AMP-activated Protein Kinase Attenuates Nitric Oxide-induced ��-Cell Death

During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by β-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct β-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of β-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of β-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the β-cell to recover from nitric oxide-mediated cellular stress.     

Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.     

AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-�� Peptide Metabolism

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

Inflammatory bowel diseases are associated with dysregulated electrolyte andwater transport and resultant diarrhea. Aquaporins are transmembrane proteinsthat function as water channels in intestinal epithelial cells. We investigatedthe effect of the inflammatory cytokine, interferon-γ, which is a majorplayer in inflammatory bowel diseases, on aquaporin-1 expression in a mousecolonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mLinterferon-γ and aquaporin-1 mRNA and protein expressions were measuredby real-time PCR and western blot, respectively. In other experiments, CMT93cells were pretreated with inhibitors or were transfected with siRNA to blockthe effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2,prior to treatment with interferon-γ. Interferon-γ decreasedaquaporin-1 expression in mouse intestinal epithelial cells in a manner that didnot depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on analternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatorycytokine, interferon-γ may contribute to diarrhea associated withintestinal inflammation in part through regulation of the epithelial aquaporin-1water channel via a non-classical JAK/STAT receptor signalling pathway.     

The AP-3 �� Adaptin Mediates the Biogenesis and Function of Lytic Vacuoles in Arabidopsis

Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker–based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route.     

Protein Kinase C�� Mediates Cigarette Smoke/Aldehyde- and Lipopolysaccharide-induced Lung Inflammation and Histone Modifications

Atypical protein kinase C (PKC) ζ is an important regulator of inflammation through activation of the nuclear factor-κB (NF-κB) pathway. Chromatin remodeling on pro-inflammatory genes plays a pivotal role in cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced abnormal lung inflammation. However, the signaling mechanism whereby chromatin remodeling occurs in CS- and LPS-induced lung inflammation is not known. We hypothesized that PKCζ is an important regulator of chromatin remodeling, and down-regulation of PKCζ ameliorates lung inflammation by CS and LPS exposures. We determined the role and molecular mechanism of PKCζ in abnormal lung inflammatory response to CS and LPS exposures in PKCζ-deficient (PKCζ^−/−) and wild-type mice. Lung inflammatory response was decreased in PKCζ^−/− mice compared with WT mice exposed to CS and LPS. Moreover, inhibition of PKCζ by a specific pharmacological PKCζ inhibitor attenuated CS extract-, reactive aldehydes (present in CS)-, and LPS-mediated pro-inflammatory mediator release from macrophages. The mechanism underlying these findings is associated with decreased RelA/p65 phosphorylation (Ser³¹¹) and translocation of the RelA/p65 subunit of NF-κB into the nucleus. Furthermore, CS/reactive aldehydes and LPS exposures led to activation and translocation of PKCζ into the nucleus where it forms a complex with CREB-binding protein (CBP) and acetylated RelA/p65 causing histone phosphorylation and acetylation on promoters of pro-inflammatory genes. Taken together, these data suggest that PKCζ plays an important role in CS/aldehyde- and LPS-induced lung inflammation through acetylation of RelA/p65 and histone modifications via CBP. These data provide new insights into the molecular mechanisms underlying the pathogenesis of chronic inflammatory lung diseases.     

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

The Ca²⁺ sensor STIM1 is crucial for activation of store-operated Ca²⁺ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca²⁺ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca²⁺ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)     

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase C?-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.     

Protein Kinase Cα Mediates the Effect of Antiarrhythmic Peptide on Gap Junction Conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH₂, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (G_j) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced G_j (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCγ and -α) and CGP 54345 (PKCα). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP 10. For further analysis, we incubated HeLa-Cx43 cells with [³²P]orthophosphate (0.05 mCi/ml) for 4 h at 37°C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of ³²P into Cx43 was significantly enhanced in the presence of AAP 10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [¹⁴C]-AAP10 as radioligand was performed. We found a saturable binding of [¹⁴C]-AAP10 with a K₀ of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP 10. In conclusion; AAP 10 increases G_j in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP 10 leads to enhanced phosphorylation of Cx43 via activation of PKCα. A membrane receptor exists for antiarrhythmic peptides.     

The PKARI�� Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function

Previously, we showed that interactions between p90^RSK1 (RSK1) and the subunits of type I protein kinase A (PKA) regulate the activity of PKA and cellular distribution of active RSK1 (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell Biol. 26, 4586–4600). Here we examined the role of the PKARIα subunit of PKA in regulating RSK1 activation and cell survival. In mouse lung fibroblasts, silencing of the PKARIα increased the phosphorylation and activation of RSK1, but not of RSK2 and RSK3, in the absence of any stimulation. Silencing of PKARIα also decreased the nuclear accumulation of active RSK1 and increased its cytoplasmic content. The increased activation of RSK1 in the absence of any agonist and changes in its subcellular redistribution resulted in increased phosphorylation of its cytoplasmic substrate BAD and increased cell survival. The activity of PKA and phosphorylation of BAD (Ser-155) were also enhanced when PKARIα was silenced, and this, in part, contributed to increased cell survival in unstimulated cells. Furthermore, we show that RSK1, PKA subunits, D-AKAP1, and protein phosphatase 2A catalytic subunit (PP2Ac) exist in a complex, and dissociation of RSK1 from D-AKAP1 by either silencing of PKARIα, depletion of D-AKAP1, or by using a peptide that competes with PKARIα for binding to AKAPs, decreased the amount of PP2Ac in the RSK1 complex. We also demonstrate that PP2Ac is one of the phosphatases that dephosphorylates RSK, but not ERK1/2. Thus, in unstimulated cells, the increased phosphorylation and activation of RSK1 after silencing of PKARIα or depletion of D-AKAP1 are due to decreased association of PP2Ac in the RSK1 complex.Cyclic AMP-dependent protein kinase (PKA)³ plays a pivotal role in manifesting an array of biological actions ranging from cell proliferation and tumorigenesis to increased inotropic and chronotropic effects in the heart as well as regulation of long term potentiation and memory. The PKA holoenzyme is a heterotetramer and consists of two catalytic (PKAc) subunits bound to a dimer of regulatory subunits. To date, four isoforms of the PKAc (PKAcα, PKAcβ, PKAcγ, and PKAcδ) and four isoforms of the regulatory subunits (RIα, RIβ, RIIα, and RIIβ) have been described (1). The various isoforms of PKA subunits are expressed differently in a tissue- and cell-specific manner (2). In addition to binding and inhibiting the activity of PKAc via their pseudo substrate region (3–6), the R subunits also interact with PKA-anchoring proteins (AKAPs) and facilitate the localization of PKA in specific subcellular compartments (7, 8). More than 50 AKAP family members have been described, and although most of these have a higher affinity for the RII subunits (9), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the PKARIα subunit (10–12). Because the AKAPs also bind other signaling molecules such as phosphatases (PP2B) and kinases (protein kinase C), they act as scaffolds to organize and integrate specific signaling events within specific compartments in the cells (7, 8, 13, 14).We have shown that the PKARIα and PKAcα subunits of PKA interact with the inactive and active forms of p90^RSK1 (RSK1), respectively (15). Binding of inactive RSK1 to PKARIα decreases the interactions between PKARIα and PKAc, whereas the association of active RSK1 with PKAc increases interactions between PKARIα and PKAc such that larger amounts of cAMP are required to activate PKAc in the presence of active RSK1 (15). Moreover, the indirect (via subunits of PKA) interaction of RSK1 with AKAPs is required for the nuclear localization of active RSK1 (15), and disruption of the interactions of RSK1·PKA complex from AKAPs results in increased cytoplasmic distribution of active RSK1 with a concomitant increase in phosphorylation of its cytosolic substrates such as BAD and reduced cellular apoptosis (15). These findings show the functional and biological significance of RSK1·PKA·AKAP interactions.Besides inhibiting PKAc activity, the physiological role of PKARIα is underscored by the findings that mutations in the PKAR1A gene that result in haploinsufficiency of PKARIα are the underlying cause of Carney complex (CNC) (16, 17). CNC is an autosomal dominant multiple neoplasia syndrome in which myxomas of the skin, heart, and/or vicera are recurrent and also associated with high incidence of endocrine and ovarian tumors as well as Schwannomas (18–20). The majority of patients with the multiple neoplasia CNC syndrome harbor mutations in the PKAR1A gene (21) that result in PKARIα haploinsufficiency. Importantly, however, loss of heterozygosity or alterations in PKA activity may not contribute toward the tumorigenicity in either CNC patients or mouse model of CNC (21). This suggests that loss of function(s) of PKARIα other than inhibition of PKA activity is(are) involved in the enhanced tumorigenicity in CNC patients and in the murine CNC model.Because RSK1 regulates cell growth, survival, and tumorigenesis (22–27), and because its subcellular localization and ability to inhibit apoptosis is regulated by its interactions via PKARIα with AKAPs (15), we reasoned that in conditions such as CNC where PKARIα levels are decreased, the increase in tumorigenicity may emanate from aberrant regulation of the activity and/or subcellular localization of RSK1. Therefore, herein we have investigated whether PKARIα regulates the activation of RSK1 and its biological functions. Decreasing expression of PKARIα by small interfering RNA (siRNA) enhanced the activation of RSK1, but not RSK2 or RSK3, in the absence of an agonist such as EGF. This was accompanied by an increase in the cytoplasmic localization of the active RSK1 and enhanced cell survival in the absence of any growth factor. Silencing of PKARIα also increased PKAc activity and while part of the anti-apoptotic response could be attributed to an increase in PKAc activity, activation of RSK1 under basal conditions contributed significantly to cell survival. The elevation in RSK1 activity upon PKARIα silencing was not due to increased PKAc activity. Rather the activation of RSK1 in the absence of PKARIα was due to a decrease in PP2A in the RSK1 complex. These findings demonstrate a novel role for PKARIα in the regulation of RSK1 activation, a key enzyme that mediates the downstream actions of the ERK1/2 cascade.