Sphingosine-1-phosphate induced mTOR-activation is mediated by the E3-ubiquitin ligase PAM

The signaling pathways that are regulated by sphingosine-1-phosphate (S1P) and mammalian target of rapamycin (mTOR) modulate cell growth, mitogenesis and apoptosis in various cell types and are of major interest for the development of new cancer therapeutics. Previous reports show that S1P can cross-activate the mTOR pathway although the mechanisms that connect both pathways are still unknown. We found that S1P-treatment activates mTOR in several cancer cell lines and primary cells. The activation was independent of ERK, Akt and PI3-kinase, but instead was mediated by the E3 ubiquitin ligase Protein Associated with Myc (PAM). Increased intracellular PAM concentrations facilitated S1P- and insulin-induced mTOR activation as well as p70S6K and 4EBP1 phosphorylation while genetic deletion of PAM decreased S1P- and insulin-induced mTOR activation. PAM activated by facilitating the GDP/GTP-exchange of Rheb which is an activator of mTOR. In conclusion we show that PAM is a novel regulator of the mTOR pathway and that PAM may directly activate Rheb as a guanosine exchange factor (GEF).     

Concurrent protective and destructive signaling of JNK2 in neuroblastoma cells

Investigation of the c-Jun N-terminal kinases (JNKs) has mainly focused on their response to stress and their pro-apoptotic effects. In this regard, JNKs are crucial mediators of chemotherapy-induced killing of tumor cells. Importantly, however, JNKs also have physiological functions in cancer involving cell cycle regulation or oncogenesis. Hypothetically, the composition of JNK signalosomes determines the signaling outcome which, in turn, implies a multitude of different, sometimes opposing and interfering functions. In the present study, the well-characterized human neuroblastoma cell line SH-SY5Y served as a model system to separate physiological and pro-apoptotic JNK actions in the response to the cytoskeleton-interfering substances colchicine, cytochalasin D and taxol. Basically, JNKs mediated both cell death and proliferation. Using the chemical JNK inhibitor SP600125 as well as compartment-specific JNK-inhibiting constructs and dominant-negative isoform mutants, we show that the nuclear subgroup of JNK2 is the dominant effector in colchicine- and taxol-induced apoptosis, while cell cycle promotion is mediated by both cytoplasmic and nuclear JNK2. In contrast, cytochalasin D-triggered apoptosis is independent of JNK signaling. Interestingly, the data of the present study demonstrate for the first time that both cell protective (cell cycle progression) and destructive mechanisms (apoptosis) are simultaneously controlled by a single JNK isoform in the same cell system even under the influence of one stimulus. This has implications for the therapeutic application of JNK inhibitors and cytoskeleton-interfering substances in oncologic disorders.     

Chemo-enzymatically induced copolymerization of phenolics with acrylate compounds

Initiation of copolymerization of lignin-like phenolic and acrylic compounds by the phenoloxidase laccase (EC 1.10.3.2) and a peroxide species (t-butylhydroperoxide, t-BHP) was compared to a Fenton-like system (ferrous ion, t-BHP). Initially, the relative activity of laccase towards different phenolic compounds and the optimum pH of some characteristic phenolics were determined. The polymer yield and the average molecular weight (Mw) of chemo-enzymatically produced polymers were dependent both on the type of each phenolic tested and on the phenol/monomer ratio. Furthermore, the success of copolymerization of the phenolics was dependent both on their redox potential and on the type of acrylic monomer applied. The extent of phenol incorporation into the polymer chain was enhanced by the presence of laccase in the reaction mixture and was significantly higher than in polymerization initiated by a Fenton-like reaction.     

P2X5 subunit assembly requires scaffolding by the second transmembrane domain and a conserved aspartate

Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.     

Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O.

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding Theta-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-beta-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the Theta-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.     

Identification of STN7/STN8 kinase targets reveals connections between electron transport,metabolism and gene expression

The thylakoid‐associated kinases STN7 and STN8 are involved in short‐ and long‐term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA‐binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin‐NADP reductase as targets for the thylakoid‐associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7‐dependent phosphorylation site are similar to those of phosphorylated light‐harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the ?1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long‐term adaptation processes affecting metabolite accumulation and gene expression.