Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons.

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.

Deficient gene expression in protein kinase inhibitor alpha Null mutant mice.

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

Implications of a simple mathematical model to cancer cell population dynamics.

Recent research in cancer progression and treatment indicates that many forms of cancer arise from the development of a small subpopulation of abnormal cancer stem cells (CSCs) that promote cancer growth and spread. Many potential treatments preferentially interact with cells at certain stages of the cell cycle by either selective killing or halting the cell cycle, such as intense, nanosecond-duration pulsed electric fields (nsPEFs). Simple mathematical models of unfed cancer cell populations at the plateau of their growth characteristics may estimate the long-term consequences of these treatments on proliferating and quiescent cell populations. Applying such a model with no transition from the quiescent to proliferating state shows that it is possible for the proliferating cell population to fall below 1 if the quiescent cell population obtains a sufficient competitive advantage with respect to nutrient consumption and/or survival rate. Introducing small, realistic transition rates did not appreciably alter short-term or long-term population behaviour, indicating that the predicted small cell population behaviour (< 1 cell) is not an artefact of the simpler model. Experimental observations of nsPEF-induced effects on the cell cycle suggest that such a model may serve as a first step in assessing the viability of a given cancer treatment in vitro prior to clinical application.

Inhibition of protein kinase-A by overexpression of the cloned human protein kinase inhibitor.

Human cDNA clones for a heat-stable protein kinase inhibitor (PKI) protein of the cAMP-dependent protein kinase (PKA) were isolated using a mouse PKI cDNA fragment. Two human cDNA clones of 1.7 and 2.0 kb were sequenced and shown to encode the entire open reading frame of 228 nucleotides. Together these clones comprised 2147 nucleotides of the mRNA. The deduced amino acid sequence of the human clones showed 100% identity to the rabbit skeletal muscle PKI protein and 97% identity to the mouse brain PKI. The mouse and human PKI cDNAs shared nucleotide homology in their 3' untranslated regions as well as in the 32 nucleotides immediately 5' of the translation initiation site. Northern blot analysis of human skeletal muscle RNA with a human cRNA probe detected a major mRNA of approximately 4.0 kb. Transient overexpression in COS cells verified that a heat-stable inhibitor of protein kinase was produced by he human PKI cDNA, and protein extracts from the transfected COS cells inhibited both the C alpha and C beta isoforms of the PKA catalytic subunit with equal efficacy. Functional expression of the human PKI protein was further studied by assaying the ability of PKI expression vectors to inhibit PKA catalytic subunit stimulation of transcription from the human enkephalin promoter. In these studies, elimination of a conserved alternative translation start site in the 5' untranslated region of PKI was shown to potentiate the inhibitory activity of the PKI expression vector.

Sertoli cells are the primary site of prodynorphin gene expression in rat testis: regulation of mRNA and secreted peptide levels by cyclic adenosine 3' ,5'-monophosphate analogs in cultured cells.

Prodynorphin is one of three endogenous opioid peptide genes expressed in testis. Through the use of cell fractionation procedures and Northern blot analysis, Sertoli cells were found to be the primary site of prodynorphin mRNA synthesis in rat testis. In situ hybridization of a prodynorphin cRNA probe to fixed adult tissue confirmed this result. Treatment of primary cultures of rat Sertoli cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP, resulted in a transient 5.6-fold increase in steady state prodynorphin mRNA levels relative to those in control cells. This increase was maximal at 48 h of treatment, after which mRNA levels gradually declined. Treatment of Sertoli cells with cAMP analogs resulted in concurrent 2.6-fold decreases in sulfated glycoprotein-2 mRNA levels. Culture medium from Sertoli cells showed a 3.1-fold increase in secreted dynorphin immunoreactivity after treatment with 8-(4-chlorophenylthio)cAMP. Chromatographic analysis indicates that the majority of the immunoreactive dynorphin peptide synthesized in Sertoli cells is present as high mol wt species, with some processing to bioactive peptides.

Genetic alteration of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunit expression affects calcium currents and beta-endorphin release in AtT-20 clonal pituitary cells.

The role of the cAMP-dependent kinase (AK) in neurotransmission was investigated by genetic alteration of AK subunit expression in AtT-20 clonal pituitary cells. We characterized and compared wild-type [AK(wt)] cells and two clones with different AK activities. The first stably expresses a gene for a mutant AK regulatory subunit (RI) that does not bind cAMP [AK(-)]; the second stably expresses a gene for the catalytic subunit (C) of AK [AK(+)]. Western blot analysis of RI and C subunit expression showed increased expression of both subunits in AK(+) and AK(-) cells relative to AK(wt), with the transfection-induced expression of one subunit producing a compensatory increase in the expression of the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) cells, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 microM 8-(4-chlorophenylthio)-cAMP increased AK activity approximately 4-fold in AK(wt) and AK(+) cells, but was without effect in AK(-) cells. Subsequent addition of 1 microM cAMP in vitro increased AK activity an additional 2- to 3-fold in all cell types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (approximately 43% and approximately 75% larger than in AK(-) cells, respectively) and faster rates of current rundown. The currents from each cell line had similar voltage-dependent and pharmacological properties, however, and [3H]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated approximately 30% in the steady state at holding potentials of -40 mV compared to -80 mV, and currents were reduced approximately 45% in the presence of nifedipine at -40 mV, but were insensitive to omega-conotoxin GVIA. AK(wt) and AK(+) cells also had higher basal and cAMP-stimulated release of beta-endorphin; control rates were approximately 50% greater, but stimulated rates were approximately 400% greater compared to those in AK(-) cells. We conclude that a greater number of calcium channels were activated by depolarization in the phosphorylated state, that current rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase.

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.

Localization of cGMP-dependent protein kinase isoforms in mouse eye.

PURPOSE: To examine the expression of the major isoforms of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) in mouse eye. METHODS: Immunohistochemical localization of cGMP in mouse eye cryosections was performed using an anti-cGMP antibody, followed by visualization with indirect fluorescence microscopy. The presence of types Ialpha, Ibeta, and II cGK mRNAs in mouse eye extracts was determined initially by RNase protection analysis. Further localization of cGK I and II mRNAs on cryosections was accomplished by in situ hybridization using digoxigenin-labeled cRNA probes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Finally, cGK I protein was localized to subcellular areas within the retina using an anti-cGK I-specific primary antibody. RESULTS: In initial immunohistochemical experiments cGMP was present in numerous regions and layers within the eye and retina. Subsequent RNase protection studies demonstrated that cGK Ialpha, Ibeta, and II mRNAs were present in mouse eye and that type Ibeta mRNA were 6.6 and 30 times more abundant than type Ialpha and type II, respectively. By in situ hybridization, cGK I mRNA was localized to photoreceptor inner segments and the ganglion cell and inner nuclear layers of the retina, and lesser amounts were found in the ciliary epithelium, lens, and cornea. The cGK II mRNA expression pattern was similar but not identical with that of cGK I. Finally, within the retina, cGK I protein was most abundant in the inner plexiform layer, with significant amounts in ganglion cells and photoreceptor inner segments as well. CONCLUSIONS: The presence of these cGK isoforms in discrete areas throughout the eye suggests multiple roles for the cGMP-dependent signal transduction system in the regulation of physiologic and pathologic ocular processes.

The reduction of neuronal calcium currents by ATP-gamma-S is mediated by a G protein and occurs independently of cyclic AMP-dependent protein kinase.

We studied the effects of ATP-gamma-S on the T, N and L calcium current components of nodose ganglion neurons using the whole cell variation of the patch clamp technique. ATP-gamma-S can serve as a phosphate donor in kinase-mediated reactions, the donated phosphate group being resistant to the action of phosphatases. We therefore compared the effect of ATP-gamma-S to that of the catalytic subunit of the cyclic AMP-dependent protein kinase (AK-C), included in the recording pipette with 5 mM ATP. AK-C (50 micrograms/ml) had no effect on the T current, and caused a approximately 30% increase in currents containing the N and L components during a 20-min recording, as compared to a approximately 45% decrease in control currents. In contrast, in the presence of 2.5 mM ATP-gamma-S, T currents declined approximately 30%, and currents containing the N and L components declined to a greater extent than control currents, about 65%. In addition, the time to peak current was increased from approximately 14 ms to approximately 40 ms. This effect of ATP-gamma-S on calcium currents was similar to that of certain neurotransmitters or GTP-gamma-S, an activator of G proteins, except that the effects of ATP-gamma-S were delayed 5-7 min relative to GTP-gamma-S. The effects of both ATP-gamma-S and GTP-gamma-S were reduced or abolished in neurons treated with pertussis toxin. We conclude that AK-C regulates neuronal calcium currents, presumably by phosphorylation of channels or associated proteins, and that the ATP-gamma-S-induced reduction of calcium currents cannot be due to its serving as a phosphate donor for endogenous AK.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a novel cyclic GMP-dependent protein kinase from mouse brain.

Synthetic oligonucleotides were used to amplify mouse brain cDNA sequences homologous to conserved regions of known cGMP-dependent protein kinases, and two classes of products were identified. The first class (CGKI) of amplification products contained approximately 1.0 kilobase (kb) of DNA sequence between the oligonucleotide primers, and this sequence showed a high degree of homology (90% identity) with the known bovine and human type I cDNA sequences for cGMP-dependent protein kinase. The second class (CGKII) of amplification products contained approximately 1.1 kb of DNA sequence between the oligonucleotide primers, and this sequence showed a much lower homology (65% identity) with the bovine and human type I cDNA sequences. Northern blot analysis showed that CGKI transcripts of 8.5 kb were abundant in brain and lung, whereas a 7-kb transcript could be detected in testis. CGKII transcripts of 6 kb were also abundant in brain and lung but could be detected at lower levels in kidney. The CGKII amplification product was used to screen a mouse brain cDNA library, and four overlapping cDNA clones were isolated which comprised the entire CGKII coding region. The predicted CGKII protein consists of 761 amino acids and has a molecular mass of 87 kDa. The CGKII protein shows highest homology to the catalytic (66% amino acid identity) and regulatory domains (45% identity) of bovine and human CGKI. Little homology is observed at the amino terminus or in the region linking the regulatory and catalytic domains. An expression vector for mouse CGKII was constructed and transfected into COS-1 cells where it directed the expression of a protein kinase which was activated by cGMP with an apparent K alpha of 300 nM cGMP.

The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation.

For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design.

Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.

The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase.

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.

Isoform-specific differences in the potencies of murine protein kinase inhibitors are due to nonconserved amino-terminal residues.

We provide here a detailed characterization of two isoforms of the protein kinase inhibitor (PKI) protein of cAMP-dependent protein kinase that have dramatically different inhibition constants. Murine PKI beta 1 possesses a 32-fold higher Ki than murine PKI alpha as determined by Henderson analysis. This finding led to the investigation of C subunit.PKI interactions involving nonconserved regions in the carboxyl and amino termini of murine PKI alpha and PKI beta 1. Chimeric cDNAs coding for amino acid sequences from both PKI isoforms were constructed and expressed in bacteria. Surprisingly, exchanging the carboxyl-terminal two-thirds of PKI alpha and PKI beta 1 has relatively little effect on the inhibition constants of the two isoforms. Similarly, introducing amino acid residues corresponding to a beta-turn region of PKI alpha into PKI beta 1 fails to lower PKI beta 1 inhibition constants. However, introducing the amino-terminal alpha-helical region of PKI alpha into PKI beta 1 reduces the Ki and IC50 of PKI beta 1 to values identical with full length PKI alpha. Site-directed mutagenesis of specific residues within this region implicates the presence of a tyrosine at position 7 in PKI alpha as a major contributor to its enhanced inhibitory potency. The results of this study suggest that variations in C subunit.PKI interactions within an amino-terminal alpha-helix provide a major mechanism for altering the inhibitory properties of PKI isoforms.

Expression of cDNAs for two isoforms of the catalytic subunit of cAMP-dependent protein kinase.

We have previously reported the cloning of cDNAs coding for two isoforms (termed C alpha and C beta) of the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M., Chrivia, J., and McKnight, G. S. (1986) J. Biol. Chem. 261, 15360-15363). We have constructed cDNA expression vectors for the C alpha and C beta proteins using the mouse metallothionein promoter so that mRNA transcripts from the expression vectors are inducible with Zn. In stable transformants of NIH 3T3 and AtT-20 cells, the induction of both C alpha and C beta mRNA with Zn increases the cAMP-dependent as well as the cAMP-independent kinase activity. The C alpha and C beta proteins synthesized from these expression vectors can be detected by Western blot analysis and have slightly different molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Interestingly, the levels of RI protein are also shown to increase in cells expressing high levels of the C alpha or C beta subunits although there is no change in RI mRNA. In contrast, the levels of RII protein are not significantly affected by increasing either C alpha or C beta expression in these cell lines. We conclude that cells can compensate for the increased levels of either C alpha or C beta subunits with a corresponding elevation of RI protein implying that both isoforms of C can interact with RI to form a holoenzyme. The relevance of these experimental results to the coordinate regulation of cAMP-dependent protein kinase subunits is discussed.

Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase.

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 PKIbeta1. Residues important for the high affinity of PKIalpha and PKIbeta1 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.

Cyclic AMP- and cyclic GMP-dependent protein kinases differ in their regulation of cyclic AMP response element-dependent gene transcription.

The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Ibeta cGMP-dependent protein kinase (cGKIbeta) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulated phosphoprotein) to a similar extent, cyclic nucleotide regulation of CRE-dependent transcription was at least 10-fold higher in cAKII-transfected cells than in cGKIbeta-transfected cells. Overexpression of each kinase in mammalian cells resulted in a cytoplasmic localization of the unactivated enzyme. As reported previously, the catalytic (C) subunit of cAKII translocated to the nucleus following activation by 8-bromo-cyclic AMP. However, cGKIbeta did not translocate to the nucleus upon activation by 8-bromo-cyclic GMP. Replacement of an autophosphorylated serine (Ser79) of cGKIbeta with an aspartic acid resulted in a mutant kinase with constitutive kinase activity in vitro and in vivo. The cGKIbetaS79D mutant localized to the cytoplasm and was only a weak activator of CRE-dependent gene transcription. However, an amino-terminal deletion mutant of cGKIbeta was found in the nucleus as well as the cytoplasm and was a strong activator of CRE-dependent gene transcription. These data suggest that the inability of cGKs to translocate to the nucleus is responsible for the differential ability of cAKs and cGKs to activate CRE-dependent gene transcription and that nuclear redistribution of cGKs is not required for NO/cGMP regulation of gene transcription.

Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase.

Synthetic oligonucleotides were designed to amplify DNA sequences related to the heat-stable protein kinase inhibitor (PKI) isolated from rabbit skeletal muscle. Using these oligonucleotides, a 167-base pair fragment was isolated and shown to code for a portion of the mouse protein kinase inhibitor gene. This amplified DNA sequence was used to isolate three cDNA clones from a mouse brain cDNA library. A composite sequence was derived from these clones and contained a 228-nucleotide open reading frame encoding a protein of 76 amino acids. In addition, the sequence contained 29 nucleotides of 5'-untranslated and 2022 nucleotides of 3'-untranslated regions of the mouse PKI mRNA. Northern blot analysis of various mouse tissues indicated that the 3.8-kilobase pair mRNA is present at high levels in skeletal muscle and brain but is present at lower levels in heart, testis, and liver. RNase protection experiments also suggested that skeletal muscle and brain represent tissues of highest expression and that similar nucleotide sequences are found in the skeletal muscle, brain, and testicular transcripts. Southern blot analysis indicated a single prominent species of genomic DNA sequence related to the mouse PKI cDNA clones but a minor species was also detected.