Lessons learned from the real-world diagnosis and management of hereditary hypophosphatemic rickets.

Hypophosphatemic rickets, which is often hereditary, is still under- or misdiagnosed in both children and adults, denying these individuals access to optimal management and genetic counseling. There have been recent calls to compile real-world data and share best practice on these rare conditions to guide clinical decision-making. Here we present eight clinical vignettes of patients with hypophosphatemic rickets encountered in our tertiary pediatric endocrinology practice. We describe the clinical features, genetics, and management of four cases of X-linked hypophosphatemia (PHEX mutations), one each of autosomal recessive hypophosphatemic rickets (DMP1 mutation) and autosomal recessive vitamin D-dependent rickets type 1A (CYP27B1 mutation), and two cases of distal renal tubular acidosis with FOXI1 mutation-associated hypophosphatemic rickets. Our cases prompt consideration of the (i) frequent misdiagnosis of hypophosphatemic rickets in clinical practice and the importance of comprehensive genetic testing; (ii) variable expressivity of the causative mutations; and (iii) a lack of responsiveness and/or compliance to conventional therapy and the value of burosumab in modern management, provided access is equitable. These cases highlight common real-world themes and challenges to managing patients presenting with these diverse conditions, especially the burden of disease hidden by misdiagnosis. In sharing these cases, we hope to raise awareness of these conditions, promote best practice in genetic diagnosis and management, and further advocate for reimbursement equity for the best available therapies.

Identification of genomic regions associated with cereal cyst nematode (Heterodera avenae Woll.) resistance in spring and winter wheat.

Cereal cyst nematode (CCN) is a major threat to cereal crop production globally including wheat (Triticum aestivum L.). In the present study, single-locus and multi-locus models of Genome-Wide Association Study (GWAS) were used to find marker trait associations (MTAs) against CCN (Heterodera avenae) in wheat. In total, 180 wheat accessions (100 spring and 80 winter types) were screened against H. avenae in two independent years (2018/2019 "Environment 1" and 2019/2020 "Environment 2") under controlled conditions. A set of 12,908 SNP markers were used to perform the GWAS. Altogether, 11 significant MTAs, with threshold value of -log10 (p-values) ≥ 3.0, were detected using 180 wheat accessions under combined environment (CE). A novel MTA (wsnp_Ex_c53387_56641291) was detected under all environments (E1, E2 and CE) and considered to be stable MTA. Among the identified 11 MTAs, eight were novel and three were co-localized with previously known genes/QTLs/MTAs. In total, 13 putative candidate genes showing differential expression in roots, and known to be involved in plant defense mechanisms were reported. These MTAs could help us to identify resistance alleles from new sources, which could be used to identify wheat varieties with enhanced CCN resistance.

GWAS scans of cereal cyst nematode (Heterodera avenae) resistance in Indian wheat germplasm.

Significant yield losses in major cereal-growing regions around the world have been linked to cereal cyst nematodes (Heterodera spp.). Identifying and deploying natural sources of resistance is of utmost importance due to increasing concerns associated with chemical methods over the years. We screened 141 diverse wheat genotypes collected from pan-Indian wheat cultivation states for nematode resistance over two years, alongside two resistant (Raj MR1, W7984 (M6)) and two susceptible (WH147, Opata M85) checks. We performed genome-wide association analysis using four single-locus models (GLM, MLM, CMLM, and ECMLM) and three multi-locus models (Blink, FarmCPU, and MLMM). Single locus models identified nine significant MTAs (-log10 (P) > 3.0) on chromosomes 2A, 3B, and 4B whereas, multi-locus models identified 11 significant MTAs on chromosomes 1B, 2A, 3B, 3D and 4B. Single and multi-locus models identified nine common significant MTAs. Candidate gene analysis identified 33 genes like F-box-like domain superfamily, Cytochrome P450 superfamily, Leucine-rich repeat, cysteine-containing subtype Zinc finger RING/FYVE/PHD-type, etc., having a putative role in disease resistance. Such genetic resources can help to reduce the impact of this disease on wheat production. Additionally, these results can be used to design new strategies for controlling the spread of H. avenae, such as the development of resistant varieties or the use of resistant cultivars. Finally, the obtained results can also be used to identify new sources of resistance to this pathogen and develop novel control methods.

QTL mapping for resistance against cereal cyst nematode (Heterodera avenae Woll.) in wheat (Triticum aestivum L.).

The resistance to cereal cyst nematode (Heterodera avenae Woll.) in wheat (Triticum aestivum L.) was studied using 114 doubled haploid lines from a novel ITMI mapping population. These lines were screened for nematode infestation in a controlled environment for two years. QTL-mapping analyses were performed across two years (Y1 and Y2) as well as combining two years (CY) data. On the 114 lines that were screened, a total of 2,736 data points (genotype, batch or years, and replication combinations) were acquired. For QTL analysis, 12,093 markers (11,678 SNPs and 415 SSRs markers) were used, after filtering the genotypic data, for the QTL mapping. Composite interval mapping, using Haley-Knott regression (hk) method in R/QTL, was used for QTL analysis. In total, 19 QTLs were detected out of which 13 were novel and six were found to be colocalized or nearby to previously reported Cre genes, QTLs or MTAs for H. avenae or H. filipjevi. Nine QTLs were detected across all three groups (Y1, Y2 and CY) including a significant QTL "QCcn.ha-2D" on chromosome 2D that explains 23% of the variance. This QTL colocalized with a previously identified Cre3 locus. Novel QTL, QCcn.ha-2A, detected in the present study could be the possible unreported homeoloci to QCcn.ha-2D, QCcn.ha-2B.1 and QCcn.ha-2B.2. Six significant digenic epistatic interactions were also observed. In addition, 26 candidate genes were also identified including genes known for their involvement in PPNs (plant parasitic nematodes) resistance in different plant species. In-silico expression of putative candidate genes showed differential expression in roots during specific developmental stages. Results obtained in the present study are useful for wheat breeding to generate resistant genetic resources against H. avenae.

Patients' Perception of the Use of the EasyPod™ Growth Hormone Injector Device and Impact on Injection Adherence: A Multi-Center Regional Study.

Objective: This study aimed to assess patient perceptions of the use of the EasyPod™ growth hormone delivery device and its association with compliance. Methods: This cross-sectional, multicenter study was conducted in six centers from three countries (United Arab Emirates, Oman, and Saudi Arabia,) between March 2020 and June 2020. Children and adolescents aged 3-18 years, diagnosed with growth disorders and receiving rhGH through the EasyPod™ device were enrolled. Patients and caregivers were given a pre-set questionnaire that evaluated patient satisfaction, preference for technical and personalized features, and device drawbacks. The results were analyzed using independent measures of analysis of variance to evaluate the association of higher satisfaction with device features and better compliance. Results: A total of 186 patients were enrolled in the study. Of these, 45.7% had GH deficiency. The mean age (±SD) of patients was 11.8 (±2.76) years; 117 (62.90%) were males. Average compliance was 87%. One hundred patients (53.76%) had injection compliance of ≥90%. Amongst these patients, 74%, 68%, and 77% top-scored (5/5) the technical features of hidden needle, skin sensor, and pre-set dosing, respectively, compared to top scores by 39%, 34%, and 51% patients in the <90% compliance group (p-value <0.05). Similarly, a statistically significant difference was observed between the groups (p-value <0.05) in the perception of the usefulness of the tracking features such as display of history of injected doses (78% vs. 47.7%), a reminder for medicine remaining (46% vs. 23.3%) and battery power indicator (48% vs. 20.9%). Personal screen messages were associated with higher compliance while the requirement to keep the device in the fridge was reported as the most inconvenient feature by 56% of patients in the higher compliance group as against 39.5% in the lower compliance group (p-value <0.05). There was no statistically significant difference in the intensity of pain reported in the two compliance groups. Conclusion: Our study showed that there is a statistically significant association between better perception of device features and higher compliance.

WheatQTLdb: a QTL database for wheat.

During the last three decades, QTL analysis in wheat has been conducted for a variety of individual traits, so that thousands of QTL along with the linked markers, their genetic positions and contribution to phenotypic variation (PV) for concerned traits are now known. However, no exhaustive database for wheat QTL is currently available at a single platform. Therefore, the present database was prepared which is an exhaustive information resource for wheat QTL data from the published literature till May, 2020. QTL data from both interval mapping and genome-wide association studies (GWAS) have been included for the following classes of traits: (i) morphological traits, (ii) N and P use efficiency, (iii) traits for biofortification (Fe, K, Se, and Zn contents), (iv) tolerance to abiotic stresses including drought, water logging, heat stress, pre-harvest sprouting and salinity, (v) resistance to biotic stresses including those due to bacterial, fungal, nematode and insects, (vi) quality traits, and (vii) a variety of physiological traits, (viii) developmental traits, and (ix) yield and its related traits. For the preparation of the database, literature was searched for data on QTL/marker-trait associations (MTAs), curated and then assembled in the form of WheatQTLdb. The available information on metaQTL, epistatic QTL and candidate genes, wherever available, is also included in the database. Information on QTL in this WheatQTLdb includes QTL names, traits, associated markers, parental genotypes, crosses/mapping populations, association mapping panels and other useful information. To our knowledge, WheatQTLdb prepared by us is the largest collection of QTL (11,552), epistatic QTL (107) and metaQTL (330) data for hexaploid wheat to be used by geneticists and plant breeders for further studies involving fine mapping, cloning, and marker-assisted selection (MAS) during wheat breeding.

Genome-wide association study in hexaploid wheat identifies novel genomic regions associated with resistance to root lesion nematode (Pratylenchus thornei).

Root lesion nematode (RLN; Pratylenchus thornei) causes extensive yield losses in wheat worldwide and thus pose serious threat to global food security. Reliance on fumigants (such as methyl bromide) and nematicides for crop protection has been discouraged due to environmental concerns. Hence, alternative environment friendly control measures like finding and deployment of resistance genes against Pratylenchus thornei are of significant importance. In the present study, genome-wide association study (GWAS) was performed using single-locus and multi-locus methods. In total, 143 wheat genotypes collected from pan-Indian wheat cultivation states were used for nematode screening. Genotypic data consisted of  > 7K SNPs with known genetic positions on the high-density consensus map was used for association analysis. Principal component analysis indicated the existence of sub-populations with no major structuring of populations due to the origin. Altogether, 25 significant marker trait associations were detected with - log10 (p value) > 4.0. Three large linkage disequilibrium blocks and the corresponding haplotypes were found to be associated with significant SNPs. In total, 37 candidate genes with nine genes having a putative role in disease resistance (F-box-like domain superfamily, Leucine-rich repeat, cysteine-containing subtype, Cytochrome P450 superfamily, Zinc finger C2H2-type, RING/FYVE/PHD-type, etc.) were identified. Genomic selection was conducted to investigate how well one could predict the phenotype of the nematode count without performing the screening experiments. Prediction value of r = 0.40 to 0.44 was observed when 56 to 70% of the population was used as a training set. This is the first report where GWAS has been conducted to find resistance against root lesion nematode (P. thornei) in Indian wheat germplasm.

Identification and characterization of SET domain family genes in bread wheat (Triticum aestivum L.).

SET domain genes (SDGs) that are involved in histone methylation have been examined in many plant species, but have never been examined in bread wheat; the histone methylation caused due to SDGs is associated with regulation of gene expression at the transcription level. We identified a total of 166 bread wheat TaSDGs, which carry some interesting features including the occurrence of tandem/interspersed duplications, SSRs (simple sequence repeats), transposable elements, lncRNAs and targets for miRNAs along their lengths and transcription factor binding sites (TFBS) in the promoter regions. Only 130 TaSDGs encoded proteins with complete SET domain, the remaining 36 proteins had truncated SET domain. The TaSDG encoded proteins were classified into six classes (I-V and VII). In silico expression analysis indicated relatively higher expression (FPKM > 20) of eight of the 130 TaSDGs in different tissues, and downregulation of 30 TaSDGs under heat and drought at the seedling stage. qRT-PCR was also conducted to validate the expression of seven genes at the seedling stage in pairs of contrasting genotypes in response to abiotic stresses (water and heat) and biotic stress (leaf rust). These genes were generally downregulated in response to the three stresses examined.

Folding Determinants of Transmembrane ß-Barrels Using Engineered OMP Chimeras.

Transmembrane ß-barrel proteins (OMPs) are highly robust structures for engineering and development of nanopore channels, surface biosensors, and display libraries. Expanding the applications of designed OMPs requires the identification of elements essential for ß-barrel scaffold formation and stability. Here, we have designed chimeric 8-stranded OMPs composed of strand hybrids of Escherichia coli OmpX and Yersinia pestis Ail, and identified molecular motifs essential for ß-barrel scaffold formation. For the OmpX/Ail chimeras, we find that the central hairpin strands ß4-ß5 in tandem are vital for ß-barrel folding. We also show that the central hairpin can facilitate OMP assembly even when present as the N- or C-terminal strands. Further, the C-terminal ß-signal and strand length are important but neither sufficient nor mutually exclusive for ß-barrel assembly. Our results point to a nonstochastic model for assembly of chimeric ß-barrels in lipidic micelles. The assembly likely follows a predefined nucleation at the central hairpin only when presented in tandem, with some influence from its absolute position in the barrel. Our findings can lead to the design of engineered barrels that retain the OMP assembly elements necessary to attain well-folded, stable, yet malleable scaffolds, for bionanotechnology applications.

Position-Specific contribution of interface tryptophans on membrane protein energetics.

Interface tryptophans are key residues that facilitate the folding and stability of membrane proteins. Escherichia coli OmpX possesses two unique interface tryptophans, namely Trp76, which is present at the interface and is solvent-exposed, and Trp140, which is relatively more lipid solvated than Trp76 in symmetric lipid membranes. Here, we address the requirement for tryptophan and the consequences of aromatic amino acid substitutions on the folding and stability of OmpX. Using spectroscopic measurements of OmpX-Trp/Tyr/Phe mutants, we show that the specific mutation W76→Y allows barrel assembly >1.5-fold faster than native OmpX, and increases stability by ~0.4kcalmol-1. In contrast, mutating W140→F/Y lowers OmpX thermodynamic stability by ~0.4kcalmol-1, without affecting the folding kinetics. We conclude that the stabilizing effect of tryptophan at the membrane interface can be position-and local environment-specific. We propose that the thermodynamic contributions for interface residues be interpreted with caution.

Transmembrane ß-barrels: Evolution, folding and energetics.

The biogenesis of transmembrane ß-barrels (outer membrane proteins, or OMPs) is an elaborate multistep orchestration of the nascent polypeptide with translocases, barrel assembly machinery, and helper chaperone proteins. Several theories exist that describe the mechanism of chaperone-assisted OMP assembly in vivo and unassisted (spontaneous) folding in vitro. Structurally, OMPs of bacterial origin possess even-numbered strands, while mitochondrial ß-barrels are even- and odd-stranded. Several underlying similarities between prokaryotic and eukaryotic ß-barrels and their folding machinery are known; yet, the link in their evolutionary origin is unclear. While OMPs exhibit diversity in sequence and function, they share similar biophysical attributes and structure. Similarly, it is important to understand the intricate OMP assembly mechanism, particularly in eukaryotic ß-barrels that have evolved to perform more complex functions. Here, we deliberate known facets of ß-barrel evolution, folding, and stability, and attempt to highlight outstanding questions in ß-barrel biogenesis and proteostasis.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Juxtamembrane tryptophans have distinct roles in defining the OmpX barrel-micelle boundary and facilitating protein-micelle association.

Defining the span of the transmembrane region, a key requirement to ensure correct folding, stability and function of bacterial outer membrane ß-barrels, is assisted by the amphipathic property of tryptophan. We demonstrate the unique and distinctive properties of the interface Trp76 and Trp140 of outer membrane protein X, and map their positional relevance to the refolding process, barrel formation and the resulting stability in dodecylphosphocholine micelles. The solvent-exposed Trp76 displays a rigid interfacial localization, whereas Trp140 is relatively micelle-solvated and contributes to barrel folding and global OmpX stability. Kinetic contribution to OmpX stability is influenced by the two tryptophans. Differential associations of the indoles with the detergent milieu therefore contribute to micelle-assisted ß-barrel folding and concomitant OmpX stability.

Methionine mutations of outer membrane protein X influence structural stability and beta-barrel unfolding.

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane ß-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.

Modulating lipid dynamics and membrane fluidity to drive rapid folding of a transmembrane barrel.

Lipid-protein interactions, critical for the folding, stability and function of membrane proteins, can be both of mechanical and chemical nature. Mechanical properties of lipid systems can be suitably influenced by physical factors so as to facilitate membrane protein folding. We demonstrate here that by modulating lipid dynamics transiently using heat, rapid folding of two 8-stranded transmembrane ß-barrel proteins OmpX and OmpA(1-171), in micelles and vesicles, can be achieved within seconds. Folding kinetics using this 'heat shock' method shows a dramatic ten to several hundred folds increase in refolding rate along with ~100% folding efficiency. We establish that OmpX thus folded is highly thermostable even in detergent micelles, and retains structural characteristics comparable to the protein in bilayers.

Different mechanisms of intestinal calcium absorption at different life stages: therapeutic implications and long-term responses to treatment in patients with hereditary vitamin D-resistant rickets.

BACKGROUND: Intestinal calcium absorption is regulated principally by 1,25-dihydroxyvitamin D, but other regulators are also involved. CASE REPORT: The 3 children studied were born with unaffected bones. Two were referred at 21 months of age, with clinical features of severe vitamin D-resistant rickets. They were treated with intravenous calcium for 12-18 months, following an initial lack of response to oral calcium and vitamin D. The third patient, who was exclusively breast-fed, was diagnosed at 4 months of age, due to alopecia. His condition was successfully managed with high doses of oral calcium and vitamin D. All 3 patients were homozygous for a mutation in the DNA-binding domain of vitamin D receptor. At the most recent evaluation of these patients, currently maintained on oral calcium and vitamin D, clinical findings were normal. CONCLUSION: During gestation, calcium flux across the placenta is normal, preventing bone diseases in affected fetuses. High calcium intake early in life and, perhaps, the maintenance of breastfeeding for several months may constitute an effective approach to ensuring adequate absorption and preventing severe rickets. During childhood, after parenteral calcium treatment to bypass intestinal calcium absorption, it is possible to maintain normal bone through long-term oral calcium supplementation.

Localization and retention of p90 ribosomal S6 kinase 1 in the nucleus: implications for its function.

Ribosomal S6 kinase 1 (RSK1) belongs to a family of proteins with two kinase domains. Following activation in the cytoplasm by extracellular signal-regulated kinases (ERK1/2), it mediates the cell-proliferative, cell-growth, and survival-promoting actions of a number of growth factors and other agonists. These diverse biological actions of RSK1 involve regulation of both cytoplasmic and nuclear events. However, the mechanisms that permit nuclear accumulation of RSK1 remain unknown. Here, we show that phosphorylation of RSK1 on S221 is important for its dissociation from the type Iα regulatory subunit of protein kinase A (PKA) in the cytoplasm and that RSK1 contains a bipartite nuclear localization sequence that is necessary for its nuclear entry. Once inside, the active RSK1 is retained in the nucleus via its interactions with PKA catalytic subunit and AKAP95. Mutations of RSK1 that do not affect its activity but disrupt its entry into the nucleus or expression of AKAP95 forms that do not enter the nucleus inhibit the ability of active RSK1 to stimulate DNA synthesis. Our findings identify novel mechanisms by which active RSK1 accumulates in the nucleus and also provide new insights into how AKAP95 orchestrates cell cycle progression.

p90 ribosomal S6 kinase 1 (RSK1) and the catalytic subunit of protein kinase A (PKA) compete for binding the pseudosubstrate region of PKAR1alpha: role in the regulation of PKA and RSK1 activities.

Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIalpha, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIalpha. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIalpha. This explains the lack of an interaction between active RSK1 and PKARIalpha. Full-length RSK1 bound to PKARIalpha with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93-99) of PKARIalpha. Overexpressed RSK1 dissociated PKAc from PKARIalpha, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIalpha interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIalpha for their interactions, RSK1/PKARIalpha association requires all four Arg residues (Arg-93-96) in the pseudosubstrate site of PKARIalpha. A peptide (Wt-PS) corresponding to residues 91-99 of PKARIalpha competed for binding of RSK1 with PKARIalpha both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIalpha, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIalpha, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIalpha by associating with RSK1 regulates its activation and its biological functions.

The PKARIalpha 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 PKARIalpha subunit of PKA in regulating RSK1 activation and cell survival. In mouse lung fibroblasts, silencing of the PKARIalpha increased the phosphorylation and activation of RSK1, but not of RSK2 and RSK3, in the absence of any stimulation. Silencing of PKARIalpha 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 PKARIalpha 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 PKARIalpha, depletion of D-AKAP1, or by using a peptide that competes with PKARIalpha 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 PKARIalpha or depletion of D-AKAP1 are due to decreased association of PP2Ac in the RSK1 complex.