Marius Ueffing

Latent membrane protein 1 of Epstein–Barr virus mimics a constitutively active receptor molecule

Latent membrane protein 1 (LMP1) of Epstein–Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B‐cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF‐R) ‐associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF‐κB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF‐R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to β‐adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy‐terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild‐type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF‐2 receptor. In EBV, immortalized B cells cross‐linking in conjunction with membrane targeting of the carboxy‐terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand‐independent.

Latent membrane protein 1 of Epstein-Barr virus interacts with JAK3 and activates STAT proteins.

Latent membrane protein 1 (LMP1) acts like a permanently activated receptor of the tumor necrosis factor (TNF)‐receptor superfamily and is absolutely required for B cell immortalization by Epstein–Barr virus. Molecular and biochemical approaches demonstrated that LMP1 usurps cellular signaling pathways resulting in the induction of NF‐κB and AP‐1 via two C‐terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of LMP1s C‐terminus is required for the activation of Janus kinase 3 (JAK3). The interaction of LMP1 and JAK3 leads to the enhanced tyrosine auto/transphosphorylation of JAK3 within minutes after crosslinking of a conditional NGF‐R:LMP1 chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the LMP1 C‐terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.

Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-κB

Reactive oxygen species (ROS) play an important role in cell death induced by many different stimuli. This study shows that hydrogen peroxide-induced apoptosis in T-cells did not require tyrosine kinase p56lck, phosphatase CD45, the CD95 receptor and its associated Caspase-8. H2O2-triggered cell death led to the induced cleavage and activation of Caspase-3. Hydrogen peroxide-treatment of T-cells resulted in the formation of mitochondrial permeability transition pores, a rapid decrease of the mitochondrial transmembrane potential ΔΨm and the release of Cytochrome C. Inhibition of the mitochondrial permeability transition by bongkrekic acid (BA), or interference with the mitochondrial electron transport system by rotenone or menadione prevented the cytotoxic effect of H2O2. Antimycin A, a mitochondrial inhibitor that increases the release of mitochondrial ROS (MiROS), enhanced apoptosis. Overexpression of Bcl-2 and the viral anti-apoptotic proteins BHRF-1 and E1B 19K counteracted H2O2-induced apoptosis. Pharmacological and genetic inhibition of transcription factor NF-κB protected cells from hydrogen peroxide-elicited cell death. This detrimental effect of NF-κB mediating hydrogen peroxide-induced cell death presumably relies on the induced expression of death effector genes such as p53, which was NF-κB-dependently upregulated in the presence of H2O2.

Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade.

The Epstein–Barr virus latent membrane protein‐1 (LMP‐1) is an integral membrane protein which transforms fibroblasts and is essential for EBV‐mediated B‐cell immortalization. LMP‐1 has been shown to trigger cellular NF‐κB activity which, however, cannot fully explain the oncogenic potential of LMP‐1. Here we show that LMP‐1 induces the activity of the AP‐1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP‐1 effects on AP‐1 are mediated through activation of the c‐Jun N‐terminal kinase (JNK) cascade, but not the extracellular signal‐regulated kinase (Erk) pathway. Consequently, LMP‐1 triggers the activity of the c‐Jun N‐terminal transactivation domain which is known to be activated upon JNK‐mediated phosphorylation. Deletion analysis indicates that the 55 C‐terminal amino acids of the LMP‐1 molecule, but not its TRAF interaction domain, are essential for AP‐1 activation. JNK‐mediated transcriptional activation of AP‐1 is the direct output of LMP‐1‐triggered signaling, as shown by an inducible LMP‐1 mutant. Using a tetracycline‐regulated LMP‐1 allele, we demonstrate that JNK is also an effector of non‐cytotoxic LMP‐1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP‐1, the SEK/JNK/c‐Jun/AP‐1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP‐1.

MECHANISM OF INHIBITION OF RAF-1 BY PROTEIN KINASE A

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Integrative Analysis of the Mitochondrial Proteome in Yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the single most common cause of inherited Parkinsons disease. Little is known about its involvement in the pathogenesis of Parkinsons disease mainly because of the lack of knowledge about the physiological role of LRRK2. To determine the function of LRRK2, we studied the impact of short hairpin RNA-mediated silencing of LRRK2 expression in cortical neurons. Paired recording indicated that LRRK2 silencing affects evoked postsynaptic currents. Furthermore, LRRK2 silencing induces at the presynaptic site a redistribution of vesicles within the bouton, altered recycling dynamics, and increased vesicle kinetics. Accordingly, LRRK2 protein is present in the synaptosomal compartment of cortical neurons in which it interacts with several proteins involved in vesicular recycling. Our results suggest that LRRK2 modulates synaptic vesicle trafficking and distribution in neurons and in consequence participates in regulating the dynamics between vesicle pools inside the presynaptic bouton.

The Parkinson disease-associated protein kinase LRRK2 exhibits MAPKKK activity and phosphorylates MKK3/6 and MKK4/7, in vitro

Autosomal dominant mutations in the human Leucine‐Rich Repeat Kinase 2 (LRRK2) gene represent the most common monogenetic cause of Parkinson disease (PD) and increased kinase activity observed in pathogenic mutants of LRRK2 is most likely causative for PD‐associated neurotoxicity. The sequence of the LRRK2 kinase domain shows similarity to MAP kinase kinase kinases. Furthermore, LRRK2 shares highest sequence homology with mixed linage kinases which act upstream of canonical MAPKK and are involved in cellular stress responses. Therefore, we addressed the question if LRRK2 exhibits MAPKKK activity by systematically testing MAPKKs as candidate substrates, in vitro. We demonstrate that LRRK2 variants phosphorylate mitogen‐activated protein kinase kinases (MAPKK), including MKK3 ‐4, ‐6 and ‐7. MKKs act upstream of the MAPK p38 and JNK mediating oxidative cell stress, neurotoxicity and apoptosis. The disease‐associated LRRK2 G2019S and I2020T mutations show an increased phosphotransferase activity towards MKKs correlating with the activity shown for its autophosphorylation. Our findings present evidence of a new class of molecular targets for mutant LRRK2 that link to neurotoxicity, cellular stress, cytoskeletal dynamics and vesicular transport.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.