates nuclear entry of the C subunit (rather than exclusively its export) or
mediates interaction with transcription factors or other nuclear proteins. In these scenarios, a PKI knockout would have reductions in CREB phosphorylation and PKA-regulated gene expression in the absence of any significant change in total kinase activity, precisely the phenotype we observed. However, neither of the characterized PKI
activities (inhibition and nuclear export of the C subunit) currently
lends support to these ideas. Further studies of these knockouts and
other PKI isoform knockouts are clearly needed to explore whether the
changes in CREB phosphorylation and gene expression derive from an as
yet undiscovered PKI function.
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ACKNOWLEDGMENTS |
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This work was funded by grants from the National Institutes of Health (HD 33057 to R.L.I. and GM32875 to G.S.M.). E.A.G. was supported by the Andrew Mellon Foundation and NIH Training Grant T32 DK07247.
We thank Thong Su and R. Scott Frayo for valuable technical assistance.
We also thank D. Granner for the PEPCK cDNA and S. Taylor for the C antibody.
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FOOTNOTES |
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* Corresponding author. Mailing address: V.A. Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108. Phone: (206) 768-5490. Fax: (206) 764-2598. E-mail: idzerda{at}u.washington.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
| 1. |
Amieux, P. S.,
D. E. Cummings,
K. Motamed,
E. P. Brandon,
L. A. Wailes,
K. Le,
R. L. Idzerda, and G. S. McKnight.
1997.
Compensatory regulation of RIalpha protein levels in protein kinase A mutant mice.
J. Biol. Chem.
272:3993-3998 |
| 2. |
Baude, E. J.,
S. S. Dignam,
E. M. Reimann, and M. D. Uhler.
1994.
Evidence for the importance of hydrophobic residues in the interactions between the cAMP-dependent protein kinase catalytic subunit and the protein kinase inhibitors.
J. Biol. Chem.
269:18128-18133 |
| 3. |
Beale, E. G.,
J. R. Dedman, and A. R. Means.
1977.
Isolation and characterization of a protein from rat testis which inhibits cyclic AMP-dependent protein kinase and phosdiesterase.
J. Biol. Chem.
252:6322-6327 |
| 4. |
Beebe, S. J.,
S. R. Koch,
D. T. Chu,
J. D. Corbin, and D. K. Granner.
1987.
Regulation of phosphoenolpyruvate carboxykinase gene transcription in H4IIE hepatoma cells: evidence for a primary role of the catalytic subunit of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.
Mol. Endocrinol.
1:639-647 |
| 5. | Brandon, E. P., K. A. Gerhold, M. Qi, G. S. McKnight, and R. L. Idzerda. 1995. Derivation of novel embryonic stem cell lines and targeting of cyclic AMP-dependent protein kinase genes. Recent Prog. Horm. Res. 50:403-408. |
| 6. |
Brandon, E. P.,
S. F. Logue,
M. R. Adams,
M. Qi,
S. P. Sullivan,
A. M. Matsumoto,
D. M. Dorsa,
J. M. Wehner,
G. S. McKnight, and R. L. Idzerda.
1998.
Defective motor behavior and neural gene expression in RIIbeta-protein kinase A mutant mice.
J. Neurosci.
18:3639-3649 |
| 7. |
Brandon, E. P.,
M. Zhuo,
Y. Y. Huang,
M. Qi,
K. A. Gerhold,
K. A. Burton,
E. R. Kandel,
G. S. McKnight, and R. L. Idzerda.
1995.
Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
92:8851-8855 |
| 8. |
Burton, K. A.,
B. D. Johnson,
Z. E. Hausken,
R. E. Westenbroek,
R. L. Idzerda,
T. Scheuer,
J. D. Scott,
W. A. Catterall, and G. S. McKnight.
1997.
Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
94:11067-11072 |
| 9. |
Clegg, C. H.,
L. A. Correll,
G. G. Cadd, and G. S. McKnight.
1987.
Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit.
J. Biol. Chem.
262:13111-13119 |
| 10. |
Collins, S. P., and M. D. Uhler.
1997.
Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase.
J. Biol. Chem.
272:18169-18178 |
| 11. |
Fantozzi, D. A.,
A. T. Harootunian,
W. Wen,
S. S. Taylor,
J. R. Feramisco,
R. Y. Tsien, and J. L. Meinkoth.
1994.
Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus.
J. Biol. Chem.
269:2676-2686 |
| 12. |
Ginty, D. D.,
J. M. Kornhauser,
M. A. Thompson,
H. Bading,
K. E. Mayo,
J. S. Takahashi, and M. E. Greenberg.
1993.
Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock.
Science
260:238-241 |
| 13. | Gonzalez, G. A., and M. R. Montminy. 1989. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675-680[CrossRef][Medline]. |
| 14. | Grove, J. R., and J. Avruch. 1991. Probing cAMP gene regulation with a recombinant protein kinase inhibitor, p. 173-196. In P. Cohen, and J. G. Foulkes (ed.), The hormonal control of gene transcription. Elsevier Science Publishers, New York, N.Y. |
| 15. |
Hagiwara, M.,
P. Brindle,
A. Harootunian,
R. Armstrong,
J. Rivier,
W. Vale,
R. Tsien, and M. R. Montminy.
1993.
Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A.
Mol. Cell. Biol.
13:4852-4859 |
| 16. |
Hanson, R. W., and A. J. Garber.
1972.
Phosphoenolpyruvate carboxykinase. I. Its role in gluconeogenesis.
Am. J. Clin. Nutr.
25:1010-1021 |
| 17. | Hanson, R. W., and L. Reshef. 1997. Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu. Rev. Biochem. 66:581-611[CrossRef][Medline]. |
| 18. | Hauer, J. A., S. S. Taylor, and D. A. Johnson. 1999. Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study. Biochemistry 38:6774-6780[CrossRef][Medline]. |
| 19. |
Knighton, D. R.,
J. H. Zheng,
L. F. Ten Eyck,
N. H. Xuong,
S. S. Taylor, and J. M. Sowadski.
1991.
Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.
Science
253:414-420 |
| 20. | Montminy, M. R., and L. M. Bilezikjian. 1987. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328:175-178[CrossRef][Medline]. |
| 21. |
Olsen, S. R., and M. D. Uhler.
1991.
Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase.
J. Biol. Chem.
266:11158-11162 |
| 22. | Pilkis, S. J., and D. K. Granner. 1992. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu. Rev. Physiol. 54:885-909[CrossRef][Medline]. |
| 23. |
Scott, J. D.,
E. H. Fischer,
J. G. Demaille, and E. G. Krebs.
1985.
Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
82:4379-4383 |
| 24. |
Scott, J. D.,
E. H. Fischer,
K. Takio,
J. G. Demaille, and E. G. Krebs.
1985.
Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle.
Proc. Natl. Acad. Sci. USA
82:5732-5736 |
| 25. |
Seasholtz, A. F.,
D. M. Gamm,
R. P. Ballestero,
M. A. Scarpetta, and M. D. Uhler.
1995.
Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.
Proc. Natl. Acad. Sci. USA
92:1734-1738 |
| 26. | Snell, K., and D. A. Duff. 1979. Muscle phosphoenolpyruvate carboxykinase activity and alanine release in progressively starved rats. Int. J. Biochem. 10:423-426[CrossRef][Medline]. |
| 27. | Tash, J. S., M. J. Welsh, and A. R. Means. 1980. Protein inhibitor of cAMP-dependent protein kinase: production and characterization of antibodies and intracellular localization. Cell 21:57-65[CrossRef][Medline]. |
| 28. |
Van Patten, S. M.,
L. F. Donaldson,
M. P. McGuinness,
P. Kumar,
A. Alizadeh,
M. D. Griswold, and D. A. Walsh.
1997.
Specific testicular cellular localization and hormonal regulation of the PKIalpha and PKIbeta isoforms of the inhibitor protein of the cAMP-dependent protein kinase.
J. Biol. Chem.
272:20021-20029 |
| 29. |
Van Patten, S. M.,
P. Howard,
D. A. Walsh, and R. A. Maurer.
1992.
The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.
Mol. Endocrinol.
6:2114-2122 |
| 30. | Walsh, D. A., and C. D. Ashby. 1973. Protein kinases: aspects of their regulation and diversity. Recent Prog. Horm. Res. 29:329-359. |
| 31. |
Walsh, D. A.,
C. D. Ashby,
C. Gonzalez,
D. Calkins,
E. H. Fischer, and E. G. Krebs.
1971.
Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependent protein kinases.
J. Biol. Chem.
246:1977-1985 |
| 32. |
Wen, W.,
A. T. Harootunian,
S. R. Adams,
J. Feramisco,
R. Y. Tsien,
J. L. Meinkoth, and S. S. Taylor.
1994.
Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal.
J. Biol. Chem.
269:32214-32220 |
| 33. |
Wiley, J. C.,
L. A. Wailes,
R. L. Idzerda, and G. S. McKnight.
1999.
Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.
J. Biol. Chem.
274:6381-6387 |
| 34. | Zimmer, D. B., and M. A. Magnuson. 1990. Immunohistochemical localization of phosphoenolpyruvate carboxykinase in adult and developing mouse tissues. J. Histochem. Cytochem. 38:171-178[Abstract]. |
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