Protein Kinase Inhibitor Alpha Research Articles

Dronedarone Attenuates Ang II-Induced Myocardial Hypertrophy Through Regulating SIRT1/FOXO3/PKIA Axis.

Long-term pathological myocardial hypertrophy (MH) seriously affects the normal function of the heart. Dronedarone was reported to attenuate left ventricular hypertrophy of mice. However, the molecular regulatory mechanism of dronedarone in MH is unclear. Angiotensin II (Ang II) was used to induce cell hypertrophy of H9C2 cells. Transverse aortic constriction (TAC) surgery was performed to establish a rat model of MH. Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. Reverse transcription quantitative polymerase chain reaction and western blot were performed to detect the mRNA and protein expressions of genes. JASPAR and luciferase activity were conducted to predict and validate interaction between forkhead box O3 (FOXO3) and protein kinase inhibitor alpha (PKIA) promoter. Ang II treatment induced cell hypertrophy and inhibited sirtuin 1 (SIRT1) expression, which were reversed by dronedarone. SIRT1 overexpression or PKIA overexpression enhanced dronedarone-mediated suppression of cell hypertrophy in Ang II-induced H9C2 cells. Mechanistically, SIRT1 elevated FOXO3 expression through SIRT1-mediated deacetylation of FOXO3 and FOXO3 upregulated PKIA expression through interacting with PKIA promoter. Moreover, SIRT1 silencing compromised dronedarone-mediated suppression of cell hypertrophy, while PKIA upregulation abolished the influences of SIRT1 silencing. More importantly, dronedarone improved TAC surgery-induced MH and impairment of cardiac function of rats via affecting SIRT1/FOXO3/PKIA axis. Dronedarone alleviated MH through mediating SIRT1/FOXO3/PKIA axis, which provide more evidences for dronedarone against MH.

A grooved porous hydroxyapatite scaffold induces osteogenic differentiation via regulation of PKA activity by upregulating miR-129-5p expression.

Hydroxyapatite scaffolds with different morphologies have been widely used in bone tissue engineering. Moreover, microRNAs (miRNAs) have been proven to be extensively involved in regulating bone regeneration. We developed grooved porous hydroxyapatite (HAG) scaffolds with good osteogenic efficiency. However, little is known about the role of miRNAs in HAG scaffold-mediated promotion of bone regeneration. The objective of this study was to reveal the mechanism from the perspective of differential miRNA expression. Scanning electron microscopy (SEM) was used to perform the coculture of cells and scaffolds. The miRNA profiles were generated by a microarray assay. A synthetic miR-129-5p mimic and inhibitor were used for overexpression or inhibition. The expression of osteogenic marker mRNAs and proteins was detected by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. An ALP activity kit and alizarin red staining (ARS) were used to measure ALP activity and mineral deposition formation. Cell migration ability was examined by wound healing and transwell assays. Protein kinase A (PKA) activity was measured by enzyme-linked immunosorbent assay (ELISA) after miR-129-5p transfection. Target genes were identified by a dual-luciferase reporter assay. H89 preculture evaluated the cross talk between miR-129-5p and PKA activity. Heterotopic implantation models, hematoxylin-eosin (HE), immunohistochemistry staining, and micro-CT were used to evaluate miR-129-5p osteogenesis in vivo. miRNAs were differentially expressed during osteogenic differentiation induced by HAG in vitro and in vivo. miR-129-5p was the only highly expressed miRNA both in vitro and in vivo. miR-129-5p overexpression promoted osteoblast differentiation and cell migration, while its inhibition weakened the effect of HAG. Moreover, miR-129-5p activated PKA to regulate the phosphorylation of β-catenin and cAMP-response element binding protein (CREB) by inhibiting cAMP-dependent protein kinase inhibitor alpha (Pkia). H89 prevented the effects of miR-129-5p on osteogenic differentiation and cell migration. HE, immunohistochemistry staining and micro-CT results showed that miR-129-5p promoted in vivo osteogenesis of the HAG scaffold. The HAG scaffold activates Pka by upregulating miR-129-5p and inhibiting Pkia, resulting in CREB-dependent transcriptional activation and accumulation of β-catenin and promoting osteogenic marker expression.

An Epigenetic Signature is Associated with Serum 25-Hydroxyvitamin D in Colorectal Cancer Tumors.

Vitamin D has been widely associated with colorectal cancer (CRC) through different insights. This study aims to explore the association between serum 25-hydroxyvitamin D (25(OH)D) and the global DNA methylation in tumor from CRC patients. A genome-wide DNA methylation analysis is conducted in 20 CRC patients under categorical (10 patients have 25(OH)D <30ng mL-1 ; 10 patients with 25(OH)D ≥30 ng mL-1 )and continuous models of 25(OH)D. A total of 95 differentially methylated CpGs (DMCpGs) are detected under the categorical model (false discovery rate (FDR) <0.05), while 16 DMCpGs are found under the continuous model. Regional analysis showed eight vitamin D-associated differentially methylated regions (DMR). Between them, a DMR is the most significant at cAMP-Dependent Protein Kinase Inhibitor Alpha (PKIA) locus. Furthermore, seven genes, including PKIA gene, have more or equal than two significant DMCpGs. The protein networking analysis found pathways implicated in cell adhesion and extracellular matrix, as well as signaling transduction. This study identifies novel epigenetic loci associated with serum 25(OH)D status. Interestingly, also, a positive association between vitamin D and DNA methylation in the CRC context is found, suggesting a role in CRC. Further studies are warranted to clarify and replicate these results.

The role of nucleus accumbens CREB attenuation in rescuing low voluntary running behavior in female rats.

Given the integral role of nucleus accumbens (NAc) cAMP response element binding protein (CREB) activity in motivational processes, the goal of the current study was to determine whether blunting chronic NAc CREB activity could rescue the low physical activity motivation of female, low voluntary running (LVR) rats. NAc CREB phosphorylation is elevated in these rats, a state previously attributed to deficits in reward valuation. It was recently shown that overexpression of the upstream CREB inhibitor, protein kinase inhibitor alpha (PKIα), increased LVR nightly running by ~threefold. Therefore, the current study addresses the extent to which NAc CREB attenuation influences female LVR and wild-type (WT) wheel-running behavior. Inducible reductions in NAc neuronal activity using Gi-coupled hM4Di DREADDs increased running behavior in LVR, but not in WT, rats. Similarly, site-directed pharmacological inhibition of NAc CREB activity significantly increased LVR nightly running distance and time by ~twofold, with no effect in WT rats. Finally, environmentally enriched LVR rats exhibit higher levels of running compared to socially isolated rats in what appeared to be a CREB-related manner. Considering the positive outcomes of upstream CREB modulation and environmental enrichment on LVR behavior, we believe that blunting NAc CREB activity has the neuromolecular potential to partially reverse low physical activity motivation, as exemplified by the LVR model. The positive physical activity outcome of early life enrichment adds translatable value to human childhood enrichment and highlights its importance on motivational processes later in life.

Backbone (1H, 13C and 15N) Chemical Shift Assignments and 15N Relaxation Parameters for protein kinase Inhibitor alpha (PKIa) bound to cAMP-dependent protein kinase A

Backbone (1H, 13C and 15N) Chemical Shift Assignments and 15N Relaxation Parameters for protein kinase Inhibitor alpha (PKIa) bound to cAMP-dependent protein kinase A

PO-131 Impact of short-term inhibition of PKA in Nucleus Accumbens on voluntary wheel running

Objective Based upon a Booth lab goal of establishing molecular regulators of physical activity motivation, my current study focuses on the effects of short-term inhibition of protein kinase A (PKA) activity in the nucleus accumbens (NAc). The NAc is a brain region integral to motivated behaviors. Downstream immediate-early gene (IEG) expression from PKA has been shown to exhibit rapid responses to acute stimuli, such as voluntary wheel-running behavior. According to previous work in our lab, long-term NAc overexpression of the endogenous PKA inhibitor, Protein Kinase Inhibitor Alpha (PKIα), increased nightly running distance in rats selectively bred for low voluntary running (LVR) behavior (Mol Neurobiol 2018 Jun 21). However, paradoxically, the same PKIα overexpression failed to increase running distance in wild-type (WT) rats. It is known that chronic manipulation of the NAc PKA pathway produces different molecular (gene expression profiles) and behavioral outcomes from that of acute manipulations. Given the above, the goal of the current work is to determine how short-term inhibition of PKA in the NAc influences its downstream gene networks and the nightly voluntary running behavior in WT rats.&#x0D; Methods An ex vivo preparation of the NAc was utilized to determine the effects of Rp-cAMPS, a selective protein kinase A inhibitor, upon its stimulation of dopamine D1-like receptor agonist SKF 38393 on downstream gene expression level in sedentary WT female rats. Further, real-time PCR was implemented to analyze the transcriptional expression of IEGs (Homer-1, Arc, Zif268) following Rp-cAMPS administration. &#x0D; Results Data showed that there were no significant difference of mRNA level for Homer-1, Arc or Zif268 among the vehicle, 50uM, 100uM and 200uM Rp-cAMPS treatment groups upon the stimulation of 10uM SKF 38393.&#x0D; Conclusions In addition to the PKA, other protein kinases such as Ca++ activated and growth factor activated kinases have both been shown to phosphorylate CREB at Ser133, and thus, lead to activation of gene transcription. Given the above results of the ex vivo experiment, in which NAc slices were treated with multiple dosages of Rp-cAMPS concurrent with the stimulation of SKF 38393, it is possible that other protein kinase pathways could be compensating the effects of short-term inhibition of PKA and, in turn, lead to no difference of IEG expression. Further experiments will need to be performed in order to testify this hypothesis.

Overexpression of Protein Kinase Inhibitor Alpha Reverses Rat Low Voluntary Running Behavior.

A gene was sought that could reverse low voluntary running distances in a model of low voluntary wheel-running behavior. In order to confirm the low motivation to wheel-run in our model does not result from defects in reward valuation, we employed sucrose preference and conditioned place preference for voluntary wheel-access. We observed no differences between our model and wild-type rats regarding the aforementioned behavioral testing. Instead, low voluntary runners seemed to require less running to obtain similar rewards for low voluntary running levels compared to wild-type rats. Previous work in our lab identified protein kinase inhibitor alpha as being lower in low voluntary running than wild-type rats. Next, nucleus accumbens injections of an adenoviral-associated virus that overexpressed the protein kinase inhibitor alpha gene increased running distance in low voluntary running, but not wild-type rats. Endogenous mRNA levels for protein kinase inhibitor alpha, dopamine receptor D1, dopamine receptor D2, and Fos were all only lower in wild-type rats following overexpression compared to low voluntary runners, suggesting a potential molecular and behavioral resistance in wild-type rats. Utilizing a nucleus accumbens preparation, three intermediate early gene mRNAs increased in low voluntary running slices after dopamine receptor agonist SKF-38393 exposure, while wild-type had no response. In summary, the results suggest that protein kinase inhibitor alpha is a promising gene candidate to partially rescue physical activity in the polygenic model of low voluntary running. Importantly, there were divergent molecular responses to protein kinase inhibitor alpha overexpression in low voluntary runners compared to wild-type rats.

Molecular rescue of low voluntary wheel‐running behavior in rats selectively bred to run low distances

Physical inactivity is associated with onset of 40 known chronic diseases and stands as the fifth leading risk factor for death in the US. Importantly, it has been determined that ~30% of human sedentary behavior is genetic, which is reflected in our unique selectively bred rat model for low (LVR) voluntary running behavior. This model highlights the genetic contributions to the low motivation to be physically active. RNA sequencing of the NAc, a brain region believed to drive motivated behaviors, from our model has revealed a target molecule which is more lowly expressed in LVR compared to WT rats and which is positively correlated with nightly running distance; Protein Kinase Inhibitor Alpha (PKIα). This molecule functions to inhibit dopamine receptor 1 (Drd1) signaling by modulating protein kinase A (PKA) activity, an action thought to modulate the “desire” or motivation to receive reward signaling from dopamine and by extension wheel‐running.ObjectiveAddress the effect of increasing PKIα rexpression in the NAc of female WT and LVR rats on 1.) Voluntary running behavior and 2.) Dopaminergic downstream signaling events specific to PKA to better evaluate the function of PKIα on the motivation to be physically active.MethodsBilateral micoinfusion of an AAV for the overexpression of PKIα was injected into the NAc core of female LVR and WT rats and wheel‐running behavior was monitored. Overexpression of PKIα overexpression was confirmed via qPCR. An ex vivo preparation of NAc punches from WT and LVR animals was used to evaluate the effect of Drd1 stimulation with agonist SKF3893 (20 μM and 100 μM for 10 min) on immediate early gene (homer1, arc, psd‐95 and zif268) induction to act as a molecular marker of reward and motivation.ResultsOverexpression of PKIα significantly increased nightly running for 5 continuous estrous cycles (~3 wks) post injection in female LVR rats, but not in female WT rats. A 4‐fold increase in PKIα mRNA expression was confirmed in NAc punches taken from LVR rats compared to pair matched empty vector injected controls. WT punches showed a near significant (p=0.058) 2.5 fold increase in PKIα mRNA expression compared to empty vector injected controls. Evoked expression of homer1, arc, psd‐95 and zif268 significantly increased in LVR punches following the 20 μM dose of SKF38393 compared to control values, whereas WT punches decreased IEG expression following both SKF38393 concentrations. Interestingly, control values of all IEG mRNAs observed were inherently lower in LVR compared to WT punches.Conclusions &amp; SignificanceCollectively, the above suggests that differences in dopamine downstream signaling, attributed to PKIα, may underlie inherent differences in voluntary running behavior. I have shown that increasing the genetic expression of PKIα increases nightly running distance in a polygenic model of low voluntary running behavior. Additionally I have shown that the NAc of LVR animals may confer increased dopamine signaling following Drd1 stimulation, suggesting a potential link between low levels of PKIα and signaling events related to reward and motivation. However, further work is needed to better elucidate PKIα's role in the context of reward signaling and the motivation to be physically active.Support or Funding InformationPhi Zeta Research Grant and the College of Veterinary Medicine, University of Missouri Development OfficeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic Evaluation to Identify Biomarkers for Carpal Tunnel Syndrome: A Comparative Serum Analysis

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment, causing pain, impairment, and disability. To identify proteins of CTS comprehensively, a comparative serum analysis of CTS patients and normal control subjects was performed. The two-dimensional electrophoresis patterns of serum obtained from six CTS patients and six normal control subjects were compared. We found 10 proteins that were significantly altered in the serum of CTS patients, among which four were upregulated and six were downregulated. The upregulated spots were identified as Chain A, heat shock 70-kDa protein, 42-kDa ATPase N-terminal domain; glutathione-insulin transhydrogenase (216AA); cAMP-dependent protein kinase inhibitor alpha; and mutant β-globin. The downregulated spots were identified as vitamin D-binding protein (VDBP), fibrinogen gamma chain, apolipoprotein A-IV (ApoA-IV), clusterin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), and one unidentified protein. The information obtained from this proteomic analysis will be very useful in understanding the pathophysiology of CTS and in finding suitable proteins that can serve as new diagnostic biomarkers of CTS.

MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

Pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase Cδ (PKCδ), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-κB and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in the generation of PKA C-α; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection.

Characterization and expression analysis of an interleukin‐7 homologue in the Japanese pufferfish, Takifugu rubripes

An interleukin-7 (IL-7) gene has been cloned and sequenced within the Japanese pufferfish, Takifugu rubripes (Fugu), providing the first conclusive evidence for the existence of IL-7 in teleosts. The IL-7 cDNA was composed of 1438 bp, with a 117 bp 5'-UTR, an 826 bp 3'-UTR containing two mRNA instability motifs, and a 498 bp ORF which translates into a peptide of 166 amino acid residues. The Fugu IL-7 peptide contained a predicted signal peptide of 19 amino acid residues and the IL-7/9 family signature (Arg140-Val149). Homology analysis of Fugu IL-7 with other known IL-2 family members showed some similarity to the known mammalian IL-7 and Fugu IL-21 molecules, and phylogenetic tree analysis showed that the Fugu IL-7 clustered with the chicken and mammalian IL-7 and mammalian IL-9, away from other IL-2 family members (IL-2, IL-4, IL-15 and IL-21). The organization of the Fugu IL-7 gene was the same as in mouse, and consisted of five exons and four introns, but differed from the human gene, which is composed of six exons and five introns. Comparison of the Fugu and human genomes showed that some synteny existed around the IL-7 gene, with the presence of both the protein kinase inhibitor-alpha and chromosome 8 ORF 70 (C8orf70) genes, with IL-7 and C8orf70 having the same transcriptional orientation. The Fugu IL-7 gene was expressed within unstimulated tissues, such as the head kidney, spleen, liver, intestine, gill and muscle. Stimulation of head kidney cells with lipopolysaccharide, polyinosinic polycytidylic acid or phytohaemagglutinin increased this expression. This discovery of IL-7 in Fugu will allow a more complete analysis of fish immune responses in the future.

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.

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway.

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

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.

Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.

Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many regions of the adult mouse brain but particularly in cerebellum, hypothalamus, hippocampus, and cortex. In contrast, PKI beta is present at much lower levels in most brain regions but is found in significant amounts in the cerebellum, as well as in distinct nuclei within the pons, medulla, and hypothalamus. These results are consistent with a regulatory role of endogenous PKI in PKA-mediated signal transduction in brain and suggest differential functions for the two isoforms of PKI within the central nervous system.

The Expression and Intracellular Distribution of the Heat-stable Protein Kinase Inhibitor Is Cell Cycle Regulated

The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. We report the isolation of a polyclonal antibody raised to purified recombinant PKI alpha. Using this antibody, the intracellular distribution of endogenous PKI alpha was assessed by immunostaining. The PKI alpha expression and intracellular distribution varied as a function of cell cycle progression. PKI alpha expression appeared low in serum-starved cells and in cells in G1 and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G2/M. Nuclear levels of PKI alpha remained high through cell division and decreased again as cells reentered G1. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G2/M phases of the cell cycle. Consistent with this, microinjection of PKI alpha antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G1/S boundary prevented their subsequent division. Together these results support the idea that PKI alpha plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis.

Evidence for two additional isoforms of the endogenous protein kinase inhibitor of cAMP-dependent protein kinase in mouse

Oligonucleotides derived from the previously published rat testicular protein kinase inhibitor (PKI) sequence (Van Patten, S. M., Ng, D. C., Th'ng, J. P. H., Angelos, K. L., Smith, A. J., and Walsh, D. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5383-5387) were used to isolate a DNA fragment coding for a mouse homologue of the rat testicular PKI. This DNA fragment was then used to screen a mouse brain cDNA library, and two cDNA clones related to the testicular PKI (PKI beta) were isolated. Sequencing and comparison showed that the two cDNAs differ only in the presence of a 105-base pair insert, which results in an amino-terminal extension of the predicted PKI beta 2 protein by 20 residues relative to the PKI beta 1 protein. By using the appropriate primers, PCR was used to amplify specific regions of both of these clones from mouse brain cDNA. When both clones were expressed in vitro, the mRNA for PKI beta 2 produced a protein product that was larger and much more effectively translated, suggesting a functional role for the inserted sequence. Both isoforms were transiently expressed in COS-1 cells to evaluate their ability to inhibit the catalytic subunit of PKA in vivo. Extracts from cells expressing the PKI beta 2 isoform showed greater inhibition of catalytic subunit kinase activity than extracts expressing the PKI beta 1 isoform. Northern blot analysis of poly(A)+ RNA from various mouse tissues showed the presence of transcripts of 1.8 kilobases related to these cDNAs. Analysis of brain RNA from several species indicated that expression of these PKI mRNAs is evolutionarily conserved. Together with previous studies, these results indicate the presence of at least three PKI proteins in mouse. The skeletal muscle isoform has been designated PKI alpha, while the testicular isoform is PKI beta. We propose to designate the two testicular isoforms described here PKI beta 1 and PKI beta 2.