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In brief: February 2007Lipids Membrane phosphatidylserine regulates surface charge and protein localization The contribution of the anionic phospholipid phosphatidylserine (PtdSer) to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents for analysis of its distribution in intact cells. Yeung et al. have now developed a biosensor to study the subcellular distribution of PtdSer and have found that it binds the cytosolic leaflets of the plasma membrane, and of endosomes and lysosomes. The presence of anionic PtdSer directed proteins with moderately positive charges to the endocytic pathway. The presence of pools of PtdSer on endosomes and lysosomes implies that these compartments can dock proteins with PtdSer-binding domains, which include important signalling and fusogenic effectors. Cell proliferation c-IAP1 cooperates with Myc by acting as a ubiquitin ligase for Mad1 The mechanism by which the E3 ubiquitin ligase c-IAP1 (a member of the inhibitor of apoptosis protein (IAP) family) promotes cell proliferation and tumorigenesis is poorly understood. A team led by Junying Yuan now shows that c-IAP1 catalyses the ubiquitylation of MAX-dimerization protein-1 (MAD1), an important cellular antagonist of the tumour-promoting factor MYC. Once ubiquitylated, MAD1 is targeted for degradation by the 26S proteasome pathway and the repression of its target genes is released. Knockdown of c-IAP1 decreases the rate of cell proliferation and oncogenic phenotypes, whereas knockdown of MAD1 promotes cell proliferation and transformation. The authors suggest that inhibition of the E3 ligase activity of c-IAP1 and stabilization of the MYC antagonist MAD1 could provide a powerful approach towards cancer treatment. Inflammation Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis A subpopulation of patients with Crohn's disease have mutations in the gene encoding nucleotide-binding oligomerization domain 2 (NOD2). One potential mechanism to explain this association is that NOD2 negatively regulates Toll-like receptor 2 (TLR2) signalling and that loss-of-function mutations in NOD2 lead to an increased inflammatory response to the intestinal microflora through TLR2. In support of this model, this study reports that pre-stimulation of wild-type mice, but not of those with mutated NOD2, with bacterial muramyl dipeptide can protect against chemically induced colitis. The protective effect was associated with the suppression of pro-inflammatory cytokine production through TLR3, TLR4, TLR5 and TLR9 pathways, in addition to the TLR2 pathway, as a result of the induction of interferon-regulatory factor 4 (IRF4), which is a negative regulator of TLR signalling. T-cell activation A scaffold protein, AHNAK1, is required for calcium signaling during T cell activation Calcium signalling has a crucial role in T-cell activation and proliferation, and the calcium response involves the influx of calcium through protein channels in the plasma membrane. A role for the scaffold protein AHNAK1 in calcium signalling has been previously suggested and the expression of AHNAK1 is linked with cell proliferation. This study shows that AHNAK1 is expressed by peripheral T cells and that proliferation and interleukin-2 production following T-cell receptor (TCR) stimulation is impaired in AHNAK1-deficient CD4+ T cells. In addition, AHNAK1 is required for TCR-induced calcium signalling in CD4+ T cells through its regulation of the expression at the plasma membrane of the L-type calcium 1 channels. So, this study identifies AHNAK1 as a new factor in T-cell calcium signalling. Mouse models TORC1 is essential for NF1-associated malignancies The authors show that the mTOR inhibitor rapamycin suppresses tumour growth in a mouse model of neurofibromatosis type 1 (NF1). Unlike in xenograft models, the microvasculature initially withstood rapamycin treatment, suggesting that rapamycin was instead acting in a cell-autonomous way. Interestingly, rapamycin did not affect either hypoxia-inducible factor 1 Tumour suppressors NUMB controls p53 tumour suppressor activity NUMB, a protein involved in cell-fate determination, has been identified as a candidate breast cancer tumour-suppressor gene. In addition to its regulation of the receptor NOTCH, this paper shows that NUMB can interact with the tumour suppressor p53 and its ubiquitin ligase MDM2, preventing degradation of p53 and stabilizing p53 levels. Loss of NUMB in breast cancer cells results in reduced levels of p53 and increased resistance to chemotherapy. Moreover, loss of NUMB increases NOTCH activity, producing an oncogenic signal. These biological changes might explain the poor prognosis seen in patients with breast cancers that have lost NUMB expression. Repair Targeting axon growth from neuronal transplants along preformed guidance pathways in the adult CNS Neuronal transplants need guidance factors to direct axon growth. The authors delivered such factors using viral vectors injected along the relevant growth pathway 1 week before transplantation of neurons into the corpus callosum. Pathways expressing fibroblast growth factor 2 (FGF2) with or without nerve growth factor (NGF) induced axon growth into the NGF-expressing target in the contralateral hemisphere. Expression of the chemorepulsant semaphorin 3A adjacent to the pathway's turning point improved axon turning. These findings might be used in the development of future transplantation paradigms. Local protein synthesis Intra-axonal translation and retrograde trafficking of CREB promotes neuronal survival Axons of developing neurons contain both mRNAs and the machinery required for protein translation; however, the role of axonally translated mRNAs is in many cases unclear. Here the authors showed that, in dorsal root ganglion neurons, nerve growth factor (NGF) stimulates the translation of cAMP-responsive-element-binding protein (CREB) in axons and its retrograde transport to the soma. By knocking down axonal CREB mRNA, the authors showed that this signalling function is required for the promotion of neuronal survival by NGF. Kinase inhibitors Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling
A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases
There are few studies describing the selectivity of kinase inhibitors across panels of diverse kinases and/or cancer cell lines. McDermott and colleagues established a high-throughput screening platform to profile 500 cell lines derived from diverse epithelial cancers for sensitivity to 14 kinase inhibitors. Although most cell lines recapitulated known tumour-associated genotypes, the screen revealed low-frequency drug-sensitizing genotypes in tumour types not previously associated with drug susceptibility. Furthermore, comparison of drugs that were thought to target the same kinase revealed unsuspected functional relationships. Fedorov and colleagues evaluated the specificity of 156 kinase inhibitors against 60 human serine/threonine kinases using a thermal stability shift assay. The analysis revealed many unexpected cross-reactivities for inhibitors that were thought to be specific for certain targets. For example, a PKC Cell biology Separating cells by cell-cycle phase
Methods for separating cells according to cell-cycle phase are important for many biotechnology applications, but gentle ways to do so without introducing cell stress are lacking. Kim et al. describe a dielectrophoresis-based microfluidic device that gently separates mammalian cells by taking advantage of the relationship between cell-cycle phase and the cell volume—G2/M phase cells are much larger than G1/S phase cells. Biophysics Dynamic protein interactions in live cells
Understanding signaling pathways requires analysis of their dynamic and interacting protein components. Fluorescence fluctuation spectroscopy methods can be used for such studies, but have not been extensively applied to live cells. In recent work, Slaughter et al. used such fluctuation techniques, along with imaging, to study the diffusion dynamics, stoichiometries and interactions between components of the MAP kinase cascade in living yeast. Structural biology Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase Type three secretion systems (T3SSs) are used by pathogenic bacteria to deliver effectors directly into host cells. Some T3SS effectors, including OspF (Shigella spp.), SpvC (Salmonella spp.) and HopA1 (Pseudomonas syringae), irreversibly inactivate plant and animal host mitogen-activated protein kinase (MAPK) pathways to downregulate host innate immune responses. MAPKs are activated by dual phosphorylation of a threonine-variable-tyrosine (T-X-Y) motif (present in an activation loop) to yield pT-X-pY. Using mass spectrometry, Zhu et al. now reveal that OspF and SpvC are phosphothreonine lyases. Analysis of a crystal structure of the complex formed by SpvC with a phosphopeptide substrate, together with kinetic studies to determine the catalytic activity of SpvC mutants, enabled them to propose an acid/base mechanism for a | |
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protein levels or Akt phosphorylation, indicating that in this tumour type the effect of rapamycin is mediated by TORC1 rather than TORC2.
inhibitor also inhibited PIM1 kinase, a suggested target for leukaemia treatment; indeed, this inhibitor suppressed growth of leukaemic cells. These studies reveal additional tumour types that could respond to established cancer therapies.