Systematic large-scale application of ClinGen InSiGHT APC -specific ACMG/AMP variant classification criteria substantially alleviates the burden of variants of uncertain significance in ClinVar and LOVD databases.

Background: Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUS), APC-specific ACMG/AMP variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP). Methods: A streamlined algorithm using the APC -specific criteria was developed and applied to assess all APC variants in ClinVar and the InSiGHT international reference APC LOVD variant database. Results: A total of 10,228 unique APC variants were analysed. Among the ClinVar and LOVD variants with an initial classification of (Likely) Benign or (Likely) Pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUS were reclassified into clinically actionable classes, the vast majority as (Likely) Benign. The total number of VUS was reduced by 37%. In 21 out of 36 (58%) promising APC variants that remained VUS despite evidence for pathogenicity, a data mining-driven work-up allowed their reclassification as (Likely) Pathogenic. Conclusions: The application of APC -specific criteria substantially reduced the number of VUS in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalisable model for other gene-/disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUS that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

MGMT promoter methylation status in Merkel cell carcinoma: in vitro versus invivo.

PURPOSE: Expression of O6-methylguanine-DNA methyltransferase (MGMT) in Merkel cell carcinoma (MCC) is very variable; thus, we tested whether this may be due to differential methylation of the MGMT gene promoter. METHODS: Quantitative analysis of MGMT mRNA and protein expression, as well as MGMT promoter methylation status, was performed in a series of tissue samples of MCC tumors, representing both primary and metastatic lesions, as well as in six MCC cell lines. RESULTS: These analyses revealed a very heterogeneous MGMT mRNA and protein expression in MCC both in vivo and in vitro. However, neither the MGMT mRNA nor protein expression correlated with the sensitivity of MCC cell lines toward the alkylating agent dacarbazine in vitro. Notably, increased methylation at the promoter of the MGMT gene was observed in 2/6 (33%) of the MCC cell lines; however, MGMT promoter methylation was absent in all MCC tissue samples. According to our results, albeit aberrant methylation of MGMT gene promoter can be observed in in vitro propagated MCC cell lines, it seems to be absent or very rare in MCC lesions in situ. CONCLUSION: Thus, the evaluation of this marker has no or only little significance for predicting response to therapy or for improving efficacy of demethylating agents in the treatment of MCC. Microenvironmental factors may play a role in explaining the different results between MCC cell lines and MCC samples.

c-Myc modulation: a key role in melanoma drug response.

Understanding molecular mechanisms involved in melanoma resistance to drugs is a big challenge. Experimental evidences suggested a correlation between mutational status in B-RAF and melanoma cell susceptibility to drugs, such as paclitaxel, doxorubicin and temozolomide, which generate an accumulation of hydrogen peroxide (H2O2) in the cells. We investigated the survival phenotype and the protein level of c-myc, a B-RAF target molecule, in melanoma cells, carrying a different mutational status in B-RAF, upon paclitaxel, doxorubicin and H2O2 treatment. For the first time, we reported c-myc modulation is critical for melanoma drug response. It appeared drug-specific and post-transcriptionally driven through PP2A; in correlation, cell pre-treatment with okadaic acid (OA), a specific PP2A inhibitor, as well as PP2A silencing of melanoma cells, was able to increase melanoma cell drug-sensitivity and c-myc protein level. This is relevant for designing efficacious therapeutic strategies in melanoma.

Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly.

OBJECTIVE: Mutations of ATP1A3 have been associated with rapid onset dystonia-parkinsonism and more recently with alternating hemiplegia of childhood. Here we report one child with catastrophic early life epilepsy and shortened survival, and another with epilepsy, episodic prolonged apnea, postnatal microcephaly, and severe developmental disability. Novel heterozygous mutations (p.Gly358Val and p.Ile363Asn) were identified in ATP1A3 in these children. METHODS: Subjects underwent next-generation sequencing under a research protocol. Clinical data were collected retrospectively. The biochemical effects of the mutations on ATP1A3 protein function were investigated. Postmortem neuropathologic specimens from control and affected subjects were studied. RESULTS: The mutations localized to the P domain of the Na,K-ATPase α3 protein, and resulted in significant reduction of Na,K-ATPase activity in vitro. We demonstrate in both control human brain tissue and that from the subject with the p.Gly358Val mutation that ATP1A3 immunofluorescence is prominently associated with interneurons in the cortex, which may provide some insight into the pathogenesis of the disease. SIGNIFICANCE: The findings indicate these mutations cause severe phenotypes of ATP1A3-related disorder spectrum that include catastrophic early life epilepsy, episodic apnea, and postnatal microcephaly.

A Korean Case of ß-Ureidopropionase Deficiency Presenting with Intractable Seizure, Global Developmental Delay, and Microcephaly.

ß-Ureidopropionase deficiency (OMIM #613161) is a rare autosomal recessive inborn error of metabolism due to mutations in the UPB1 gene, which encodes the third enzyme involved in the pyrimidine degradation pathway. A total of 28 cases have been reported, mainly presenting with seizures, microcephaly, and intellectual disabilities. However, 11 of them were asymptomatic cases (Nakajima et al., J Inherit Metab Dis 37(5):801-812, 2014). We report on a 9-year-old female presenting with intractable epilepsy, microcephaly, and global developmental delay. She was homozygous for p.R326Q (c.977G>A) and heterozygous for p.G31S (c.91G>A) in the UPB1 gene, detected by targeted next-generation sequencing test and subsequently confirmed by biochemical analysis of urine, plasma, and cerebrospinal fluid (CSF) using reversed-phase HPLC, combined with electrospray tandem mass spectrometry. We report a first Korean female case with ß-ureidopropionase deficiency.

Molecular genetic testing of patients with monogenic diabetes and hyperinsulinism.

Genetic sequencing has become a critical part of the diagnosis of certain forms of pancreatic beta cell dysfunction. Despite great advances in the speed and cost of DNA sequencing, determining the pathogenicity of variants remains a challenge, and requires sharing of sequence and phenotypic data between laboratories. We reviewed all diabetes and hyperinsulinism-associated molecular testing done at the Seattle Children's Molecular Genetics Laboratory from 2009 to 2013. 331 probands were referred to us for molecular genetic sequencing for Neonatal Diabetes (NDM), Maturity-Onset Diabetes of the Young (MODY), or Congenital Hyperinsulinism (CHI) during this period. Reportable variants were identified in 115 (35%) patients with 91 variants in one of 6 genes: HNF1A, GCK, HNF4A, ABCC8, KCNJ11, or INS. In addition to identifying 23 novel variants, we identified unusual mechanisms of inheritance, including mosaic and digenic MODY presentations. Re-analysis of all reported variants using more recently available databases led to a change in variant interpretation from the original report in 30% of cases. These results represent a resource for molecular testing of monogenic forms of diabetes and hyperinsulinism, providing a mutation spectrum for these disorders in a large North American cohort. In addition, they highlight the importance of periodic review of molecular testing results.

Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders.

BACKGROUND: Neuromuscular disorders are a clinically, pathologically, and genetically heterogeneous group. Even for the experienced clinician, an accurate diagnosis is often challenging due to the complexity of these disorders. Here, we investigated the utility of next generation sequencing (NGS) in early diagnostic algorithms to improve the diagnosis for patients currently lacking precise molecular characterisation, particularly for hereditary myopathies. METHODS: 43 patients presenting with early onset neuromuscular disorders from unknown genetic origin were tested by NGS for 579 nuclear genes associated with myopathy. RESULTS: In 21 of the 43 patients, we identified the definite genetic causes (48.8%). Additionally, likely pathogenic variants were identified in seven cases and variants of uncertain significance (VUS) were suspected in four cases. In total, 19 novel and 15 known pathogenic variants in 17 genes were identified in 32 patients. Collagen VI related myopathy was the most prevalent type in our cohort. The utility of NGS was highlighted in three cases with congenital myasthenia syndrome, as early diagnosis is important for effective treatment. CONCLUSIONS: A targeted NGS can offer cost effective, safe and fairly rapid turnaround time, which can improve quality of care for patients with early onset myopathies and muscular dystrophies; in particular, collagen VI related myopathy and congenital myasthenia syndromes. Nevertheless, a substantial number of patients remained without molecular diagnosis in our cohort. This may be due to the intrinsic limitation of detection for some types of mutations by NGS or to the fact that other causative genes for neuromuscular disorders are yet to be identified.

Targeted exome sequencing for mitochondrial disorders reveals high genetic heterogeneity.

BACKGROUND: Mitochondrial disorders are difficult to diagnose due to extreme genetic and phenotypic heterogeneities. METHODS: We explored the utility of targeted next-generation sequencing for the diagnosis of mitochondrial disorders in 148 patients submitted for clinical testing. A panel of 447 nuclear genes encoding mitochondrial respiratory chain complexes, and other genes inducing secondary mitochondrial dysfunction or that cause diseases which mimic mitochondrial disorders were tested. RESULTS: We identified variants considered to be possibly disease-causing based on family segregation data and/or variants already known to cause disease in twelve genes in thirteen patients. Rare or novel variants of unknown significance were identified in 45 additional genes for various metabolic, genetic or neurogenetic disorders. CONCLUSIONS: Primary mitochondrial defects were confirmed only in four patients indicating that majority of patients with suspected mitochondrial disorders are presumably not the result of direct impairment of energy production. Our results support that clinical and routine laboratory ascertainment for mitochondrial disorders are challenging due to significant overlapping non-specific clinical symptoms and lack of specific biomarkers. While next-generation sequencing shows promise for diagnosing suspected mitochondrial disorders, the challenges remain as the underlying genetic heterogeneity may be greater than suspected and it is further confounded by the similarity of symptoms with other conditions as we report here.

Next-generation sequencing for mitochondrial diseases: a wide diagnostic spectrum.

BACKGROUND: The current diagnostic approach for mitochondrial disorders requires invasive procedures such as muscle biopsy and multiple biochemical testing but the results are often inconclusive. Clinical sequencing tests are available only for a limited number of genes. Recently, massively parallel sequencing has become a powerful tool for testing genetically heterogeneous conditions such as mitochondrial disorders. METHODS: Targeted next-generation sequencing was performed on 26 patients with known or suspected mitochondrial disorders using in-solution capture for the exons of 908 known and candidate nuclear genes and an Illumina genome analyzer. RESULTS: None of the 18 patients with various abnormal respiratory chain complex (RCC) activities had molecular defects in either subunits or assembly factors of mitochondrial RCC enzymes except a reference control sample with known mutations in SURF1. Instead, several variants in known pathogenic genes including CPT2, POLG, PDSS1, UBE3A, SDHD, and a few potentially pathogenic variants in candidate genes such as MTO1 or SCL7A13 were identified. CONCLUSIONS: Sequencing only nuclear genes for RCC subunits and assembly factors may not provide the diagnostic answers for suspected patients with mitochondrial disorders. The present findings indicate that the diagnostic spectrum of mitochondrial disorders is much broader than previously thought, which could potentially lead to misdiagnosis and/or inappropriate treatment. Overall analytic sensitivity and precision appear acceptable for clinical testing. Despite the limitations in finding mutations in all patients, the present findings underscore the considerable clinical benefits of targeted next-generation sequencing and serve as a prototype for extending the clinical evaluation in this clinically heterogeneous patient group.

Assay to measure oxidized and reduced forms of CoQ by LC-MS/MS.

The redox status of mitochondrial coenzyme Q (CoQ) is an important marker for oxidative stress associated with several disorders such as Parkinson disease and Alzheimer disease. Altered redox status may be present in mitochondrial electron transport complex disorders. Intracellular CoQ levels reflect the functional status of the mitochondrial electron transport complex better than plasma levels. Here, we describe the method to determine the reduced and oxidized form of CoQ in white blood cells using LC-MS/MS.

Characterization of human melanoma cell lines and melanocytes by proteome analysis.

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma.

The role of DMQ(9) in the long-lived mutant clk-1.

INTRODUCTION: Ubiquinone (UQ) is a redox active lipid that transfers electrons from complex I or II to complex III in the electron transport chain (ETC). The long-lived Caenorhabditis elegans mutant clk-1 is unable to synthesize its native ubiquinone, and accumulates high amounts of its precursor, 5-demethoxyubiquinone-9 (DMQ(9)). In clk-1, complexes I-III activity is inhibited while complexes II-III activity is normal. We asked whether the complexes I-III defect in clk-1 was caused by: (1) a defect in the ETC; (2) an inhibitory effect of DMQ(9); or (3) a decreased amount of ubiquinone. METHODS: We extracted the endogenous quinones from wildtype (N2) and clk-1 mitochondria, replenished them with exogenous ubiquinones, and measured ETC activities. RESULTS: Replenishment of extracted mutant and wildtype mitochondria resulted in equal enzymatic activities for complexes I-III and II-III ETC assays. Blue native gels showed that supercomplex formation was indistinguishable between clk-1 and N2. The addition of a pentane extract from clk-1 mitochondria containing DMQ(9) to wildtype mitochondria specifically inhibited complexes I-III activity. UQ in clk-1 mitochondria was oxidized compared to N2. DISCUSSION: Our results show that no measurable intrinsic ETC defect exists in clk-1 mitochondria. The data indicate that DMQ(9) specifically inhibits electron transfer from complex I to ubiquinone.

Next generation sequence analysis for mitochondrial disorders.

BACKGROUND: Mitochondrial disorders can originate from mutations in one of many nuclear genes controlling the organelle function or in the mitochondrial genome (mitochondrial DNA (mtDNA)). The large numbers of potential culprit genes, together with the little guidance offered by most clinical phenotypes as to which gene may be causative, are a great challenge for the molecular diagnosis of these disorders. METHODS: We developed a novel targeted resequencing assay for mitochondrial disorders relying on microarray-based hybrid capture coupled to next-generation sequencing. Specifically, we subjected the entire mtDNA genome and the exons and intron-exon boundary regions of 362 known or candidate causative nuclear genes to targeted capture and resequencing. We here provide proof-of-concept data by testing one HapMap DNA sample and two positive control samples. RESULTS: Over 94% of the targeted regions were captured and sequenced with appropriate coverage and quality, allowing reliable variant calling. Pathogenic mutations blindly tested in patients' samples were 100% concordant with previous Sanger sequencing results: a known mutation in Pyruvate dehydrogenase alpha 1 subunit (PDHA1), a novel splicing and a known coding mutation in Hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) were correctly identified. Of the additional variants recognized, 90 to 94% were present in dbSNP while 6 to 10% represented new alterations. The novel nonsynonymous variants were all in heterozygote state and mostly predicted to be benign. The depth of sequencing coverage of mtDNA was extremely high, suggesting that it may be feasible to detect pathogenic mtDNA mutations confounded by low level heteroplasmy. Only one sequencing lane of an eight lane flow cell was utilized for each sample, indicating that a cost-effective clinical test can be achieved. CONCLUSIONS: Our study indicates that the use of next generation sequencing technology holds great promise as a tool for screening mitochondrial disorders. The availability of a comprehensive molecular diagnostic tool will increase the capacity for early and rapid identification of mitochondrial disorders. In addition, the proposed approach has the potential to identify new mutations in candidate genes, expanding and redefining the spectrum of causative genes responsible for mitochondrial disorders.

Tryptic peptide analysis of ceruloplasmin in dried blood spots using liquid chromatography-tandem mass spectrometry: application to newborn screening.

BACKGROUND: Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods. METHODS: We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. CP-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). RESULTS: The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for CP concentrations of 25, 35, and 55 mg/dL (250, 350, and 550 mg/L) was 9.2%, 10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L). CONCLUSIONS: Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers.

Foxp3-dependent programme of regulatory T-cell differentiation.

Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such 'anergy' and Tr suppressive capacity are intimately linked. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.

Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A.

Leydig cells produce testosterone in the testes under the pulsatile control of pituitary luteinizing hormone (LH). cAMP is the intracellular messenger for LH action on steroidogenesis, and pharmacological evidence indicates that the response to LH can be modulated by cyclic nucleotide phosphodiesterases (PDEs). However the types and roles of the PDEs present in Leydig cells have not been fully defined. We report here that PDE8A is expressed in Leydig cells, and using PDE8A knockout mice we provide evidence that PDE8A is a key regulator of LH signaling and steroidogenesis. A 4-fold increase in the sensitivity to LH for testosterone production was detected in Leydig cells isolated from PDE8A knockout mice. In Leydig cells from wild-type mice, 3-isobutyl-1-methylxanthine, a compound that inhibits all cAMP PDEs except PDE8A, elicited only a small increase in the sensitivity of testosterone production to LH. However, in the PDE8-null mice, the effect of this inhibitor is much more pronounced. These observations indicate that PDE8A and at least one other PDE control the same or a complementary pool of cAMP that mediates LH-regulated steroidogenesis. Overall, these results suggest that pharmacological manipulation of PDE8A, alone or in combination with other PDEs present in Leydig cells, may be exploited to modulate testosterone synthesis and possibly to treat various conditions where the local levels of this androgen need to be altered.

Identification of a new variant of PDE1A calmodulin-stimulated cyclic nucleotide phosphodiesterase expressed in mouse sperm.

In mature sperm, cAMP plays an important role as a second messenger regulating functions that include capacitation, the acrosome reaction, motility, and, in some cases, chemosensing. We have cloned from mouse testis a novel calmodulin-stimulated cyclic nucleotide phosphodiesterase 1A isoform, Pde1a_v7 (mmPDE1A7), which arises from an alternative transcription start in the cyclic nucleotide phosphodiesterase 1A gene. The open reading frame is predicted to encode a polypeptide with a molecular mass of 52 kDa. Two further variants of this form, which contain two additional new exons, arise from alternative splicing. Analysis of testis cDNA by real-time polymerase chain reaction (PCR) indicates that the Pde1A_v7 transcript variant is the most abundant. The PDE1A_v7 protein uniquely lacks the first amino-terminal calmodulin-binding domain, but does possess an inhibitory domain and a second calmodulin-binding site shared with other variants. In vitro translation of the corresponding Pde1a_v7 cDNA produced a 52-kDa polypeptide having cyclic nucleotide hydrolytic activity, which was stimulated threefold by calcium-bound calmodulin. Immunoprecipitation of cyclic nucleotide phosphodiesterase 1 activity from detergent extracts of mouse sperm revealed a major protein of the size expected for PDE1A_v7, and the immunocytochemical staining for cyclic nucleotide phosphodiesterase 1A in mouse sperm showed intense immunoreactivity in the tail only. These observations, along with the PCR data, strongly suggest that this new variant PDE1A_v7 is the major form of cyclic nucleotide phosphodiesterase 1A expressed in mature sperm and is therefore likely to play an important role in cyclic nucleotide regulation of mature sperm function.

A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines.

The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.

Activation of phospholipase D by bradykinin and sphingosine 1-phosphate in A549 human lung adenocarcinoma cells via different GTP-binding proteins and protein kinase C delta signaling pathways.

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.