Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1

Reevaluation of Lte1’s involvement in the mitotic exit network reveals that it is involved in localizing Bfa1 to the spindle pole body but does not function as a GEF for Tem1

HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications

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Binding of Kif23-iso1/CHO1 to 14-3-3 Is Regulated by Sequential Phosphorylations at Two LATS Kinase Consensus Sites

<div><p>Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases <i>in vitro</i>, and this requires the presence of an upstream -5 histidine residue. We further show that these sites are phosphorylated <i>in vivo</i> and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.</p></div

<i>In vivo</i> phosphorylation of S716 and S814 on endogenous and exogenous Kif23.

<p>A. Detection of S814 phosphorylation by Western blot on whole cell extracts (WCE) of HeLa cells treated or not with Kif23 siRNA (left) or WCE of HEK293T cells transfected with WT and mutated Flag-tagged Kif23-iso1 (right). B. Detection of S716 phosphorylation on material immunoprecipitated with anti-Kif23 antibody from HeLa cells (left) or HEK293T cells transfected with WT and mutated GFP-tagged Kif23-iso1 (right).</p

pS814–Kif23 levels are reduced after depletion of LATS1,2.

<p>A. Unsynchronised HeLa cells were treated with LATS1,2 siRNAs (set1, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117857#sec002" target="_blank">Materials and methods</a>) for 72 hrs and analysed for pS814–Kif23 levels. Depletion of Lats1 was monitored by Western blot and that of LATS2 by RT-PCR (right panel). Ability of LATS2 siRNA (set 1) to deplete LATS2 protein was verified indirectly on exogenous myc-LATS2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117857#pone.0117857.s006" target="_blank">S6 Fig</a>.). B. Quantification of pS814 levels corrected for the amounts of Kif23 (pS814/Kif23) from A and three other similar experiments.</p

Midbody remnants show low level of Kif23 phosphorylation at S814.

<p>A, B. Micrographs of HeLa cells stained with DAPI and antibodies against pS814, Kif23 and tubulin, and representative of cytokinetic MBs (A) or MB^Rs (B). Yellow arrows point to cytokinetic MBs, while red and green arrows point to MB^Rs with low and high pS814 relative staining, respectively. Scale bar: 15 μM. C. Relative phosphorylation at S814 (pS814/Kif23 ratio) was calculated on individual midbodies either engaged in late cytokinesis (MB) or not (MB^R), and represented as box and whiskers plots. Outliers represent values found outside the 2.5 to 97.5 percentile range. The red horizontal line represents the threshold ratio value above which lie 90% of cytokinetic midbodies. D. Bars represent the percentage of pS814/Kif23 ratio values that are higher than the threshold value shown in C and counted as pS814 positive.</p

Analysis of pS716 and pS814 levels on WT and mutant Kif23 in HEK293T cells.

<p>A. WT and mutant Flag-tagged Kif23-iso1 were expressed in HEK293T cells and analysed for S814 phosphorylation by Western blot. B. Histograms of pS814/Kif23 ratios calculated from A and three other experiments. ND: not detected; NS: not significant. P values refer to comparisons between WT and specified mutant Kif23. C. WT and mutant GFP-tagged Kif23-iso1 were expressed in HEK293T cells, immunoprecipitated with anti-Kif23 antibody and analysed for S716 phosphorylation.</p

ERK5 Knockdown generates mouse leukemia cells with low MHC class i levels that activate NK cells and block tumorigenesis.

International audienceTumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine

Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity

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