Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.     

Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication

Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.     

The Inotropic Effect of the Active Metabolite of Levosimendan,OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

Aims

We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca²⁺ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors.

Methods

Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.

Results

OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca²⁺. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α₁-AR response.

Conclusion

The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca²⁺ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.     

ClusTrack: Feature Extraction and Similarity Measures for Clustering of Genome-Wide Data Sets

Clustering is a popular technique for explorative analysis of data, as it can reveal subgroupings and similarities between data in an unsupervised manner. While clustering is routinely applied to gene expression data, there is a lack of appropriate general methodology for clustering of sequence-level genomic and epigenomic data, e.g. ChIP-based data. We here introduce a general methodology for clustering data sets of coordinates relative to a genome assembly, i.e. genomic tracks. By defining appropriate feature extraction approaches and similarity measures, we allow biologically meaningful clustering to be performed for genomic tracks using standard clustering algorithms. An implementation of the methodology is provided through a tool, ClusTrack, which allows fine-tuned clustering analyses to be specified through a web-based interface. We apply our methods to the clustering of occupancy of the H3K4me1 histone modification in samples from a range of different cell types. The majority of samples form meaningful subclusters, confirming that the definitions of features and similarity capture biological, rather than technical, variation between the genomic tracks. Input data and results are available, and can be reproduced, through a Galaxy Pages document at http://hyperbrowser.uio.no/hb/u/hb-superuser/p/clustrack. The clustering functionality is available as a Galaxy tool, under the menu option "Specialized analyzis of tracks", and the submenu option "Cluster tracks based on genome level similarity", at the Genomic HyperBrowser server: http://hyperbrowser.uio.no/hb/.     

Molecular Subtypes in Stage II-III Colon Cancer Defined by Genomic Instability: Early Recurrence-Risk Associated with a High Copy-Number Variation and Loss of RUNX3 and CDKN2A

Objective

We sought to investigate various molecular subtypes defined by genomic instability that may be related to early death and recurrence in colon cancer.

Methods

We sought to investigate various molecular subtypes defined by instability at microsatellites (MSI), changes in methylation patterns (CpG island methylator phenotype, CIMP) or copy number variation (CNV) in 8 genes. Stage II-III colon cancers (n = 64) were investigated by methylation-specific multiplex ligated probe amplification (MS-MLPA). Correlation of CNV, CIMP and MSI, with mutations in KRAS and BRAFV600E were assessed for overlap in molecular subtypes and early recurrence risk by uni- and multivariate regression.

Results

The CIMP phenotype occurred in 34% (22/64) and MSI in 27% (16/60) of the tumors, with noted CIMP/MSI overlap. Among the molecular subtypes, a high CNV phenotype had an associated odds ratio (OR) for recurrence of 3.2 (95% CI 1.1-9.3; P = 0.026). Losses of CACNA1G (OR of 2.9, 95% CI 1.4-6.0; P = 0.001), IGF2 (OR of 4.3, 95% CI 1.1-15.8; P = 0.007), CDKN2A (p16) (OR of 2.0, 95% CI 1.1-3.6; P = 0.024), and RUNX3 (OR of 3.4, 95% CI 1.3-8.7; P = 0.002) were associated with early recurrence, while MSI, CIMP, KRAS or BRAF V600E mutations were not. The CNV was significantly higher in deceased patients (CNV in 6 of 8) compared to survivors (CNV in 3 of 8). Only stage and loss of RUNX3 and CDKN2A were significant in the multivariable risk-model for early recurrence.

Conclusions

A high copy number variation phenotype is a strong predictor of early recurrence and death, and may indicate a dose-dependent relationship between genetic instability and outcome. Loss of tumor suppressors RUNX3 and CDKN2A were related to recurrence-risk and warrants further investigation.     

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.     

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-protein kinase A (PKA)-Csk inhibitory pathway that prevents full T-cell activation in the absence of a coreceptor stimulus. Here, we demonstrate that stimulation of CD28 leads to recruitment to lipid rafts of a β-arrestin/phosphodiesterase 4 (PDE4) complex that serves to degrade cAMP locally. Redistribution of the complex from the cytosol depends on Lck and phosphatidylinositol 3-kinase (PI3K) activity. Protein kinase B (PKB) interacts directly with β-arrestin to form part of the supramolecular complex together with sequestered PDE4. Translocation is mediated by the PKB plextrin homology (PH) domain, thus revealing a new role for PKB as an adaptor coupling PI3K and cAMP signaling. Functionally, PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production, leading to recruitment of the supramolecular PKB/β-arrestin/PDE4 complex to the membrane via the PKB PH domain, results in degradation of the TCR-induced cAMP pool located in lipid rafts, thereby allowing full T-cell activation to proceed.T-cell receptor (TCR) stimulation alone is insufficient for activation of T cells, and sustainable T-cell immune responses require a second signal in addition to the TCR-mediated signal. The second signal is typically elicited by ligands B7-1 or B7-2 on antigen-presenting cells engaging the coreceptor CD28 to prevent anergy and apoptosis and enhancing interleukin-2 (IL-2) production and clonal expansion (4). Although CD28 plays a central role in T-cell activation in vivo (5), relatively little is known about the molecular basis for the increased efficacy of T-cell activation upon TCR and CD28 costimulation. Involvement of Lck, Itk, phosphatidylinositol 3-kinase (PI3K), SLP-76, Vav-1, and phospholipase C-γ (PLC-γ) has, however, been reported (43). CD28-mediated signals are transmitted via a short intracellular stretch in the receptor containing a conserved YMNM motif (32). Phosphorylation of Tyr173 in this motif by Lck and Fyn following CD28 ligation is key to efficient signal transduction (41), generating a binding site for the SH2 domain of the p85 regulatory subunit of PI3K (37, 40). CD28 may also contribute to TCR-dependent PI3K activity without recruiting PI3K directly (18). Whether engagement of CD28 alone can also induce PI3K activity has been a matter of controversy. However, recent reports confirming phosphorylation of the protein kinase B (PKB) substrate glycogen synthase kinase 3 (GSK3) upon CD28 ligation has demonstrated that this is indeed the case (6, 15). In addition, CD28 can recruit growth factor receptor-bound protein 2 (Grb2), and such association of Grb2 occurs via the phosphorylated YMNM motif as well as via the C-terminal PXXP motif (22, 35). The PXXP motif also binds and regulates Src family kinases (SFKs) (21, 47), and knock-in mice mutated in this motif were recently reported to have impaired IL-2 secretion (16).Ligation of the TCR induces cyclic AMP (cAMP) production (27). However, the significance of this observation is still not fully understood, as it is well established that cAMP potently inhibits T-cell function and proliferation (2, 45, 46, 50). The spatiotemporal dynamics of the activation-induced cAMP gradient also are not completely appreciated. We have previously shown that cAMP is rapidly produced in lipid rafts following engagement of the TCR in primary T cells (3). This activates a pool of PKA type I targeted to rafts by association with the anchoring protein Ezrin, forming part of a supramolecular complex where Ezrin, EBP50, and PAG provide a scaffold that is able to coordinate PKA phosphorylation and activation of Csk, thereby inhibiting T-cell activation (44, 50). In addition, we have demonstrated that CD3/CD28 costimulation leads to recruitment of type 4 phosphodiesterase (PDE4) isoforms to rafts, resulting in degradation of the TCR-induced cAMP pool (3). Thus, we envisage that TCR-induced cAMP production constitutes a negative feedback loop capable of abrogating T-cell activation in the absence of a second signal. In order then to allow full T-cell activation to proceed, cAMP-mediated inhibition must be lifted. This appears to occur in the presence of a costimulus involving CD28 acting to trigger recruitment of PDE4 to lipid rafts, thereby degrading cAMP at this spatially critical location and resulting in an overriding positive feed-forward signal rather than the negative feedback loop activated from the TCR. In addition, a recent publication by Conche et al. has also found a possible stimulatory effect of cAMP, as the paper surprisingly showed that a transient cAMP increase shortly after TCR triggering may potentiate the calcium component of the TCR signaling. This could constitute a positive feed-forward in addition to the negative feedback signal by cAMP (12).Spatial organization and recruitment of mediators of specific pathways as outlined above are essential to ensure signaling specificity and amplification. Among the many protein scaffolds linking effector molecules into linear pathways, β-arrestins have been reported to confer cross talk with a growing list of molecules important in cellular trafficking and signal transduction, including Src family members and mitogen-activated protein (MAP) kinases (reviewed in reference 14). The arrestins were first identified as having a role in desensitization of G protein-coupled receptors (GPCRs) (9); later, they were discovered to be involved in receptor internalization by interacting with clathrin and AP-2, thereby bringing activated receptors to clathrin-coated pits for endocytosis (19, 26). A role for β-arrestin in the spatially localized degradation of cAMP by scaffolding PDE4 isoforms to the proximity of cAMP generation at the plasma membrane has also been suggested (3, 7, 30, 38).In the present study, we uncover a novel pathway that defines how T-cell costimulation elicits recruitment of PDE4 to lipid rafts to overcome cAMP-mediated inhibition of T-cell activation. This pathway is initiated by CD28 engagement leading to PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production and resulting in recruitment of a supramolecular complex of PKB/β-arrestin/PDE4 targeted to the plasma membrane due to sequestration via the PKB plextrin homology (PH) domain. Functionally, this pathway is essential for CD28 costimulation to strengthen and sustain T-cell immune responses.     

Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

Background  

The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH₄ prolactin producing cell line from rat, and primary cell culture from fish pituitaries.