(Received for publication, April 23, 1997)
From the Department of Biological Chemistry and the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109
Attempts to understand the physiological roles of
the protein kinase inhibitor (PKI) proteins have been hampered by a
lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKI cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKI
and
30% identity to PKI
1. Residues important for the high affinity of
PKI
and PKI
1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be
conserved in PKI
. Northern blot analysis showed that a 1.3-kilobase
PKI
message is widely expressed, with highest levels in heart,
skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKI
is expressed at levels equal to or higher
than the other known PKI isoforms and that in several mouse-derived cell lines, PKI
is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKI
and PKI
identified these inhibitory activities to be PKI
and PKI
. A comparison of
inhibitory potencies of PKI
and PKI
expressed in
Escherichia coli revealed that PKI
was a potent
competitive inhibitor of C
phosphotransferase activity in
vitro (Ki = 0.44 nM) but is
6-fold less potent than PKI
(Ki = 0.073 nM). Like PKI
, PKI
was capable of blocking the
nuclear accumulation of Flag-tagged C subunit in transiently
transfected mammalian cells. Finally, the murine PKI
gene was found
to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKI
is a novel, functional PKI
isoform that accounts for the previously observed discrepancy between
PKI activity and PKI mRNA levels in several mammalian tissues.
The cAMP-dependent protein kinases (PKAs)1 comprise a subfamily of serine/threonine kinases that are activated by increases in intracellular concentrations of cAMP. Members of this family play a central role in the coordination of cellular responses to both hormones and neurotransmitters. Upon activation of receptors coupled to adenylate cyclase, intracellular concentrations of cAMP rise, and cAMP binds to each of two regulatory (R) subunits of the inactive tetrameric holoenzyme complex, releasing C subunit. Once released, C subunit phosphorylates both cytoplasmic and nuclear substrates that can alter the rate of cell division, cellular morphology, membrane ion permeability, metabolic enzyme activity, or levels of gene transcription (1-3).
Extensive biochemical characterization and molecular cloning studies
have identified three C subunit (C, C
, and C
) and four R
subunit isoforms (RI
, RI
, RII
, and RII
) (4, 5). Although
the amino acid sequences of C
and C
are highly similar (6), they
differ significantly in their tissue distributions and interactions
with R subunits. C
is widely expressed in mammalian tissues, and
C
is expressed in cells of the nervous, endocrine, and reproductive
systems (6, 7). Importantly, PKA holoenzymes formed with RII
and
C
have a 5-fold lower Ka value for cAMP than
RII
and C
containing complexes (8). Like the C subunits, the R
subunit isoforms also show heterogeneity due to differences in their
tissue distributions, subcellular localizations, and interactions with
C subunit. The RII isoforms are localized to different parts of the
cytosol via their interactions with cAMP-dependent protein
kinase-anchoring proteins (9, 10), and RI isoforms are generally
cytoplasmic. Furthermore, holoenzymes containing RI
are less
sensitive to increases in cAMP than those containing RI
(11, 12).
Several of these isoform-specific differences have been verified in
whole animal studies (13-15).
In addition to the R subunits that inhibit the activity of the C subunit in a cAMP-regulated manner, there is a second level of regulation of PKA activity by protein kinase inhibitor (PKI) proteins. The PKIs are specific and potent inhibitors of the C subunit; however, unlike R subunits, PKI inhibition of C subunit is not relieved by cAMP. Due to the low levels of PKI activity found previously in tissues relative to C subunit activity and the high binding affinity of the PKIs for C subunit, it has been proposed that the PKIs may regulate basal PKA activity (16, 17).
To date, biochemical characterization and molecular cloning of
cDNAs encoding PKIs have demonstrated that at least two distinct PKI genes are expressed in mammals, PKI and PKI
(18-21). The PKI
isoform is highly expressed in heart, skeletal muscle, cerebral cortex, and cerebellum (18, 21), whereas the PKI
isoform is most
highly expressed in testis (21). Similar to R subunits, both PKI
isoforms are pseudosubstrate, competitive inhibitors (19, 22, 23) and
inhibit the C subunit through interactions within the substrate binding
site of the C subunit (24). Specific amino acids conserved between
PKI
and PKI
1 (Phe10, Arg15, and the
pseudosubstrate sequence
(Arg18-Arg19-Asn20-Ala21))
(25, 26) have been demonstrated to be important for binding and
inhibition of the C subunit. Although both isoforms have a high
affinity for C subunit, the murine PKI
and PKI
1 isoforms differ
significantly in their inhibitory potency, and individual residues
important for this difference have been identified (16).
In addition to inhibiting C subunit phosphotransferase activity, the
PKIs also serve to localize C subunit in the cell. It has been
demonstrated that C·PKI complexes are more rapidly exported out of
the nucleus than C subunit alone and that this process is both
temperature- and ATP-dependent (27). Specifically, a nuclear export signal (NES) has been identified on PKI corresponding to a leucine-rich sequence conserved between PKI
and PKI
(28).
Previous attempts to understand the cellular roles of the PKIs have
been complicated by the heterogeneity of PKI activity in mammalian
tissues. To help resolve this problem, we sought to identify the nature
of PKI activity in tissues that expressed low levels of PKI and
PKI
. In this report we describe the identification and
characterization of a cDNA sequence that encodes a novel PKI isoform which is abundant, widely expressed, and a potent inhibitor of
the C subunit of PKA. Because of its similarity to the known murine PKI
isoforms, we have named it PKI
. Like other members of the PKI
family, PKI
inhibits cAMP-dependent gene transcription and nuclear accumulation of the C subunit. The results of this study
suggest that PKI
is found at physiologically significant levels in
many tissues and functions in a manner similar to previously characterized PKI isoforms.
A full-length cDNA sequence coding for
murine PKI (I.M.A.G.E. Consortium Clone
419982/GenBank accession W91205) (29) was identified in a search of the
expressed sequence tag data base for protein sequences homologous to
murine PKI
using the basic local alignment search tool algorithm
(30). This I.M.A.G.E. Consortium (LLNL) cDNA clone was obtained
from Research Genetics Inc. It was sequenced in both directions by
manual sequencing using Sequenase DNA polymerase (U.S. Biochemical
Corp.). Sequence analyses were performed using DNASTAR software. The
murine PKI
sequence has been submitted to the GenBank data base.
Plasmids AR-1 and MtPKI.pcr were
linearized with BamHI and BglII, respectively,
and used to generate antisense RNA probes for PKI and PKI
,
respectively, as described (31). The template for the PKI
antisense
RNA probe was constructed by inserting the 231-bp
BamHI/BglII fragment of pGEM-T.mPKI
(described
below) into the BamHI/BglII site of pSP73
(Promega) to create pSP73.mPKI
. This construct was linearized with
BglII and used to generate PKI
antisense RNA probes.
Mouse multiple tissue Northern blots (CLONTECH,
Palo Alto, CA) were hybridized at 60 °C with antisense cRNA probes
for 10-14 h. Following hybridization, blots were washed for 2 h
at 70 °C in 0.5 × SET containing 0.1% sodium pyrophosphate, dried, and autoradiographed as described previously (31).
RNase protection analysis was
performed essentially as described previously (31). The polymerase
chain reaction (PCR) was performed with oligonucleotides 5 GGA GAT CTC
CAC CAT GAC TGA TGT GGA AAC TAC G 3
and 5
GGG AGA TCT TTA CTT GTC ATC
GTC GTC CTT GTA GTC CCC GCT TTC AGA CTT GGC TGC 3
(Biomedical Core
Facilities, University of Michigan) with pMAL-PKI
(32) as a template
to amplify a fragment consisting of the full ORF of murine PKI
flanked by BglII sites. This amplified fragment was digested
with BglII, isolated, and ligated into the
BamHI/BglII sites of pSP73 to create pSP73.mPKI
. The resulting plasmid was linearized with
BglII or HindIII and used as a template to
synthesize antisense PKI
ORF RNA probes or sense RNA standards,
respectively. MtPKI.pcr was used as a template to synthesize antisense
murine PKI
ORF RNA probes or sense RNA standards as described (31).
pSP73.mPKI
was linearized with BglII or BamHI
and used as a template to synthesize antisense PKI
ORF RNA probes or
sense RNA standards, respectively. Total RNA was isolated from mouse
tissues and cell lines by using an acid guanidinium
isothiocyanate/phenol/chloroform protocol (33). T7 RNA polymerase was
used to generate [
-32P]UTP-radiolabeled antisense RNA
probes. The radiolabeled probes were incubated with 10 µg of total
RNA or varying amounts of sense RNA (0, 0.3, 1, 3, 10, or 30 pg) for
16 h at 50 °C. Yeast tRNA was added to sense RNA samples to
bring the RNA total to 10 µg, and the samples were then treated with
RNase A (20 µg/ml) and RNase T1 (200 units/ml) (Sigma). The protected
fragments were isolated and electrophoresed through 6% polyacrylamide
sequencing gels. PhosphorImager quantitation was performed in a
PhosphorImager apparatus and analyzed with IMAGEQUANT software
(Molecular Dynamics).
A mammalian expression plasmid encoding a
carboxyl-terminal hemagglutinin (HA)-tagged murine PKI protein was
constructed by PCR. A PCR fragment encoding PKI
with a
carboxyl-terminal 12CA5 epitope (YPYDVPDYA) and a one amino acid
glycine linker was generated using primers 5
GGA GAT CTC CAC CAT GAC
TGA TGT GGA AAC TAC G 3
and 5
GGG AGA TCT TTA AGC GTA GTC TGG GAC GTC GTA TGG GTA CCC GCT TTC AGA CTT GGC TGC 3
with pMAL-PKI
as a template. The resulting PCR fragment was digested with
BglII, isolated, and ligated into BglII digested
pCMV.Neo (34) to create pCMV.HA-PKI
. Likewise, the pCMV.mPKI
mammalian expression vector was constructed by PCR using the
oligonucleotides 5
GGG AGA TCT CCA CCA TGA TGG AAG TCG AGT CCC 3
and
5
GGG GGA TCC TCA GGA TGA GGT GTT CGC ATC 3
with clone 419982 as a
template. The resulting PCR fragment containing the coding region of
PKI
flanked by a BglII site and a BamHI site
was ligated into pGEM-T (Promega) to create pGEM-T.mPKI
.
pGEM-T.mPKI
was digested with BamHI and BglII,
and a 231-bp fragment coding for mPKI
was isolated and ligated into
the BglII site of pCMV.Neo. Both pCMV.HA-PKI
and pCMV.mPKI
were sequenced to confirm the coding region sequence. The
human PKI
and murine PKI
1 mammalian expression plasmids have been
described previously (20, 35). The human PKI
protein is 97%
identical to the murine PKI
amino acid sequence (35). HEK293 cells
at 50% confluency in 10-cm plates were transfected using a calcium
phosphate co-precipitation method (36) with 25 µg per plate of either
pCMV.hPKI
, pCMV.mPKI
1, pCMV.mPKI
, or pCMV.Neo. Forty-eight
hours after application of DNA precipitates, plates were washed twice
with ice-cold phosphate-buffered saline (PBS). Following the addition
of 200 µl of homogenization buffer (10 mM sodium
phosphate (pH 7.0), 1 mM EDTA, 1 mM
dithiothreitol, 250 mM sucrose) containing 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 1 µg/ml
leupeptin (Sigma), cells were scraped into separate tubes and sonicated
twice for 10 s. C subunit inhibitory activity was determined
essentially as described (35). Extracts were heated to 95 °C for 5 min. Various concentrations of heat-denatured extract were added to
recombinant C
(1 nM) (32) in a phosphotransferase assay
mix for 10 min at 30 °C. The assay was initiated by addition of
Kemptide substrate (30 µM), incubated for an additional
20 min, and then terminated.
PKI activities from mouse heart were partially purified by a modification of previously described procedures (19, 37, 38). Mouse hearts (2.5 g) (Pel-Freeze) were frozen in liquid nitrogen, pulverized, and added to 8 ml of homogenization buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The ice-cold suspension was homogenized with 20 strokes in a Dounce homogenizer (Wheaton), sonicated for 10 s at 4 °C, and placed in a boiling water bath for approximately 5 min until the suspension temperature rose to 95 °C. The resulting precipitate was removed by centrifugation for 1 h at 50,000 rpm in a TV-1665 rotor (Sorvall). The supernatant was adjusted to pH 4.0 by addition of glacial acetic acid and incubated on ice for 1 h. The resulting precipitate was removed by centrifugation for 15 min at 13,000 rpm in an HB-6 rotor (Sorvall). The supernatant was adjusted to pH 7.0 by addition of 1 M Tris base and dialyzed in dialysis tubing (molecular weight cut-off, 3500) against three 2-liter changes of 5 mM KPO4, 1 mM EDTA, pH 7.0. The dialysate (25 ml) was adjusted to pH 5.0 and absorbed to a 2-ml Bio-Scale-DEAE2 column equilibrated with 5 mM KPO4, pH 5.0, using the BioLogic system (Bio-Rad). The column was washed with 15 ml of 5 mM KPO4, pH 5.0, and 5 ml of 5 mM NaOAc, pH 5.0. Proteins were eluted with a 40-ml linear gradient of 5-1000 mM NaOAc, pH 5.0, as 1-ml fractions were collected. Fractions were adjusted to pH 7.4 with 2 M Tris, pH 8.0, and assayed for PKI inhibitory activity as described above.
Generation of Polyclonal Antibodies to PKIThe pET9d.His6PKI and
pET9d.His6PKI
prokaryotic expression vectors were
generated using PCR. Specific sense and antisense oligonucleotides were
used to generate PCR fragments encoding the full-length ORFs of murine
PKI
and PKI
each with an amino-terminal hexahistidine tag.
pMAL-PKI
and clone 419982 were used as templates in the PKI
and
PKI
PCR reactions, respectively. Fragments were digested with
NcoI and BamHI, isolated, and ligated into pET9d (Novagen) that had been NcoI- and BamHI-digested.
Escherichia coli (BL21(DE3)/pLysS strain) were
transformed with pET9d.His6PKI
and
pET9d.His6PKI
. E. coli cultures (1 liter)
were grown and induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C. Pellets were resuspended in 20 ml of buffer A (20 mM Tris, pH 8.0, 300 mM NaCl, 0.1% Triton
X-100) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Each suspension was
sonicated three times for 1 min, and imidazole was added to a final
concentration of 20 mM. The bacterial lysate was
centrifuged for 1 h at 100,000 × g, and the supernatant was loaded onto a 2-ml nickel affinity resin (Qiagen) column. The column was washed with 10 column volumes of buffer A
containing 20 mM imidazole and eluted with a step gradient
of imidazole in buffer A. The majority of each His-tagged PKI eluted in
the 80 mM imidazole elution. Purified
His6PKI
and His6PKI
were separately
conjugated to keyhole limpet hemocyanin and used to immunize rabbits
for antibody production (Research Genetics Inc.).
Antisera raised against His-tagged PKI
and PKI
were affinity purified on MBP-PKI
(32) and MBP-PKI
(see below) nitrocellulose blots essentially as described (39, 40).
Affinity purified anti-PKI
antibody recognized PKI
(14 kDa) but
did not react with PKI
or PKI
on Western blots of HEK293 cell
extracts from cells transfected with PKI expression constructs.
Likewise, affinity purified anti-PKI
recognized PKI
(16 kDa), but
no signal was detected in extracts from PKI
- or PKI
-transfected
cells. Fractions from the DEAE column were concentrated in
microconcentrators (Microcon-3, Amicon), denatured in SDS-PAGE buffer
at 95 °C for 5 min, resolved on 15% SDS-PAGE gels, and transferred
to 0.2-µm nitrocellulose membranes (BA-83, Schleicher and Schuell).
Membranes were blocked for 4 h in PBS supplemented with 5%
non-fat dried milk, 2% polyvinylpyrrolidone (PVP-40), and 0.1% Triton
X-100 and subsequently incubated with either a 1:10 dilution of
affinity purified anti-PKI
or anti-PKI
in PBS supplemented with
0.5% bovine serum albumin and 0.1% Triton X-100 for 2 h. Filters
were washed three times for 10 min with TBST (50 mM Tris,
pH 7.5, 150 mM NaCl, and 0.05% Tween 20), and then they
were incubated with a 1:10,000 dilution of goat anti-rabbit alkaline
phosphatase (Life Technologies, Inc.) in TBST supplemented with 5%
non-fat dried milk as the secondary antibody for 2 h. Following
the final set of three 10-min washes with TBST, the blots were
developed with the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate system (Life Technologies, Inc.).
To create an MBP-PKI prokaryotic expression vector, the
murine PKI
coding region was amplified by PCR using oligonucleotides 5
GGG GAA TTC ATG GAA GTC GAG TCC TCC 3
and 5
GGG GAA TTC TTA GGA
TGA GGT GTT CGC ATC 3
designed to create EcoRI recognition sites. The resulting PCR fragment was cut with EcoRI,
isolated, and ligated into the EcoRI site of pMAL.cRI (New
England Biolabs). This vector was sequenced and transformed into
E. coli (XLI-Blue, Stratagene). The MBP-PKI
fusion
protein was expressed and purified over a amylose affinity column as
described previously (32). Protein concentrations of the purified
fusion proteins were determined by the Bradford method (Bio-Rad Protein
Assay). MBP-PKI
and MBP-PKI
were greater than 95% pure as
determined by scanning densitometry of Coomassie Blue-stained SDS-PAGE
gels. IC50 values and Ki values for
MBP-PKI
and MBP-PKI
were determined essentially as described
(16). Fifty pM recombinant C
was used in all
phosphotransferase activity assays. Ki values for
murine PKI
and PKI
were determined using the Henderson method for
tightly bound inhibitors (41).
Mammalian expression plasmids encoding amino-terminal
Flag-tagged murine C and C
Y235S/F239S (42) proteins were
constructed using the PCR method. An amino-terminal C
PCR fragment
containing an amino-terminal Flag epitope (DYKDDDDK) was generated
using the primers 5
GGG GGA TCC ACC ATG GAC TAC AAG GAC GAC GAT GAC AAG GGC AAC GCC GCG GCC GCC AAG AA 3
and 5
AAG TAC TCC GGA GTC CCA C
3
with pGEM-4.C
(43) as a template. The resulting PCR fragment was
digested with BamHI and BglII, isolated, and
ligated into BglII-digested pCMV.Neo to create
pCMV.Flag-C
1. pGem-4.C
was digested with BamHI and
BglII. An approximately 450-bp fragment encoding the
carboxyl-terminal of C
was isolated and ligated into
BglII digested pCMV.Flag-C
1 to generate pCMV.Flag-C
2.
A 240-bp BglII fragment coding the central one-third of C
was isolated from the same digest and ligated into BglII
digested pCMV.Flag-C
2 to generate the wild type Flag-tagged C
expression vector pCMV.Flag-C
3. To create the C
Y235S/F239S mutant
expression vector, pET9d.C
Y235S/F239S (42) was digested with
BglII, and the 240-bp fragment containing the mutated sites
was ligated into BglII-digested pCMV.Flag-C
2 to generate
pCMV.Flag-C
Y235S/F239S. Both expression plasmids were restriction
mapped and sequenced.
NIH 3T3 cells were grown on 10-cm plates to 50%
confluency and transfected using a standard calcium phosphate method
(36) with 1 µg of pCMV.Flag-C3 or pCMV.Flag-C
Y235S/F239S, 1 µg of human chorionic gonadotropin-luciferase (HCG.Luciferase), 5 µg of pRSV.
gal, and the indicated amounts of pCMV.mPKI
. The
total amount of plasmid DNA was brought to 25 µg with the parental
vector pCMV.Neo. Twenty-one hours after transfection, cells were washed twice with ice-cold PBS, scraped into homogenization buffer, sonicated, and assayed for luciferase and
-galactosidase activities as
described (8).
COS-1 cells or CV-1 cells were grown in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
in 8-well tissue culture chambers on poly-L-lysine-coated
glass slides (Lab-Tek) to 30% confluency and transfected using a
standard calcium phosphate method (36). Cells were transfected with 2 µg of pCMV.Flag-C3, 8 µg of pCMV.RII
, and either no PKI
expression vector, 8 µg of pCMV.HA-PKI
, or 4 µg of pCMV.mPKI
.
Total plasmid concentration was maintained at 25 µg by addition of
the parental vector, pCMV.Neo. Following a 12-h incubation with DNA
precipitates, cells were washed once with Dulbecco's modified Eagle's
medium and grown for 24 h. Indicated cells were then stimulated
with forskolin (25 µM) and 3-isobutyl-1-methyl-xanthine
(500 µM) in Dulbecco's modified Eagle's medium for 40 min at 37 °C. Following stimulation, cells were washed twice with
ice-cold PBS and fixed with 4% formaldehyde in PBS for 10 min at room
temperature followed by a 1:1 mixture of methanol and acetone for 5 min. After washing three times with PBS, cells were incubated with an
anti-Flag epitope antibody (M2) (Eastman Kodak) at a 1:2000 dilution in
PBS supplemented with 1% bovine serum albumin, 1% horse serum, and
0.1% saponin (Sigma). After four washes with PBS supplemented with
0.1% saponin, a 1:3000 dilution of Cy3-F(ab
)2 fragment
goat anti-mouse IgG (Jackson) was incubated with the cells for 1 h
in the dark in PBS supplemented with 1% bovine serum albumin, 1%
horse serum, and 0.1% saponin. Prior to examination by fluorescence
microscopy (Olympus), cells were washed four times for 2 min in PBS
plus 0.1% saponin and twice for 2 min in PBS. In control experiments,
no fluorescence was detected in non-transfected cells.
A
comparison of PKI activity of tissue extracts with PKI and PKI
tissue mRNA levels supported the idea that novel PKI isoforms might
exist (18, 20, 21). For example, tissues such as kidney and liver show
low levels of PKI
and PKI
mRNA but significant amounts of PKI
inhibitory activity (data not shown). To determine if evidence for
other PKI isoforms existed, the amino acid sequence for murine PKI
(44) was used to search for homologous sequences in the NCBI GenBank
expressed sequence tag data base using the basic local alignment search
tool program. This search identified a murine full-length cDNA
clone (I.M.A.G.E. consortium clone 419982) derived from a mouse embryo
(embryonic day 13.5-14.5) cDNA library which encoded a protein
having statistically significant homology (p < 0.001)
to the murine PKI
amino acid sequence. The cDNA clone was fully
sequenced (Fig. 1), shown to contain 1064 base pairs (bp), and characterized further.
Assuming that the first of two methionine codons encodes the initiator
methionine, the murine PKI cDNA contains an ORF of 231 nucleotides and a 3
-untranslated sequence with a putative polyadenylation signal AATAAA-(1042-1047) and a poly(A) tail (Fig. 1).
The PKI
protein predicted by the ORF is 75 amino acids in length
with a calculated molecular mass of 7.8 kDa and pI of 3.9. PKI
shows
relatively low amino acid homology to the other known murine PKI
isoforms: PKI
(35% identity) and PKI
1 (30% identity). However,
most of the residues demonstrated to play a role in the high affinity
of PKI
for the C subunit are conserved. Not only is the inhibitory
pseudosubstrate sequence
(Arg18-Arg19-Asn20-Ala21)
found, but also Arg15, Phe10 and
Tyr7 are conserved (16, 25, 26). Likewise, a sequence
highly similar to the consensus PKI nuclear export signal
"L37XL39XL41XXL44XHy46"
(where X is any amino acid and Hy is any hydrophobic amino
acid) is present in PKI
(28) (Fig. 2). Based on these
elements of sequence homology, the new protein was designated PKI
.
Most of the homology between PKI
and the other known PKI isoforms
occurs in the amino-terminal two-thirds of the protein, whereas little homology is seen in the carboxyl-terminal portion of the protein (Fig.
2).
Expression of PKI Isoforms in Mouse Tissues
To compare
directly the expression patterns of PKI isoforms in mouse tissues,
poly(A)+ Northern blots were probed using antisense RNA
probes specific for the known murine PKI isoforms (PKI, PKI
, and
PKI
). The PKI
probe detected a single 4.3-kb transcript in heart,
brain, and skeletal muscle with lower levels in lung and kidney (Fig. 3A). Prolonged exposure showed barely
detectable levels of message in spleen, liver, and testis (data not
shown). These data are consistent with the previously reported Northern
blot analysis of mouse and rat tissues (18, 21). Hybridization with a
PKI
probe detected two messages, a strongly hybridizing species of approximately 700 bp in testis and a weakly hybridizing 1.8-kb RNA in
brain, spleen, lung, and testis (Fig. 3B). Low levels of the
1.8-kb message could also be detected in heart, liver, skeletal muscle,
and kidney with longer exposures (data not shown). Previously, it was
reported that a 1.8-kb PKI
transcript was expressed in all mouse
tissues with highest levels in skeletal muscle (20). The discrepancy
between these two studies may be due to differences in the age of the
mice examined. Both a 700-bp and 1.8-kb PKI
transcript have been
identified in rat testis, and the 700-bp message has been shown to be
developmentally regulated (21). It has yet to be determined whether
this 700-bp rat message is the product of a highly homologous gene, due
to alternative polyadenylation or due to alternative splicing of the
rat PKI
gene. The PKI
probe detected a single 1.3-kb transcript
in all tissues examined. Unlike the PKI
and PKI
transcripts, this
message was expressed at high levels in all tissues tested. Highest
levels were seen in heart, skeletal muscle, and testis; however,
significant levels were also seen in spleen, lung, liver, and kidney
(Fig. 3C). The presence of PKI
mRNA in these organs
is important since they have low levels of PKI
and PKI
message
(18, 20, 21).
RNase Protection Analysis of PKI Isoform Expression in Mouse Tissues and Mouse-derived Cell Lines
PKI activity has been
extensively characterized in several tissues where PKI mRNA is
detected in Northern blot analysis including skeletal muscle (21, 22),
heart (38, 45), and testis (19, 37). However, it was surprising that
PKI
had not been identified previously. One explanation could be
that PKI
transcripts are widely expressed but of low abundance
relative to PKI
and PKI
transcripts. To quantitate PKI
mRNA more accurately, RNase protection assays using
isoform-specific, antisense RNA probes of similar length were
performed. Antisense coding region probes of the PKI isoforms were
chosen for these experiments so as to generate similar size protected
fragments and to detect all possible coding region splice variants. For
PKI
, a protected fragment of 228 bp was seen prominently in heart,
brain, and skeletal muscle with weaker bands in lung, kidney, and
thyroid. Even with prolonged exposures, no fragments were detected in
spleen, liver, or testis (data not shown). After hybridization with
PKI
probe, a protected fragment of 254 nucleotides was observed in
all tissues examined. The highest level of expression was observed in
the testis with significantly lower levels in all other tissues (data
not shown). Using the same total RNAs and conditions as those used for
the PKI
and PKI
RNase protections, hybridization with the PKI
probe resulted in a protected fragment of 233 bp apparent in all mouse
tissues studied with the highest levels found in heart and testis (Fig. 4A). Significant levels of PKI
mRNA
were also seen in uterus, prostate, small intestine, and stomach (data
not shown). The tissue distributions of the PKI isoforms as determined
by RNase protection analysis are in good agreement with the Northern
blot analysis with the exception of PKI
expression in the testis.
The PKI
Northern blot analysis shows an intermediate level of PKI
expression in the testis (Fig. 3C), and the RNase protection
analysis suggests that the testis is the organ of highest PKI
mRNA expression (Fig. 4A). PhosphorImager quantitation
of sense RNA-protected fragments and tissue-protected fragments
indicates that PKI
mRNA is expressed at comparable levels to
PKI
and PKI
in all mouse tissues tested. In several tissues such
as heart, lung, liver, and kidney, PKI
is the predominant PKI
isoform transcript (Fig. 4, B-D).
To confirm the widespread nature of PKI transcripts, total RNA from
several mouse-derived cell lines was isolated and assayed for PKI
isoform transcript expression by RNase protection analysis. As for the
mouse tissues, hybridization with the PKI
probe generated a 233-bp
fragment in all cell lines tested (data not shown).
C2C12 myoblasts, N1E-115 neuroblastoma and L939
cells showed the highest levels of expression, and TM4 Sertoli cells
showed the least (Fig. 4E). Only the N1E-115 neuroblastoma
cell line displayed a significant level of PKI
transcript, whereas
no bands were detected in any of the cell lines in the PKI
RNase
protection analysis (data not shown). Of interest, no evidence of PKI
alternative splice variants was observed in these RNase protection
experiments.
Another possible explanation for why PKI was not
detected during prior PKI purifications is that it possesses
significantly different biochemical characteristics from the other two
known isoforms. Analysis of PKI
, PKI
1, and PKI
amino acid
sequences shows that the three proteins vary considerably in their
predicted isoelectric points; PKI
has a pI of 4.4, PKI
1 has a pI
of 5.1, and PKI
has a pI of 3.9. Previous purifications of PKI
and PKI
1 have involved elution from a DEAE-cellulose column with a
linear gradient of NaOAc (0-350 mM) at pH 5.0 (19, 38).
Under these conditions, PKI
1 eluted at lower ionic strengths than
PKI
. Since PKI
has a lower pI than PKI
, it would be expected
to elute later in the gradient. No inhibitory peaks were eluted after
PKI
, but it is possible that PKI
was never eluted from the
column. To test this hypothesis and to verify that the PKI
protein
is present in native mouse tissues, PKI activities from mouse heart
were partially purified by a procedure similar to a previously
described three-step purification scheme involving heat denaturation,
acid treatment, and DEAE chromatography (19, 37, 38). This purification was modified to extend the elution from 350 mM NaOAc to 1 M NaOAc. Mouse heart was chosen for this experiment due to
its high levels of both PKI
and PKI
message as determined by
Northern blot and RNase protection analysis. In vitro kinase
inhibition assays were conducted on the mouse heart fractions obtained
from the DEAE column.
Two peaks of inhibitory activity were detected, one that eluted at a
theoretical salt concentration of 250-325 mM NaOAc
(9.9-13.0 millisiemens/cm), and a second that eluted at a theoretical
salt concentration of 450-575 mM NaOAc (16.0-20.0
millisiemens/cm) (Fig. 5A). To determine if
fractions of these peaks were likely to contain PKI or PKI
,
eukaryotic expression vectors for human PKI
and murine PKI
were
transiently transfected into HEK293 cells, and PKI activities were
partially purified as for mouse heart. Assay of the fractions from
PKI
-transfected HEK293 cell extracts revealed a single inhibitory
peak at approximately 250 mM NaOAc, overlapping the first
heart inhibitory peak. Assay of the fractions from the HEK.mPKI
extracts likewise revealed a single inhibitory peak at approximately
500 mM NaOAc correlating exactly with the elution profile
of the second heart inhibitory peak. No inhibitory peaks were
identified in control HEK cells transfected with the parental
expression vector pCMV.Neo (data not shown).
To confirm the identity of the inhibitors responsible for inhibitory
peak 1 and peak 2 from mouse heart, isoform-specific PKI and PKI
antisera were prepared from rabbits using His-tagged PKI
and
His-tagged PKI
as antigens. The specificity of these antisera was
determined using HEK293 cells transfected with pCMV.hPKI
(HEK.hPKI
extracts), pCMV.mPKI
1 (HEK.mPKI
1 extracts),
pCMV.mPKI
(HEK.mPKI
extracts), and pCMV.Neo (HEK.Neo extracts).
These extracts were resolved by 15% SDS-PAGE and transferred to
nitrocellulose. Anti-PKI
sera recognized a single band with an
apparent molecular mass of 14 kDa in the HEK.hPKI
extracts. No bands
were detected in the other extracts. Similar results were obtained with
the anti-PKI
sera that detected a single band with an apparent
molecular mass of 16 kDa in the HEK.mPKI
extract but no bands in the
other lanes. Preimmune sera from the two rabbits did not detect any bands in similar experiments (data not shown).
When samples from the heart inhibitory peak fractions were analyzed by
Western blotting, the affinity purified anti-mPKI antibody detected
a single 14-kDa band in peak 1 fractions (Fig. 5B). This
band was the same apparent molecular mass as transfected PKI
.
Likewise, the affinity purified anti-PKI
antibody recognized a
single 16-kDa band in peak 2 fractions that co-migrated with transfected PKI
(Fig. 5C). Importantly, the intensity of
the 14-kDa band in peak 1 fractions and the intensity of the 16-kDa band in peak 2 fractions correlated directly with the C subunit inhibitory activity of the fractions showing that PKI
is the inhibitor responsible for the majority of inhibitory peak 1 and PKI
is the inhibitor responsible for the majority of inhibitory peak 2. These results suggest that both PKI
and PKI
are expressed at
significant levels in mouse heart and that PKI
can be distinguished from other PKI isoforms both immunologically and by its DEAE elution characteristics.
To study the
inhibitory potency of murine PKI, full-length PKI
was
PCR-amplified, cloned into pMAL.cRI, and expressed in E. coli as an MBP-PKI
fusion protein. Previously, it has been shown that the presence of an amino-terminal MBP fusion does not affect
the inhibitory efficacy of PKI
or PKI
1 (16). To assess relative
inhibitory efficacy, PKI
was compared with murine PKI
(16) in
in vitro kinase inhibition assays. C subunit
phosphotransferase activity was measured in the presence of increasing
concentrations of either PKI
or PKI
. A representative experiment
is shown in Fig. 6A, and average
IC50 values obtained from similar experiments are listed in
Table I. Measurement of IC50 values for
PKI
and PKI
at 30 µM Kemptide substrate revealed
that PKI
possesses a 14-fold greater IC50 value than
PKI
(Table I). To measure more accurately the difference in
inhibitory potencies between these two tight binding inhibitors,
Ki values were determined by Henderson analysis (41)
(Fig. 6, B and C). The Ki values for PKI
and PKI
were determined to be 0.073 and 0.44 nM, respectively (Table I). The increase in slope with
increasing Kemptide substrate concentrations in Fig. 6B
suggests that as for PKI
and PKI
1, PKI
is a competitive
inhibitor of C subunit (16, 19, 22, 23). Hence, PKI
is a potent,
competitive inhibitor of C subunit; however, it is 6-fold less potent
than PKI
. PKI
possesses all the amino acid residues previously
shown to be important in the high affinity of PKI
for C subunit
(Tyr7, Phe10, Arg15,
Arg18, and Arg19) (16, 25, 26); however, it is
possible that PKI
contains negative binding determinants.
Mutagenesis of Thr8 and Ser12 in PKI
1 to
alanines caused a 4-fold increase in the inhibitory potency of PKI
1.
It has been postulated that these residues may block formation of the
amino-terminal inhibitory
-helix of PKI
1 (16). Thus, it is
possible that Ser8 and Ser12 of PKI
are
negative determinants of C subunit binding.
|
Expression experiments were performed to demonstrate that
the PKI cDNA sequence was capable of producing a relatively
heat- and acid-stable inhibitor of PKA. In addition, these expression experiments sought to characterize the ability of PKI
to inhibit cAMP-dependent gene transcription and C subunit nuclear
accumulation. pCMV.mPKI
, a PKI
mammalian expression vector, was
constructed. Initially, HEK293 cells were transiently transfected with
pCMV.mPKI
, pCMV.hPKI
, pCMV.mPKI
1, or pCMV.Neo. Cell extracts
were heated and acid-treated, and increasing amounts were added to an
in vitro kinase inhibition assay to determine if they could
inhibit recombinant C subunit. HEK293 cells transfected with either of
the three PKI isoform expression vectors contained at least 10-fold
higher levels of C subunit inhibitory activity than those transfected
with the parental vector pCMV.Neo. The extracts from cells transfected with pCMV.mPKI
showed greater inhibitory activity than
pCMV.mPKI
1-transfected cells but less inhibitory activity than the
pCMV.hPKI
-transfected cells (data not shown). The presence of the
PKIs in cell extracts was verified with PKI-specific antibodies (data
not shown).
To verify the ability of PKI to inhibit C subunit
in vivo, NIH 3T3 cells were transiently transfected with a
constant amount of the C
expression vector, either
pCMV.Flag-C
3 or pCMV.Flag-C
Y235S/F239S, and increasing amounts of
pCMV.mPKI
. Each plate also received a constant amount of a
cAMP-responsive reporter plasmid (HCG.luciferase) (46) and pRSV.
gal
to control for transfection efficiency. In this experiment, luciferase
activity was used as an in vivo measure of free cellular C
subunit. Transient transfection of either C subunit alone produced a
35-fold increase in luciferase activity (data not shown). One to two
µg of pCMV.mPKI
was required to completely inhibit luciferase
activity in cells transfected with pCMV.Flag-C
3. NIH 3T3 cells
expressing the Flag-tagged C
Y235S/F239S mutant, a C
mutant with
reduced affinity for PKI
and PKI
1 (42), showed significantly less
inhibition of luciferase activity at all pCMV.mPKI
concentrations
(Fig. 7). It has been shown previously that three PKI
residues outside the pseudosubstrate sequence, Tyr7,
Phe10, and Arg15, contribute significantly to
the high affinity of PKI
for C subunit. Specifically,
Phe10 of PKI
interacts with a hydrophobic pocket on the
surface of the C subunit consisting of residues Tyr235 and
Phe239 (24, 42). The reduced ability of PKI
to inhibit
the C
Y235S/F239S mutant is consistent with the conservation of
Phe10 between PKI
and PKI
(Fig. 2). Interestingly,
even though C
Y235S/F239S is 2000-fold less sensitive to inhibition
by PKI in vitro (18), it is still significantly inhibited
in vivo. This is likely due to the fact that in
vivo concentrations of C subunit reach micromolar levels, whereas
nanomolar concentrations of C subunit are used in in vitro
experiments.
PKI
Both PKI
and PKI
are capable of actively exporting C subunit from the nucleus
(27). In the case of PKI
, this export of C·PKI complexes has been
shown to require a nuclear export signal (NES) consisting of a
hydrophobic sequence that is conserved between PKI
and PKI
1 (28).
Amino acid alignment of PKI
with PKI
and PKI
1 reveals the
possible presence of a NES in PKI
(Fig. 2). The most significant
difference observed was a glycine at amino acid residue 39 where both
PKI
and PKI
1 contain leucines. To test the functionality of the
NES of PKI
, a transient transfection assay was developed. An
amino-terminal Flag-tagged C
expression vector was constructed to
determine the localization of C subunit in transfected cells. To verify
that the Flag-tagged C subunit was catalytically active and that the
Flag epitope was accessible both when the C subunit was free and when
it was bound to a PKI, HEK293 cells were transiently transfected with
the Flag-tagged C subunit alone or with a combination of Flag-tagged C
subunit and PKI
. Western blot analysis of these HEK293 cell extracts with the M2 anti-Flag antibody detected a single band with an apparent
molecular mass of 41 kDa, the size expected for full-length C subunit
(data not shown). No band was seen in extracts of HEK293 cells
transfected with the parental vector alone (data not shown). When
extracts of HEK293 cells transiently transfected with or without the
Flag-tagged C subunit vector alone were subjected to
immunoprecipitation with the M2 anti-Flag antibody, only the immunoprecipitates isolated from extracts made from cells transfected with pCMV.Flag-C
3 showed significant C subunit activity (data not
shown). Importantly, boiled M2 antibody immunoprecipitates from
extracts of HEK293 cells co-transfected with Flag-tagged C subunit and
PKI
showed significant C subunit inhibitory activity (data not
shown). Moreover, immunoprecipitation of this inhibitory activity was
dependent on the presence of Mg2+ and ATP in the
immunoprecipitation buffer, both critical to the tight binding of C
subunit to PKI (47). Hence, a catalytically active Flag-tagged C
subunit is produced in cells transiently transfected with the
pCMV.Flag-C
3 expression vector, and it is recognized by the M2
anti-Flag monoclonal antibody when free in solution and when bound to
PKI
.
To study the effect of elevations in cAMP on the cellular localization
of C subunit in the presence and absence of PKI, CV-1 cells were
transiently co-transfected with expression vectors for RII
,
amino-terminal Flag-tagged C
and either no PKI, PKI
, or PKI
.
Enough RII
expression vector was used in each transfection to
completely inhibit transfected C subunit phosphotransferase activity
(data not shown). Likewise, in transfections including PKI
or
PKI
, a sufficient amount of PKI expression vector was included so as
to fully inhibit transfected cAMP-dependent kinase activity
(data not shown). Following transfections, CV-1 cells were stimulated
for 40 min with forskolin and isobutylmethylxanthine and then analyzed
by indirect immunofluorescence with the M2 anti-Flag monoclonal
antibody. Consistent with previous results, the Flag-tagged C subunit
was localized to the cytoplasm in untreated cells expressing Flag-tagged C subunit and RII
(Fig. 8A)
(48). When identically transfected cells were treated with forskolin, C
subunit was found to accumulate in the nucleus (Fig. 8B)
(48). Co-transfection of PKI
or PKI
expression vectors with the
Flag-tagged C subunit and RII
had no discernible effect on the
cytoplasmic localization of C subunit in non-stimulated cells (Fig. 8,
C and E). However, both PKI
and PKI
prevented the nuclear accumulation of C subunit in stimulated cells
(Fig. 8, D and F) (49). Similar results were
obtained in identically treated COS-1 cells (data not shown). In both
cells lines, no significant anti-Flag immunofluorescence was observed
in cells transfected with the parental vector alone (data not
shown).
This study describes the identification, expression, and
characterization of a new member of the PKI family of inhibitor
proteins. The PKI family now includes at least three members: PKI,
PKI
, and PKI
. The amino acid sequence of PKI
is 35% identical
to PKI
and 30% identical to PKI
1 suggesting that these three PKI isoforms are encoded by distinct genes. All members of the family possess an amino-terminal inhibitory region that includes a
pseudosubstrate sequence and a central region containing a leucine and
hydrophobic amino acid-rich NES. Most of the amino acid differences
between the PKI isoforms occur in the carboxyl-terminal one-third of
the molecule. All three PKI isoforms are approximately the same amino acid length suggesting that this size is important for physiological function.
The high degree of amino acid similarity between PKI and the other
known PKI isoforms in the amino-terminal inhibitory region suggests
that the PKI isoforms may also have common biochemical characteristics.
For instance, we anticipate that PKI
will require Mg2+
and ATP for high affinity interactions with C subunit (47), that it
will inhibit both C
and C
to similar extents (8), and that it
will be specific for PKA (50, 51). To understand the role of the PKIs
in the PKA signal transduction system, it will be important to
understand the differences between the PKI isoforms. Even though PKI
and PKI
1 share many of the same amino acids in their inhibitory
region, they differ significantly in their inhibitory potencies. As
determined by Henderson analysis, murine PKI
1 possesses a 32-fold
higher Ki for C
than murine PKI
(16). This
difference has been demonstrated to be due in part to the absence of a
tyrosine at position 7 of PKI
1 (16). Because Tyr7 of
PKI
is conserved in PKI
, we anticipated that PKI
would bind
and inhibit C
with a subnanomolar Ki. Data from this study demonstrate that indeed PKI
is a tight binding inhibitor of C subunit with a Ki of 0.44 nM.
Still, it is 6-fold less potent than PKI
. This decreased potency is
probably due to potential negative determinants such as
Ser8 and Ser12. It is unlikely that a 6-fold
difference in inhibitory potency could make a significant difference in
a stimulated cell where free C subunit levels are micromolar; however,
at resting cellular levels of cAMP, where there is little active kinase
in the cell, this difference in inhibitory potency between PKI isoforms
could be significant (16, 17).
Unlike the other PKI isoforms, which do not contain any cysteines,
PKI possesses a single cysteine residue at amino acid position 13. Assuming that PKI
has an overall structure similar to PKI
(24),
Cys13 of PKI
would be located at the boundary of the
amino-terminal
-helix and
-turn regions, two regions implicated
in the high binding affinity of PKI
for C subunit. Previously, the
-turn region of PKI
has been hypothesized to be important in the
proper positioning of Arg15 with Glu203 of the
C subunit (24, 32). Since the
-turn region of PKI
is believed to
be flexible and not in a fixed conformation until binding to C subunit
(24), it was possible that modification of this cysteine residue could
affect the ability of PKI
to bind and inhibit C subunit. However,
attempts at selective modification of Cys13 with selective
sulfhydryl-modifying reagents such as N-ethylmaleimide, iodoacetic acid, and iodoacetamide failed to show specific decreases in
PKI
inhibitory activity (data not shown).
The original goal of this study was to identify novel PKI isoforms from
tissues showing significant levels of PKI activity but low levels of
PKI and PKI
mRNA. Due to the relatively tissue-specific localization of PKI
and PKI
mRNAs, previous models of PKI
function had assumed that some tissues and cell types did not require
PKI for cAMP-mediated signal transduction. Assuming equivalent
translation rates, results from Northern blot analysis and RNase
protection analysis indicate that PKI
is widely expressed and may be
the predominant PKI isoform in several tissues, including kidney and liver. In addition, determination of PKI isoform message levels in
cultured cells showed that PKI
was expressed in all cell lines studied, and it was the major PKI transcript in all cell lines tested.
These cell lines could afford the opportunity to examine the role of
PKI
in the regulation of cAMP signaling.
PKI activity was first reported in 1965 as a heat-stable, trypsin-labile component of rabbit skeletal muscle extracts (52). The original observation that the inhibitory activity was stable to heat and acid treatment was used to devise a purification scheme (53). Rabbit skeletal muscle extracts were heated and loaded onto a DEAE column. PKI activity was eluted from this column using 0.25 M sodium acetate. Subsequently, this procedure was used to isolate PKI activity from a wide variety of tissues from many species. Using the results of these purifications and tissue purifications of C subunit, the level of PKI relative to C subunit in different tissues was calculated (54). For example, it was previously estimated that PKI activity in rat heart was sufficient to inhibit approximately 20% of the total heart C subunit. Similar results were obtained in other tissues with most tissues estimated to have significantly less total inhibitor than total C subunit. Importantly, this purification scheme limited the estimate of inhibitor activity from various tissues to those proteins that had the same chromatographic properties on DEAE-cellulose as the original material isolated from skeletal muscle.
To verify the existence of PKI protein in mouse tissues, PKI
activities from mouse heart were partially purified and identified in
this study. Two inhibitory peaks were resolved following heat denaturation, acid treatment, and DEAE chromatography of the heart extracts. PKI
and PKI
were identified as the proteins responsible for inhibitory peak 1 and peak 2, respectively. It is likely that the
previous purification procedure used to estimate PKI activity in
tissues did not detect PKI
due to its higher affinity for DEAE-cellulose than the other PKI isoforms. Interestingly, in the
purification of PKI activity from rat testis, the yield of inhibitory
activity after DEAE-cellulose chromatography is significantly lower
than after other steps in the purification. This loss of PKI activity
could reflect a selective loss of the PKI
isoform (37). Since the
PKI
mRNA is abundant and widely expressed, previous studies
significantly underestimated the PKI activities in several tissues.
Recent results further challenge the belief that all cells have less
total PKI than total C subunit. In situ hybridization
analysis of mouse brain suggests that there is considerable heterogeneity of both PKI
and PKI
mRNA among different mouse brain regions (31). If this heterogeneity is also true of the PKI
protein, then several regions of brain may have sufficient PKI to
inhibit all of the C subunit present (i.e. cerebellar
Purkinje cells and CA2 hippocampal neurons).
Results from this paper demonstrate that mouse heart contains
significant protein levels of at least two PKI isoforms, PKI and
PKI
. These results do not rule out the possibility of other uncharacterized PKIs, which may not bind DEAE-cellulose, or may not
elute over the 5-1000 mM NaOAc gradient used in this
study. Furthermore, multiple peaks of PKI activity have been detected by DEAE-cellulose chromatography of testicular extracts (19). Even
though the heat stability of the known PKI isoforms has greatly aided
in their purification, there is no a priori reason why all PKI isoforms should be heat stable.
During the course of these experiments the full-length PKI cDNA
nucleotide sequence was used to search the NCBI GenBank data base.
Significant sequence homology (p < 0.001) was detected
between the 3
end of the mouse PKI
gene and the 3
end of the mouse adenosine deaminase (Ada) gene (55). Direct comparison of
the two cDNA sequences demonstrated that the two genes overlapped in a tail-to-tail orientation with their coding sequences on opposite strands (Fig. 1), an uncommon occurrence in mammalian genomes. Adenosine deaminase is an important enzyme in purine metabolism, and
adenosine deaminase deficiency is a major cause of autosomal recessive
severe combined immune deficiency disease (56). Although tightly
linked, there is no obvious functional relationship between PKI
and
adenosine deaminase. To our knowledge no patients with deletions in the
3
end of the Ada gene have been identified. The mouse
Ada gene has been localized to mouse chromosome 2 approximately 94 centimorgans from the centromere (57). The mouse
PKI
gene (Prkacn2) has been localized to mouse chromosome
10 (58), and the mouse PKI
gene has not been mapped. Interestingly,
the PKI
mRNA transcript was first observed during the
characterization of the mouse Ada gene as an abundant,
widely expressed 1.3-kb transcript transcribed from the antisense DNA
strand that was co-amplified with the Ada gene during gene
amplification (55). The low levels of amino acid identity and the
distinct chromosomal localizations of the known PKI isoforms suggest
that the members of the PKI family are distantly related.
PKI mRNA is widely expressed and abundant in mammalian tissues
but has not been characterized previously due to its low sequence homology with the other known PKI isoforms and its high affinity for
DEAE-cellulose. The identification of a widely expressed, abundant PKI
isoform suggests that PKI activity may be more critical to general cell
function than previously believed. PKI
is differentially expressed
in adult tissues and shows distinct interactions with C subunit,
suggesting it may serve distinct roles. The availability of
purification methods and antibodies capable of differentiating between
PKI
and other PKI isoforms should help clarify their specific roles.
Future studies should determine what, if any, specific functions the
non-conserved carboxyl-terminal region of these proteins play.
Verification of these specific roles will require the development of
model systems selectively deficient in the expression of the three PKI
genes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U97170.
We thank Linda Harper for expert cell culture
assistance and Donna Ray, Elizabeth Leslie, and Cindy Overmyer for
assistance in the preparation of this manuscript. We acknowledge Dr.
Audrey F. Seasholtz, Rafael P. Ballestero, John W. Denninger, David M. Gamm, and the members of the Uhler lab for helpful discussions. In
addition, we acknowledge John W. Denninger for technical expertise and
assistance in the partial purification of PKI activities from mouse
heart and Dr. Michael A. Marletta for use of the BioLogic system. We
extend additional thanks to Dr. Eric J. Baude for providing recombinant
C and David M. Gamm for providing the pET9d.His6PKI
expression vector and recombinant MBP-PKI
.