PKCß phosphorylates PI3Kγ to activate it and release it from GPCR control.

All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCß phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²âº- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCß-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²âº and PKCß. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization.

The recognition of bacterial lipoproteins by toll-like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2-dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (myeloid differentiation primary response gene 88) and Mal/TIRAP (MyD88-adapter-like/TIR-domain-containing adaptor protein). TLR2-dependent, but MyD88-independent responses have not been described yet. We report here on a novel-signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3-kinase (PI3K), p85alpha, in an inducible fashion. The Mal-p85alpha interaction drives PI3K-dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP(3)) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP(3) formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP(3) generation and macrophage polarization.

Bicyclononane aldehydes and antiproliferative constituents from Amomum tsao-ko.

A racemic mixture of a new bicyclononane aldehyde, (1 RS,5 SR,6 RS)-5-hydroxybicyclo[4.3.0]non-2-ene-2-carbaldehyde (1) was isolated from the fruits of Amomum tsao-ko, together with 12 known compounds (2-13). The structure of 1 was determined on the basis of extensive spectroscopic analysis, including 1D and 2D NMR data. The antiproliferative activity of compounds 1-13 was assessed in the murine neuroblastoma cell line N2a.

Promotion of cell death or neurite outgrowth in PC-12 and N2a cells by the fungal alkaloid militarinone A depends on basal expression of p53.

The fungal alkaloid militarinone A (MiliA) was recently found to stimulate neuronal outgrowth in PC-12 cells by persistant activation of pathways that are also involved in NGF-mediated differentiation, namely the PI3-K/PKB and the MEK/ERK pathways. Application of equal concentrations of MiliA to other cells such as the murine neuroblastoma cell line N2a resulted in immediate onset of apoptosis by nuclear translocation of apoptosis inducing factor (AIF), activation of caspases and c-Jun/AP-1 transcription factor without an intermediate differentiated phenotype, although minor transient phosphorylation of PKB and MAPK as well as activation of NF-kappaB were also observed. Translocation of AIF was preceded by p53 phosphorylation at Ser15 and blocked by pifithrin alpha, a known inhibitor of p53-transcriptional activity. We here show that both cell types activate the same pathways albeit in different time scales. This is mainly due to contrasting basal expression levels of p53, which in turn regulates expression of AIF. In PC-12 cells, continuous activation of these pathways after prolonged treatment with 40 muM MiliA first led to up-regulation of p53, phosphorylation of p53, release of AIF from mitochondria and its translocation into the nucleus. Additionally, also activation of the c-Jun/AP-1 transcription factor was observed, and PC-12 cells subsequently underwent apoptosis 48-72 h post-treatment. We report that similar pathways working on different levels are able to initially shape very divergent cellular responses.

The strategies of the Theileria parasite: a new twist in host-pathogen interactions.

Theileria parasites infect and transform cells of the ruminant immune system. Continuous proliferation and survival of Theileria-transformed cells involves the well-orchestrated activation of several host-cell signalling pathways. Constitutive NF-kappa B (nuclear factor kappa B) activation is accomplished by recruiting the IKK (I kappa B kinase) complex, a central regulator of NF-kappa B pathways, to the surface of the transforming schizont, where it becomes permanently activated. Constitutive activation of the PI-3K-PKB [phosphoinositide 3-kinase-(Akt) protein kinase B] pathway is likely to be indirect and is essential for continuous proliferation. Theileria-transformed T cells express a range of anti-apoptotic proteins that can be expected to provide protection against apoptosis induced by death receptors, as well as cellular control mechanisms that are mobilised to eliminate cells that entered a cycle of uncontrolled proliferation.

Separation and detection of all phosphoinositide isomers by ESI-MS.

Phosphoinositides (PIs) play fundamental roles as signalling molecules in numerous cellular processes. Direct analysis of PIs is typically accomplished by metabolic labelling with (3)H-inositol or inorganic (32)P followed by deacylation, ion-exchange chromatography and flow scintillation detection. This analysis is laborious, time-consuming, and involves massive amounts of radioactivity. To overcome these limitations we established a robust, non-radioactive LC-ESI-MS assay for the separation and analysis of deacylated PIs that allows discrimination of all isomers without the need for radioactive labelling. We applied the method to various cell types to study the PI levels upon specific stimulation.

Regulation of secretases by all-trans-retinoic acid.

One of the emerging approaches for the treatment of Alzheimer's disease aims at reducing toxic levels of Alphabeta-species through the modulation of secretases, namely by inducing alpha-secretase or inhibiting beta-secretase and/or gamma-secretase activities, or a combination of both. Although there is increasing evidence for the involvement of retinoids in Alzheimer's disease, their significance in the regulation of Alphabeta-peptide production remains unresolved. Our work concentrated on the regulation of all secretases mediated by all-trans-retinoic acid (ATRA), and supports the hypothesis that ATRA is capable of regulating them in an antiamyloidogenic sense at the levels of transcription, translation, and activation. Apart from increased alpha-secretase activity, we show a complex chain of regulatory events, resulting in impaired beta-secretase trafficking and membrane localization upon protein kinase C (PKC) activation by ATRA. Furthermore, ATRA demonstrates substrate specificity for beta-site amyloid precursor protein-cleaving enzyme (BACE) 1 over nonamyloidogenic BACE2 in beta-secretase regulation, which probably promotes competition for amyloid precursor protein between ADAM17 and BACE1. Additionally, we report enhanced secretion of soluble amyloid precursor protein alpha after ATRA exposure, possibly due to PKC activation, as pretreatment with the PKC inhibitor Gö6976 abolished all these events.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis.

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

Hijacking of host cell IKK signalosomes by the transforming parasite Theileria.

Parasites have evolved a plethora of mechanisms to ensure their propagation and evade antagonistic host responses. The intracellular protozoan parasite Theileria is the only eukaryote known to induce uncontrolled host cell proliferation. Survival of Theileria-transformed leukocytes depends strictly on constitutive nuclear factor kappa B (NF-kappaB) activity. We found that this was mediated by recruitment of the multisubunit IkappaB kinase (IKK) into large, activated foci on the parasite surface. IKK signalosome assembly was specific for the transforming schizont stage of the parasite and was down-regulated upon differentiation into the nontransforming merozoite stage. Our findings provide insights into IKK activation and how pathogens subvert host-cell signaling pathways.