PKA-RIIß autophosphorylation modulates PKA activity and seizure phenotypes in mice.

Temporal lobe epilepsy (TLE) is one of the most common and intractable neurological disorders in adults. Dysfunctional PKA signaling is causally linked to the TLE. However, the mechanism underlying PKA involves in epileptogenesis is still poorly understood. In the present study, we found the autophosphorylation level at serine 114 site (serine 112 site in mice) of PKA-RIIß subunit was robustly decreased in the epileptic foci obtained from both surgical specimens of TLE patients and seizure model mice. The p-RIIß level was negatively correlated with the activities of PKA. Notably, by using a P-site mutant that cannot be autophosphorylated and thus results in the released catalytic subunit to exert persistent phosphorylation, an increase in PKA activities through transduction with AAV-RIIß-S112A in hippocampal DG granule cells decreased mIPSC frequency but not mEPSC, enhanced neuronal intrinsic excitability and seizure susceptibility. In contrast, a reduction of PKA activities by RIIß knockout led to an increased mIPSC frequency, a reduction in neuronal excitability, and mice less prone to experimental seizure onset. Collectively, our data demonstrated that the autophosphorylation of RIIß subunit plays a critical role in controlling neuronal and network excitabilities by regulating the activities of PKA, providing a potential therapeutic target for TLE.

Structural analyses of the PKA RIIß holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma.

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIß, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIß:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIß2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIß holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIß holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

PRKAR2B-HIF-1α loop promotes aerobic glycolysis and tumour growth in prostate cancer.

OBJECTIVES: Reprogramming of cellular metabolism is profoundly implicated in tumorigenesis and can be exploited to cancer treatment. Cancer cells are known for their propensity to use glucose-dependent glycolytic pathway instead of mitochondrial oxidative phosphorylation for energy generation even in the presence of oxygen, a phenomenon known as Warburg effect. The type II beta regulatory subunit of protein kinase A (PKA), PRKAR2B, is highly expressed in castration-resistant prostate cancer (CRPC) and contributes to tumour growth and metastasis. However, whether PRKAR2B regulates glucose metabolism in prostate cancer remains largely unknown. MATERIALS AND METHODS: Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRKAR2B in aerobic glycolysis. Real-time qPCR, Western blotting, luciferase reporter assay and chromatin immunoprecipitation were employed to determine the underlying mechanisms. RESULTS: PRKAR2B was sufficient to enhance the Warburg effect as demonstrated by glucose consumption, lactate production and extracellular acidification rate. Mechanistically, loss-of-function and gain-of-function studies showed that PRKAR2B was critically involved in the tumour growth of prostate cancer. PRKAR2B was able to increase the expression level of hypoxia-inducible factor 1α (HIF-1α), which is a key mediator of the Warburg effect. Moreover, we uncovered that HIF-1α is a key transcription factor responsible for inducing PRKAR2B expression in prostate cancer. Importantly, inhibition of glycolysis by the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) or replacement of glucose in the culture medium with galactose (which has a much lower rate than glucose entry into glycolysis) largely compromised PRKAR2B-mediated tumour-promoting effect. Similar phenomenon was noticed by genetic silencing of HIF-1α. CONCLUSIONS: Our study identified that PRKAR2B-HIF-1α loop enhances the Warburg effect to enable growth advantage in prostate cancer.

Hypoxia and the hypoxia inducible factor 1α activate protein kinase A by repressing RII beta subunit transcription.

Overactivation of the cAMP signal transduction pathway plays a central role in the pathogenesis of endocrine tumors. Genetic aberrations leading to increased intracellular cAMP or directly affecting PKA subunit expression have been identified in inherited and sporadic endocrine tumors, but are rare indicating the presence of nongenomic pathological PKA activation. In the present study, we examined the impact of hypoxia on PKA activation using human growth hormone (GH)-secreting pituitary tumors as a model of an endocrine disease displaying PKA-CREB overactivation. We show that hypoxia activates PKA and enhances CREB transcriptional activity and subsequently GH oversecretion. This is due to a previously uncharacterized ability of HIF-1α to suppress the transcription of the PKA regulatory subunit 2B (PRKAR2B) by sequestering Sp1 from the PRKAR2B promoter. The present study reveals a novel mechanism through which the transcription factor HIF-1α transduces environmental signals directly onto PKA activity, without affecting intracellular cAMP concentrations. By identifying a point of interaction between the cellular microenvironment and intracellular enzyme activation, neoplastic, and nonneoplastic diseases involving overactivated PKA pathway may be more efficiently targeted.

Transcriptional regulation of PRKAR2B by miR-200b-3p/200c-3p and XBP1 in human prostate cancer.

The cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) pathway is profoundly implicated in Prostate cancer (PCa) progression. Previously, we showed that PRKAR2B, the type II-beta regulatory subunit of PKA, is highly expressed in castration-resistant prostate cancer (CRPC) and can induce epithelial-mesenchymal transition by activating Wnt/ß-catenin signaling in PCa cells. However, the molecular mechanism of dysregulated PRKAR2B expression pattern is still largely unknown. In this study, we found that the mutation, copy number alteration, and methylation status of PRKAR2B gene have no correlation with its expression level in PCa. Then, we identified two microRNAs (miR-200b-3p and miR-200c-3p) to be critical regulators of PRKAR2B expression in PCa. Notably, miR-200b-3p and miR-200c-3p expression were significantly downregulated in metastatic CRPC and negatively correlated with the expression level of PRKAR2B in PCa tissues. Moreover, we characterized X-Box Binding Protein 1 (XBP1) as a key transcription factor responsible for PRKAR2B expression in PCa. Importantly, miR-200b-3p/200c-3p or XBP1 knockdown inhibited PCa cell proliferation and promoted cell apoptosis and these inhibitory roles could be largely restored by PRKAR2B, suggesting that PRKAR2B is a functional mediator of miR-200b-3p, miR-200c-3p, and XBP1 in PCa. Collectively, our study firstly identified miR-200b-3p/200c-3p and XBP1 as the critical upstream regulators of PRKAR2B in PCa and provided novel insights to PRKAR2B-driven PCa progression.

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIß, RIIα, RIIß), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIß, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Epigenetic regulation of diabetogenic adipose morphology.

OBJECTIVE: Hypertrophic white adipose tissue (WAT) morphology is associated with insulin resistance and type 2 diabetes. The mechanisms governing hyperplastic versus hypertrophic WAT expansion are poorly understood. We assessed if epigenetic modifications in adipocytes are associated with hypertrophic adipose morphology. A subset of genes with differentially methylated CpG-sites (DMS) in the promoters was taken forward for functional evaluation. METHODS: The study included 126 women who underwent abdominal subcutaneous biopsy to determine adipose morphology. Global transcriptome profiling was performed on WAT from 113 of the women, and CpG methylome profiling on isolated adipocytes from 78 women. Small interfering RNAs (siRNA) knockdown in human mesenchymal stem cells (hMSCs) was used to assess influence of specific genes on lipid storage. RESULTS: A higher proportion of CpG-sites were methylated in hypertrophic compared to hyperplastic WAT. Methylation at 35,138 CpG-sites was found to correlate to adipose morphology. 2,102 of these CpG-sites were also differentially methylated in T2D; 98% showed directionally consistent change in methylation in WAT hypertrophy and T2D. We identified 2,508 DMS in 638 adipose morphology-associated genes where methylation correlated with gene expression. These genes were over-represented in gene sets relevant to WAT hypertrophy, such as insulin resistance, lipolysis, extracellular matrix organization, and innate immunity. siRNA knockdown of ADH1B, AZGP1, C14orf180, GYG2, HADH, PRKAR2B, PFKFB3, and AQP7 influenced lipid storage and metabolism. CONCLUSION: CpG methylation could be influential in determining adipose morphology and thereby constitute a novel antidiabetic target. We identified C14orf180 as a novel regulator of adipocyte lipid storage and possibly differentiation.

A Genome-Wide Association Study for Susceptibility to Visual Experience-Induced Myopia.

Purpose: The rapid rise in prevalence over recent decades and high heritability of myopia suggest a role for gene-environment (G × E) interactions in myopia susceptibility. Few such G × E interactions have been discovered to date. We aimed to test the hypothesis that genetic analysis of susceptibility to visual experience-induced myopia in an animal model would identify novel G × E interaction loci. Methods: Chicks aged 7 days (n = 987) were monocularly deprived of form vision for 4 days. A genome-wide association study (GWAS) was carried out in the 20% of chicks most susceptible and least susceptible to form deprivation (n = 380). There were 304,963 genetic markers tested for association with the degree of induced axial elongation in treated versus control eyes (A-scan ultrasonography). A GWAS candidate region was examined in the following three human cohorts: CREAM consortium (n = 44,192), UK Biobank (n = 95,505), and Avon Longitudinal Study of Parents and Children (ALSPAC; n = 4989). Results: A locus encompassing the genes PIK3CG and PRKAR2B was genome-wide significantly associated with myopia susceptibility in chicks (lead variant rs317386235, P = 9.54e-08). In CREAM and UK Biobank GWAS datasets, PIK3CG and PRKAR2B were enriched for strongly-associated markers (meta-analysis lead variant rs117909394, P = 1.7e-07). In ALSPAC participants, rs117909394 had an age-dependent association with refractive error (-0.22 diopters [D] change over 8 years, P = 5.2e-04) and nearby variant rs17153745 showed evidence of a G × E interaction with time spent reading (effect size -0.23 D, P = 0.022). Conclusions: This work identified the PIK3CG-PRKAR2B locus as a mediator of susceptibility to visually induced myopia in chicks and suggests a role for this locus in conferring susceptibility to myopia in human cohorts.

FOXG1 Regulates PRKAR2B Transcriptionally and Posttranscriptionally via miR200 in the Adult Hippocampus.

Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent form of atypical Rett syndrome, called FOXG1 syndrome. FOXG1 is a key transcription factor crucial for forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using genome-wide small RNA sequencing and quantitative proteomics, we identified that FOXG1 affects the biogenesis of miR200b/a/429 and interacts with the ATP-dependent RNA helicase, DDX5/p68. Both FOXG1 and DDX5 associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. RNA-Seq analyses of Foxg1cre/+ hippocampi and N2a cells overexpressing miR200 family members identified cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) as a target of miR200 in neural cells. PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity; thus, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 syndrome. Our data suggest that FOXG1 regulates PRKAR2B expression both on transcriptional and posttranscriptional levels.

Knockdown of PRKAR2B Results in the Failure of Oocyte Maturation.

BACKGROUND/AIMS: Cyclic adenosine monophosphate (cAMP)-dependent type 2 regulatory subunit beta (Prkar2b) is a regulatory isoform of cAMP-dependent protein kinase (PKA), which is the primary target for cAMP actions. In oocytes, PKA and the pentose phosphate pathway (PPP) have important roles during the germinal vesicle (GV) stage arrest of development. Although the roles of the PKA signal pathway have been studied in the development of oocyte, there has been no report on the function of PRKAR2B, a key regulator of PKA. METHODS: Using reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), immunohistochemistry, and immunofluorescence, we determined the relative expression of Prkar2b in various tissues, including ovarian follicles, during oocyte maturation. Prkar2b-interfering RNA (RNAi) microinjection was conducted to confirm the effect of Prkar2b knockdown, and immunofluorescence, qRT-PCR, and time-lapse video microscopy were used to analyze Prkar2b-deficient oocytes. RESULTS: Prkar2b is strongly expressed in the ovarian tissues, particularly in the growing follicle. During oocyte maturation, the highest expression of Prkar2b was during metaphase I (MI), with a significant decrease at metaphase II (MII). RNAi-mediated Prkar2b suppression resulted in MI-stage arrest during oocyte development, and these oocytes exhibited abnormal spindle formation and chromosome aggregation. Expression of other members of the PKA family (except for Prkaca) were decreased, and the majority of the PPP factors were also reduced in Prkar2b-deficient oocytes. CONCLUSION: These results suggest that Prkar2b is closely involved in the maturation of oocytes by controlling spindle formation and PPP-mediated metabolism.

Crosstalk from cAMP to ERK1/2 emerges during postnatal maturation of nociceptive neurons and is maintained during aging.

Maturation of nociceptive neurons depends on changes in transcription factors, ion channels and neuropeptides. Mature nociceptors initiate pain in part by drastically reducing the activation threshold via intracellular sensitization signaling. Whether sensitization signaling also changes during development and aging remains so far unknown. Using a novel automated microscopy approach, we quantified changes in intracellular signaling protein expression and in their signaling dynamics, as well as changes in intracellular signaling cascade wiring, in sensory neurons from newborn to senescent (24 months of age) rats. We found that nociceptive subgroups defined by the signaling components protein kinase A (PKA)-RIIß (also known as PRKAR2B) and CaMKIIα (also known as CAMK2A) developed at around postnatal day 10, the time of nociceptor maturation. The integrative nociceptor marker, PKA-RIIß, allowed subgroup segregation earlier than could be achieved by assessing the classical markers TRPV1 and Nav1.8 (also known as SCN10A). Signaling kinetics remained constant over lifetime despite in part strong changes in the expression levels. Strikingly, we found a mechanism important for neuronal memory - i.e. the crosstalk from cAMP and PKA to ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) - to emerge postnatally. Thus, maturation of nociceptors is closely accompanied by altered expression, activation and connectivity of signaling pathways known to be central for pain sensitization and neuronal memory formation.

The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP.

Biochemical and structural studies demonstrate that S100A1 is involved in a Ca2+-dependent interaction with the type 2α and type 2ß regulatory subunits of protein kinase A (PKA) (RIIα and RIIß) to activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca2+-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca2+ and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.

FRET biosensor uncovers cAMP nano-domains at ß-adrenergic targets that dictate precise tuning of cardiac contractility.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

PRKAR2B plays an oncogenic role in the castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer. Despite some progresses have been made, the mechanism of CRPC development is still largely unknown, including the genes involved in its development have not been well defined. Here, we identifiedPRKAR2B to be a gene over-expressingin castration-resistant prostate cancer by analyzing the different online databases. Followed functional validation experiments showed that PRKAR2B promoted CRPC cell proliferation and invasion, and inhibited CRPC cell apoptosis. Whole genome transcriptome and GO enrichment analyses of the knock-down of PRKAR2B in CRPC cells showed that PRKAR2B mainly accelerated cell cycle biological process and modulated multiple cell cycle genes, such as CCNB1, MCM2, PLK1 and AURKB. Our study firstly identified PRKAR2B as a novel oncogenic gene involved in CRPC development and suggested it is a promising target for the future investigation and the treatment of CRPC.

Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes.

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIß via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

Hematopoietic neoplasms in Prkar2a-deficient mice.

BACKGROUND: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIß, RIIα, RIIß) and four catalytic subunits (Cα, Cß, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIß, respectively) are also viable but have not been studied for their susceptibility to any tumors. METHODS: Cohorts of Prkar1a (+/-) , Prkar2a (+/-) , Prkar2a (-/-) , Prkar2b (+/-) and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. RESULTS: Both Prkar2a (-/-) and Prkar2a (+/-) mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. CONCLUSIONS: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.

Hypothalamic PKA regulates leptin sensitivity and adiposity.

Mice lacking the RIIß regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIß knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIß KO mice. During fasting, RIIß-PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIß KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIß KO mice. Our findings suggest that RIIß-PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin.

PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.

The roles of the RIIß linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIß protein kinase A: a small angle x-ray and neutron scattering study.

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIß homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIß isoform of PKA.