Characterization of PKIgamma , a Novel Isoform of the Protein Kinase Inhibitor of cAMP-dependent Protein Kinase*

(Received for publication, April 23, 1997)

Sean P. Collins and Michael D. Uhler Dagger

From the Department of Biological Chemistry and the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta 1. Residues important for the high affinity of PKIalpha and PKIbeta 1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma . Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma 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 PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma . A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM). Like PKIalpha , PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.


INTRODUCTION

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 (Calpha , Cbeta , and Cgamma ) and four R subunit isoforms (RIalpha , RIbeta , RIIalpha , and RIIbeta ) (4, 5). Although the amino acid sequences of Calpha and Cbeta are highly similar (6), they differ significantly in their tissue distributions and interactions with R subunits. Calpha is widely expressed in mammalian tissues, and Cbeta is expressed in cells of the nervous, endocrine, and reproductive systems (6, 7). Importantly, PKA holoenzymes formed with RIIalpha and Cbeta have a 5-fold lower Ka value for cAMP than RIIalpha and Calpha 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 RIalpha are less sensitive to increases in cAMP than those containing RIbeta (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, PKIalpha and PKIbeta (18-21). The PKIalpha isoform is highly expressed in heart, skeletal muscle, cerebral cortex, and cerebellum (18, 21), whereas the PKIbeta 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 PKIalpha and PKIbeta 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 PKIalpha and PKIbeta 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 PKIalpha corresponding to a leucine-rich sequence conserved between PKIalpha and PKIbeta (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 PKIalpha and PKIbeta . 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 PKIgamma . Like other members of the PKI family, PKIgamma inhibits cAMP-dependent gene transcription and nuclear accumulation of the C subunit. The results of this study suggest that PKIgamma is found at physiologically significant levels in many tissues and functions in a manner similar to previously characterized PKI isoforms.


MATERIALS AND METHODS

Isolation and Sequencing of a cDNA Clone Encoding Murine PKIgamma

A full-length cDNA sequence coding for murine PKIgamma (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 PKIalpha 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 PKIgamma sequence has been submitted to the GenBank data base.

Northern Blot Analysis

Plasmids AR-1 and MtPKI.pcr were linearized with BamHI and BglII, respectively, and used to generate antisense RNA probes for PKIalpha and PKIbeta , respectively, as described (31). The template for the PKIgamma antisense RNA probe was constructed by inserting the 231-bp BamHI/BglII fragment of pGEM-T.mPKIgamma (described below) into the BamHI/BglII site of pSP73 (Promega) to create pSP73.mPKIgamma . This construct was linearized with BglII and used to generate PKIgamma 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

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-PKIalpha (32) as a template to amplify a fragment consisting of the full ORF of murine PKIalpha flanked by BglII sites. This amplified fragment was digested with BglII, isolated, and ligated into the BamHI/BglII sites of pSP73 to create pSP73.mPKIalpha . The resulting plasmid was linearized with BglII or HindIII and used as a template to synthesize antisense PKIalpha ORF RNA probes or sense RNA standards, respectively. MtPKI.pcr was used as a template to synthesize antisense murine PKIbeta ORF RNA probes or sense RNA standards as described (31). pSP73.mPKIgamma was linearized with BglII or BamHI and used as a template to synthesize antisense PKIgamma 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 [alpha -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).

Construction of PKI Mammalian Expression Vectors, Transient Transfection of HEK293 Cells, and Determination of C Subunit Inhibitory Activity

A mammalian expression plasmid encoding a carboxyl-terminal hemagglutinin (HA)-tagged murine PKIalpha protein was constructed by PCR. A PCR fragment encoding PKIalpha 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-PKIalpha as a template. The resulting PCR fragment was digested with BglII, isolated, and ligated into BglII digested pCMV.Neo (34) to create pCMV.HA-PKIalpha . Likewise, the pCMV.mPKIgamma 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 PKIgamma flanked by a BglII site and a BamHI site was ligated into pGEM-T (Promega) to create pGEM-T.mPKIgamma . pGEM-T.mPKIgamma was digested with BamHI and BglII, and a 231-bp fragment coding for mPKIgamma was isolated and ligated into the BglII site of pCMV.Neo. Both pCMV.HA-PKIalpha and pCMV.mPKIgamma were sequenced to confirm the coding region sequence. The human PKIalpha and murine PKIbeta 1 mammalian expression plasmids have been described previously (20, 35). The human PKIalpha protein is 97% identical to the murine PKIalpha 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.hPKIalpha , pCMV.mPKIbeta 1, pCMV.mPKIgamma , 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 Calpha (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.

Partial Purification of PKI Activities from Mouse Heart

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 PKIalpha and PKIgamma

The pET9d.His6PKIalpha and pET9d.His6PKIgamma 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 PKIalpha and PKIgamma each with an amino-terminal hexahistidine tag. pMAL-PKIalpha and clone 419982 were used as templates in the PKIalpha and PKIgamma 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.His6PKIalpha and pET9d.His6PKIgamma . E. coli cultures (1 liter) were grown and induced with 1 mM isopropyl-1-thio-beta -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 His6PKIalpha and His6PKIgamma were separately conjugated to keyhole limpet hemocyanin and used to immunize rabbits for antibody production (Research Genetics Inc.).

Western Blotting

Antisera raised against His-tagged PKIalpha and PKIgamma were affinity purified on MBP-PKIalpha (32) and MBP-PKIgamma (see below) nitrocellulose blots essentially as described (39, 40). Affinity purified anti-PKIalpha antibody recognized PKIalpha (14 kDa) but did not react with PKIbeta or PKIgamma on Western blots of HEK293 cell extracts from cells transfected with PKI expression constructs. Likewise, affinity purified anti-PKIgamma recognized PKIgamma (16 kDa), but no signal was detected in extracts from PKIalpha - or PKIbeta -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-PKIalpha or anti-PKIgamma 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.).

Construction of Prokaryotic Expression Vector for PKIgamma and Determination of PKI Isoform IC50 and Ki Values

To create an MBP-PKIgamma prokaryotic expression vector, the murine PKIgamma 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-PKIgamma 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-PKIalpha and MBP-PKIgamma were greater than 95% pure as determined by scanning densitometry of Coomassie Blue-stained SDS-PAGE gels. IC50 values and Ki values for MBP-PKIalpha and MBP-PKIgamma were determined essentially as described (16). Fifty pM recombinant Calpha was used in all phosphotransferase activity assays. Ki values for murine PKIalpha and PKIgamma were determined using the Henderson method for tightly bound inhibitors (41).

Construction of Mammalian Expression Vectors for Flag-tagged C Subunits

Mammalian expression plasmids encoding amino-terminal Flag-tagged murine Calpha and Calpha Y235S/F239S (42) proteins were constructed using the PCR method. An amino-terminal Calpha 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.Calpha (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-Calpha 1. pGem-4.Calpha was digested with BamHI and BglII. An approximately 450-bp fragment encoding the carboxyl-terminal of Calpha was isolated and ligated into BglII digested pCMV.Flag-Calpha 1 to generate pCMV.Flag-Calpha 2. A 240-bp BglII fragment coding the central one-third of Calpha was isolated from the same digest and ligated into BglII digested pCMV.Flag-Calpha 2 to generate the wild type Flag-tagged Calpha expression vector pCMV.Flag-Calpha 3. To create the Calpha Y235S/F239S mutant expression vector, pET9d.Calpha Y235S/F239S (42) was digested with BglII, and the 240-bp fragment containing the mutated sites was ligated into BglII-digested pCMV.Flag-Calpha 2 to generate pCMV.Flag-Calpha Y235S/F239S. Both expression plasmids were restriction mapped and sequenced.

Transient Transfection of NIH 3T3 Cells and Luciferase Assays

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-Calpha 3 or pCMV.Flag-Calpha Y235S/F239S, 1 µg of human chorionic gonadotropin-luciferase (HCG.Luciferase), 5 µg of pRSV.beta gal, and the indicated amounts of pCMV.mPKIgamma . 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 beta -galactosidase activities as described (8).

Immunofluorescence

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-Calpha 3, 8 µg of pCMV.RIIalpha , and either no PKI expression vector, 8 µg of pCMV.HA-PKIalpha , or 4 µg of pCMV.mPKIgamma . 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.


RESULTS

Identification of cDNA Clone Encoding Murine PKIgamma

A comparison of PKI activity of tissue extracts with PKIalpha and PKIbeta 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 PKIalpha and PKIbeta 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 PKIalpha (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 PKIalpha amino acid sequence. The cDNA clone was fully sequenced (Fig. 1), shown to contain 1064 base pairs (bp), and characterized further.


Fig. 1. Nucleotide and predicted amino acid sequence of murine PKIgamma . Amino acid sequence of murine PKIgamma inferred from the nucleotide sequence is represented below the DNA sequence with the one-letter amino acid codes. Initiator ATG indicated was selected as the translation start site due to optimal alignment with the other known PKI isoforms. Nucleotide numbers are indicated at the left of the sequence, and amino acid numbers are indicated at the right of the sequence. The nucleotide sequence homologous to the murine adenosine deaminase genomic sequence starts at base 377 and extends to the end of the sequence. The putative polyadenylation signal is underlined once.
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Assuming that the first of two methionine codons encodes the initiator methionine, the murine PKIgamma 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 PKIgamma 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. PKIgamma shows relatively low amino acid homology to the other known murine PKI isoforms: PKIalpha (35% identity) and PKIbeta 1 (30% identity). However, most of the residues demonstrated to play a role in the high affinity of PKIalpha 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 PKIgamma (28) (Fig. 2). Based on these elements of sequence homology, the new protein was designated PKIgamma . Most of the homology between PKIgamma 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).


Fig. 2. Amino acid sequence alignment of murine PKI isoforms. Predicted protein sequence of murine PKIgamma is compared with the protein sequences of murine PKIalpha and murine PKIbeta 1 in the above alignment using DNASTAR software. The numbering of the three sequences begins with the predicted or known initiator methionine and is placed on the right of the diagram. Amino acid residues identical between any two of the three sequences are boxed. Residues known to be important in high binding affinity of PKIalpha for C subunit are indicated with a plus sign on the top line (Tyr7, Phe10, Arg15, and pseudosubstrate sequence). Hydrophobic residues shown to be important in the nuclear export of C subunit by PKIalpha are marked by an asterisk on the bottom line.
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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 (PKIalpha , PKIbeta , and PKIgamma ). The PKIalpha 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 PKIbeta 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 PKIbeta 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 PKIbeta 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 PKIbeta gene. The PKIgamma probe detected a single 1.3-kb transcript in all tissues examined. Unlike the PKIalpha and PKIbeta 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 PKIgamma mRNA in these organs is important since they have low levels of PKIalpha and PKIbeta message (18, 20, 21).


Fig. 3. Expression of PKI isoforms mRNA in adult murine tissues. A single Northern blot containing 2 µg of poly(A)+ RNA isolated from adult mouse tissues was hybridized separately with antisense RNA probes (see "Materials and Methods") specific for PKIalpha (A), PKIbeta (B), and PKIgamma (C). The blot was stripped for 10 min in boiling water containing 0.5% SDS prior to reprobing. The sizes of RNA standards are indicated to the left of the figure in kilobases (kb). The positions of the mRNA transcripts, their identities, and their apparent sizes are indicated with an arrow on the right of the figure.
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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 PKIgamma mRNA is detected in Northern blot analysis including skeletal muscle (21, 22), heart (38, 45), and testis (19, 37). However, it was surprising that PKIgamma had not been identified previously. One explanation could be that PKIgamma transcripts are widely expressed but of low abundance relative to PKIalpha and PKIbeta transcripts. To quantitate PKIgamma 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 PKIalpha , 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 PKIbeta 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 PKIalpha and PKIbeta RNase protections, hybridization with the PKIgamma 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 PKIgamma 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 PKIgamma expression in the testis. The PKIgamma Northern blot analysis shows an intermediate level of PKIgamma expression in the testis (Fig. 3C), and the RNase protection analysis suggests that the testis is the organ of highest PKIgamma mRNA expression (Fig. 4A). PhosphorImager quantitation of sense RNA-protected fragments and tissue-protected fragments indicates that PKIgamma mRNA is expressed at comparable levels to PKIalpha and PKIbeta in all mouse tissues tested. In several tissues such as heart, lung, liver, and kidney, PKIgamma is the predominant PKI isoform transcript (Fig. 4, B-D).


Fig. 4. Quantitation of PKI isoform mRNA in murine tissues and cell lines. A, RNase protection analysis of PKIgamma expression in murine tissues. Total RNA (10 µg) from murine tissues or sense standard RNA (0, 0.3, 1, 3, 10, or 30 pg) was hybridized to a PKIgamma -specific antisense RNA probe, RNase-treated, and separated by denaturing gel electrophoresis (see "Materials and Methods"). The position of the RNA probe (300 base pairs (bp)) and the sense standards RNA-protected fragments (246 bp) are indicated on the left with an arrow, and the position of the tissue RNA-protected fragments (233 bp) are indicated on the right with an arrow. B-E, PhosphorImager quantitation of RNase protection analysis of PKI isoform expression in murine tissues (B--D) and PKIgamma expression in murine cell lines (E). RNase protection analyses were performed as above. Sense RNA standard assays were used to determine the number of picograms of protected fragment per microgram of total RNA. He, heart; Br, brain; Sp, spleen; Lu, lung; Li, liver; SM, skeletal muscle; Ki, kidney; Te, testis; Th, thyroid; NIH, NIH 3T3 fibroblasts; C2, C2C12 myoblasts; N1E, N1E-115 neuroblastoma; TM4, TM4 Sertoli cells; A20, AtT-20 pituitary corticotrophs; Y1, Y1 adrenal tumor cells; and L, L929 fibroblasts.
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To confirm the widespread nature of PKIgamma 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 PKIgamma 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 PKIalpha transcript, whereas no bands were detected in any of the cell lines in the PKIbeta RNase protection analysis (data not shown). Of interest, no evidence of PKI alternative splice variants was observed in these RNase protection experiments.

Separation and Identification of PKI Activities from Mouse Heart

Another possible explanation for why PKIgamma was not detected during prior PKI purifications is that it possesses significantly different biochemical characteristics from the other two known isoforms. Analysis of PKIalpha , PKIbeta 1, and PKIgamma amino acid sequences shows that the three proteins vary considerably in their predicted isoelectric points; PKIalpha has a pI of 4.4, PKIbeta 1 has a pI of 5.1, and PKIgamma has a pI of 3.9. Previous purifications of PKIalpha and PKIbeta 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, PKIbeta 1 eluted at lower ionic strengths than PKIalpha . Since PKIgamma has a lower pI than PKIalpha , it would be expected to elute later in the gradient. No inhibitory peaks were eluted after PKIalpha , but it is possible that PKIgamma was never eluted from the column. To test this hypothesis and to verify that the PKIgamma 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 PKIalpha and PKIgamma 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 PKIalpha or PKIgamma , eukaryotic expression vectors for human PKIalpha and murine PKIgamma were transiently transfected into HEK293 cells, and PKI activities were partially purified as for mouse heart. Assay of the fractions from PKIalpha -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.mPKIgamma 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).


Fig. 5. Partial purification and identification of PKI isoforms from murine heart. A, heat- and acid-treated mouse heart extract (25 ml) was chromatographed on a DEAE column (see "Materials and Methods"). Fractions (1 ml) were collected and assayed for C subunit inhibitory activity. Inhibitory activity is defined as the percent inhibition of total control kinase activity and is denoted by a solid line (bullet ). Conductivity is represented by a dashed line. The open bar spans the fractions of inhibitory peak 1 (fractions 10-15), and the hatched bar spans the fractions of inhibitory peak 2 (fractions 18-23). B and C, Western blot analysis of PKI activities eluted from DEAE column. Aliquots from fractions containing significant inhibitory activity (peak 1, fractions 10-15; peak 2, fractions 18-23) were concentrated, resolved by 15% SDS-PAGE, and immunoblotted with either affinity purified anti-PKIalpha antibody (B) or affinity purified anti-PKIgamma antibody (C) using colorimetric detection. The sizes of the protein standards are indicated on the left of the figure in kilodaltons (kDa). The identity and the apparent molecular mass of the identified bands is indicated on the right with an arrow.
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To confirm the identity of the inhibitors responsible for inhibitory peak 1 and peak 2 from mouse heart, isoform-specific PKIalpha and PKIgamma antisera were prepared from rabbits using His-tagged PKIalpha and His-tagged PKIgamma as antigens. The specificity of these antisera was determined using HEK293 cells transfected with pCMV.hPKIalpha (HEK.hPKIalpha extracts), pCMV.mPKIbeta 1 (HEK.mPKIbeta 1 extracts), pCMV.mPKIgamma (HEK.mPKIgamma extracts), and pCMV.Neo (HEK.Neo extracts). These extracts were resolved by 15% SDS-PAGE and transferred to nitrocellulose. Anti-PKIalpha sera recognized a single band with an apparent molecular mass of 14 kDa in the HEK.hPKIalpha extracts. No bands were detected in the other extracts. Similar results were obtained with the anti-PKIgamma sera that detected a single band with an apparent molecular mass of 16 kDa in the HEK.mPKIgamma 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-mPKIalpha antibody detected a single 14-kDa band in peak 1 fractions (Fig. 5B). This band was the same apparent molecular mass as transfected PKIalpha . Likewise, the affinity purified anti-PKIgamma antibody recognized a single 16-kDa band in peak 2 fractions that co-migrated with transfected PKIgamma (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 PKIalpha is the inhibitor responsible for the majority of inhibitory peak 1 and PKIgamma is the inhibitor responsible for the majority of inhibitory peak 2. These results suggest that both PKIalpha and PKIgamma are expressed at significant levels in mouse heart and that PKIgamma can be distinguished from other PKI isoforms both immunologically and by its DEAE elution characteristics.

Kinetic Analysis of Murine PKIalpha and PKIgamma

To study the inhibitory potency of murine PKIgamma , full-length PKIgamma was PCR-amplified, cloned into pMAL.cRI, and expressed in E. coli as an MBP-PKIgamma fusion protein. Previously, it has been shown that the presence of an amino-terminal MBP fusion does not affect the inhibitory efficacy of PKIalpha or PKIbeta 1 (16). To assess relative inhibitory efficacy, PKIgamma was compared with murine PKIalpha (16) in in vitro kinase inhibition assays. C subunit phosphotransferase activity was measured in the presence of increasing concentrations of either PKIalpha or PKIgamma . 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 PKIalpha and PKIgamma at 30 µM Kemptide substrate revealed that PKIgamma possesses a 14-fold greater IC50 value than PKIalpha (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 PKIalpha and PKIgamma 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 PKIalpha and PKIbeta 1, PKIgamma is a competitive inhibitor of C subunit (16, 19, 22, 23). Hence, PKIgamma is a potent, competitive inhibitor of C subunit; however, it is 6-fold less potent than PKIalpha . PKIgamma possesses all the amino acid residues previously shown to be important in the high affinity of PKIalpha for C subunit (Tyr7, Phe10, Arg15, Arg18, and Arg19) (16, 25, 26); however, it is possible that PKIgamma contains negative binding determinants. Mutagenesis of Thr8 and Ser12 in PKIbeta 1 to alanines caused a 4-fold increase in the inhibitory potency of PKIbeta 1. It has been postulated that these residues may block formation of the amino-terminal inhibitory alpha -helix of PKIbeta 1 (16). Thus, it is possible that Ser8 and Ser12 of PKIgamma are negative determinants of C subunit binding.


Fig. 6. Inhibition of recombinant Calpha by murine PKIalpha and PKIgamma . A, IC50 determinations. Protein kinase activity was measured with 30 µM Kemptide substrate in the absence or presence of increasing concentrations of PKIalpha (open circle ) and PKIgamma (bullet ). Activity was expressed as the percentage of Calpha -specific activity in the absence of inhibitor. The curves were fitted using the average values of triplicate assay points from representative experiments, and the error bars represent the standard deviation from the mean. The experiments were performed four times for each inhibitor. Average IC50 values are reported in Table I. B, Ki determination by Henderson analysis. Protein kinase activity was measured in the absence or presence of PKIalpha or PKIgamma at the following Kemptide substrate concentrations: 5 µM (open circle ), 20 µM (bullet ), 40 µM (down-triangle), and 60 µM (black-down-triangle ). The data for a representative experiment of PKIgamma are shown. It is the total inhibitor concentration, and Vi and Vo are the reaction velocities in the presence and absence of inhibitory proteins, respectively. C, replots of the slopes from Henderson analyses versus Kemptide substrate concentration for PKIalpha (open circle ) and PKIgamma (bullet ). The experiments were performed three times for each inhibitor. Average Ki values are reported in Table I.
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Table I. Inhibition constants of recombinant PKIalpha and PKIgamma

The 50% inhibitory concentrations (IC50 values) are derived from titration of recombinant C subunit with PKI isoforms in the presence of 30 µM Kemptide substrate (see "Materials and Methods"). A representative experiment is shown in Fig. 6A. The Ki values were obtained from Henderson analyses (Fig. 6, B and C). IC50 and Ki values are expressed as the average ± S.D. from at least three experiments.

PKI isoform IC50 Ki

nM nM
PKIalpha 0.085  ± 0.017 0.073  ± 0.017
PKIgamma 1.2  ± 0.2 0.44  ± 0.06

Mammalian Expression and C Subunit Inhibitory Activity of PKIgamma

Expression experiments were performed to demonstrate that the PKIgamma 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 PKIgamma to inhibit cAMP-dependent gene transcription and C subunit nuclear accumulation. pCMV.mPKIgamma , a PKIgamma mammalian expression vector, was constructed. Initially, HEK293 cells were transiently transfected with pCMV.mPKIgamma , pCMV.hPKIalpha , pCMV.mPKIbeta 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.mPKIgamma showed greater inhibitory activity than pCMV.mPKIbeta 1-transfected cells but less inhibitory activity than the pCMV.hPKIalpha -transfected cells (data not shown). The presence of the PKIs in cell extracts was verified with PKI-specific antibodies (data not shown).

In Vivo Inhibition of cAMP-dependent Gene Transcription by PKIgamma

To verify the ability of PKIgamma to inhibit C subunit in vivo, NIH 3T3 cells were transiently transfected with a constant amount of the Calpha expression vector, either pCMV.Flag-Calpha 3 or pCMV.Flag-Calpha Y235S/F239S, and increasing amounts of pCMV.mPKIgamma . Each plate also received a constant amount of a cAMP-responsive reporter plasmid (HCG.luciferase) (46) and pRSV.beta 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.mPKIgamma was required to completely inhibit luciferase activity in cells transfected with pCMV.Flag-Calpha 3. NIH 3T3 cells expressing the Flag-tagged Calpha Y235S/F239S mutant, a Calpha mutant with reduced affinity for PKIalpha and PKIbeta 1 (42), showed significantly less inhibition of luciferase activity at all pCMV.mPKIgamma concentrations (Fig. 7). It has been shown previously that three PKIalpha residues outside the pseudosubstrate sequence, Tyr7, Phe10, and Arg15, contribute significantly to the high affinity of PKIalpha for C subunit. Specifically, Phe10 of PKIalpha interacts with a hydrophobic pocket on the surface of the C subunit consisting of residues Tyr235 and Phe239 (24, 42). The reduced ability of PKIgamma to inhibit the Calpha Y235S/F239S mutant is consistent with the conservation of Phe10 between PKIalpha and PKIgamma (Fig. 2). Interestingly, even though Calpha 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.


Fig. 7. Inhibition of luciferase gene transcription by PKIgamma in vivo. NIH 3T3 cells were transiently co-transfected with 1 µg of an expression vector for either Calpha (open circle ) or Calpha Y235S/F239S (bullet ) and the indicated amounts of a PKIgamma expression vector. All plates received 1 µg of a cAMP-responsive reporter construct (HCG.luciferase) and 5 µg of pRSV.beta gal to normalize for transfection efficiency. 21 h after removal of precipitates cells were harvested and assayed for luciferase and beta -galactosidase activities. Luciferase activity was corrected for transfection efficiency by dividing by beta -galactosidase activity and expressed as the percentage of relative light units (RLU) in the absence of the inhibitor. The error bars depict the standard deviation from the mean. This experiment was repeated three times, and a representative experiment is shown above.
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PKIgamma Blocks Nuclear Accumulation of C Subunit

Both PKIalpha and PKIbeta are capable of actively exporting C subunit from the nucleus (27). In the case of PKIalpha , 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 PKIalpha and PKIbeta 1 (28). Amino acid alignment of PKIgamma with PKIalpha and PKIbeta 1 reveals the possible presence of a NES in PKIgamma (Fig. 2). The most significant difference observed was a glycine at amino acid residue 39 where both PKIalpha and PKIbeta 1 contain leucines. To test the functionality of the NES of PKIgamma , a transient transfection assay was developed. An amino-terminal Flag-tagged Calpha 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 PKIalpha . 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-Calpha 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 PKIalpha 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-Calpha 3 expression vector, and it is recognized by the M2 anti-Flag monoclonal antibody when free in solution and when bound to PKIalpha .

To study the effect of elevations in cAMP on the cellular localization of C subunit in the presence and absence of PKIgamma , CV-1 cells were transiently co-transfected with expression vectors for RIIalpha , amino-terminal Flag-tagged Calpha and either no PKI, PKIalpha , or PKIgamma . Enough RIIalpha expression vector was used in each transfection to completely inhibit transfected C subunit phosphotransferase activity (data not shown). Likewise, in transfections including PKIalpha or PKIgamma , 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 RIIalpha (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 PKIalpha or PKIgamma expression vectors with the Flag-tagged C subunit and RIIalpha had no discernible effect on the cytoplasmic localization of C subunit in non-stimulated cells (Fig. 8, C and E). However, both PKIalpha and PKIgamma 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).


Fig. 8. Inhibition of Flag-tagged C subunit nuclear accumulation by PKIalpha and PKIgamma . Indirect immunofluorescence microscopy analysis of CV-1 cells transiently co-transfected with pCMV.Flag-Calpha 3, pCMV.RIIalpha , and either no PKI expression vector (a and b), pCMV.HA-PKIalpha (c and d), or pCMV.mPKIgamma (e and f). 21-h post-transfection forskolin (Forsk.) (25 µM) and isobutylmethylxanthine (500 µM) were added to (Forsk.; b, d, and f) or omitted (Cont.; a, c, and e) for 40 min. Cells were fixed with a 4% paraformaldehyde solution and a 1:1 solution of acetone and methanol and then labeled with the anti-Flag antibody (M2) as the primary antibody and Cy3-F(ab')2 goat anti-mouse IgG as the secondary antibody.
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DISCUSSION

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: PKIalpha , PKIbeta , and PKIgamma . The amino acid sequence of PKIgamma is 35% identical to PKIalpha and 30% identical to PKIbeta 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 PKIgamma 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 PKIgamma will require Mg2+ and ATP for high affinity interactions with C subunit (47), that it will inhibit both Calpha and Cbeta 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 PKIalpha and PKIbeta 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 PKIbeta 1 possesses a 32-fold higher Ki for Calpha than murine PKIalpha (16). This difference has been demonstrated to be due in part to the absence of a tyrosine at position 7 of PKIbeta 1 (16). Because Tyr7 of PKIalpha is conserved in PKIgamma , we anticipated that PKIgamma would bind and inhibit Calpha with a subnanomolar Ki. Data from this study demonstrate that indeed PKIgamma is a tight binding inhibitor of C subunit with a Ki of 0.44 nM. Still, it is 6-fold less potent than PKIalpha . 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, PKIgamma possesses a single cysteine residue at amino acid position 13. Assuming that PKIgamma has an overall structure similar to PKIalpha (24), Cys13 of PKIgamma would be located at the boundary of the amino-terminal alpha -helix and beta -turn regions, two regions implicated in the high binding affinity of PKIalpha for C subunit. Previously, the beta -turn region of PKIalpha has been hypothesized to be important in the proper positioning of Arg15 with Glu203 of the C subunit (24, 32). Since the beta -turn region of PKIalpha 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 PKIgamma 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 PKIgamma 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 PKIalpha and PKIbeta mRNA. Due to the relatively tissue-specific localization of PKIalpha and PKIbeta 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 PKIgamma 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 PKIgamma 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 PKIgamma 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 PKIgamma 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. PKIalpha and PKIgamma 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 PKIgamma 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 PKIgamma isoform (37). Since the PKIgamma 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 PKIalpha and PKIbeta 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, PKIalpha and PKIgamma . 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 PKIgamma 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 PKIgamma 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 PKIgamma 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 PKIbeta gene (Prkacn2) has been localized to mouse chromosome 10 (58), and the mouse PKIalpha gene has not been mapped. Interestingly, the PKIgamma 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.

PKIgamma 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. PKIgamma 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 PKIgamma 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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM 38788 (to M. D. U.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U97170.


Dagger    To whom correspondence should be addressed: Neuroscience Laboratories Bldg., 1103 E. Huron St., University of Michigan, Ann Arbor, MI 48104-1687. Tel.: 313-647-3172; Fax: 313-936-2690.
1   The abbreviations used are: PKA, cAMP-dependent protein kinase; bp, base pair(s); C, catalytic; CMV, cytomegalovirus; HA, hemagglutinin; HCG, human chorionic gonadotropin; I.M.A.G.E., integrated molecular analysis of genomes and their expression; kb, kilobase; MBP, maltose binding protein; NES, nuclear export signal; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PKI, protein kinase inhibitor; R, regulatory.

ACKNOWLEDGEMENTS

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 Calpha and David M. Gamm for providing the pET9d.His6PKIalpha expression vector and recombinant MBP-PKIalpha .


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