KA-dependent kinase activity as well as cAMP-induced gene expression. Activation of the mutant RIa allele in hepatocytes using Alb-cre transgenic mice results in a substantial decrease in PKA activity only in the liver, demonstrating that the RIaB mice can be used to elicit a dominant negative PKA effect in a cell-specific manner in vivo. Although kinase activity is clearly reduced when the RIaB allele is activated, we do observe an increase in RIa and Ca protein levels, suggesting that PKAI holoenzyme is stabilized in the inactive state; this is consistent with previous work from our laboratory demonstrating that RIa and Ca proteins are more stable when incorporated into holoenzyme and also with our April 2011 | Volume 6 | Issue 4 | e18772 A Dominant Negative PKA Mutation in Mice unpublished data using other Cre Driver mouse lines crossed to the RIaB mouse. One important criterion that must be GSK1363089 site established before using the Cre/loxP system in mice is that the RIaB allele is effectively silenced prior to Cre recombination. We have demonstrated that insertion of a floxed-neor gene into intron 10 upstream of the site B mutation in exon 11 conditionally regulates expression of the mutant RIa allele. As demonstrated by RT-PCR experiments, when the floxed-neor cassette is present, the silent RIaB allele effectively renders ES cells and mice heterozygous at the RIa locus. The floxed-neor gene employed in this study contains splice donor/acceptor sites from the SV40 small T antigen and tandem poly A addition signals and is inserted in the same orientation as the gene. It is likely that the poly A addition sequence causes early termination of the mutant transcript. Once activated by Cre-mediated excision of the floxed-neor cassette, ES cells and hepatocytes expressing the RIaB protein exhibit significant reductions in basal and cAMP-stimulated PKA activity. However, this approach does not completely eliminate PKA activity as can be seen for RIaB expressing ES cells where all of the cells have activated the RIaB allele and yet 40% of the PKA activity remains. We have previously shown that overexpression of a dominant negative RIa protein, R, in cell culture completely inhibits PKA activity. This difference may be due in part to the fact that the dominant-negative R subunit is completely resistant to cAMP activation whereas the single RIaB site mutation created in our mice can still respond to high levels of cAMP. Probably a more significant difference is that stable R transformants in cell culture contain many copies of the R construct driven by robust promoters that produce a large excess of mutant protein. In our ES and mouse model systems, a weaker mutant is being transcribed from only a single allele under the control of the endogenous RIa promoter and this may not be adequate to sequester all of the available C subunits. The cAMP-dependent regulation of gene expression is thought to occur in part through the phosphorylation of CREB/ATF transcription factors bound to DNA regulatory sequences known as CREs located in the promoter of many genes. Transcriptional activation is initiated by phosphorylation of serine 133 in CREB by the C subunit of PKA and studies have shown that translocation of C into the nucleus stimulates transcription of CRE-containing genes. It has been demonstrated that expression of R in various cell lines blocks cAMP-responsive gene induction of CRE-containing promoters. In this paper we show that attenuation of PKA activitKA-dependent kinase activity as well as cAMP-induced gene expression. Activation of the mutant RIa allele in hepatocytes using Alb-cre transgenic mice results in a substantial decrease in PKA activity only in the liver, demonstrating that the RIaB mice can be used to elicit a dominant negative PKA effect in a cell-specific manner in vivo. Although kinase activity is clearly reduced when the RIaB allele is activated, we do observe an increase in RIa and Ca protein levels, suggesting that PKAI holoenzyme is stabilized in the inactive state; this is consistent with previous work from our laboratory demonstrating that RIa and Ca proteins are more stable when incorporated into holoenzyme and also with our April 2011 | Volume 6 | Issue 4 | e18772 A Dominant Negative PKA Mutation in Mice unpublished data using other Cre Driver mouse lines crossed to the RIaB mouse. One important criterion that must be established before using the Cre/loxP system in mice is that the RIaB allele is effectively silenced prior to Cre recombination. We have demonstrated that insertion of a floxed-neor gene into intron 10 upstream of the site B mutation in exon 11 conditionally regulates expression of the mutant RIa allele. As demonstrated by RT-PCR experiments, when the floxed-neor cassette is present, the silent RIaB allele effectively renders ES cells and mice heterozygous at the RIa locus. The floxed-neor gene employed in this study contains splice donor/acceptor 
sites from the SV40 small T antigen and tandem poly A addition signals and is inserted in the same orientation as the gene. It is likely that the poly A addition sequence causes early termination of the mutant transcript. Once activated by Cre-mediated excision of the floxed-neor cassette, ES cells and hepatocytes expressing the RIaB protein exhibit significant reductions in basal and cAMP-stimulated PKA activity. However, this approach does not completely eliminate PKA activity as can be seen for RIaB expressing ES cells where all of the cells have activated the RIaB allele and yet 40% of the PKA activity remains. We have previously shown that overexpression of a dominant negative RIa protein, R, in cell culture completely inhibits PKA activity. This difference may be due in part to the fact that the dominant-negative R subunit is completely resistant to cAMP activation whereas the single RIaB site mutation created in our mice can still respond to high levels of cAMP. Probably a more significant difference is that stable R transformants in cell culture contain many copies of the R construct driven by robust promoters that produce a large excess of mutant protein. In our ES and mouse model systems, a weaker mutant is being transcribed from only a single allele under the control of the endogenous RIa promoter and this may not be adequate to sequester all of the available C subunits. The cAMP-dependent regulation of gene expression is thought to occur in part through the phosphorylation of CREB/ATF transcription factors bound to DNA regulatory sequences known as CREs located in the promoter of many genes. Transcriptional activation is initiated by phosphorylation of serine 133 in CREB by the C subunit of PKA and studies have shown that translocation of C into the nucleus stimulates transcription of CRE-containing genes. It has been demonstrated that expression of R in various cell lines blocks cAMP-responsive gene induction of CRE-containing promoters. In this paper we show that attenuation of PKA activit
