Furthermore, HeLa cells also exhibit this active mechanism (our results). In short, ERK2 has an opposite effect on Sec16 from our proposed function for ERK7/MAPK15 (Supplementary Figure S8): (1) Growth factors stimulate Ras and ERK2 that directly phosphorylates Sec16 on T415. sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating Rabbit polyclonal to SCFD1 in the complex regulation of cell growth. and followed by its recruitment to ERESs, increased ERES number and anterograde ER to Golgi trafficking (Farhan et al, 2010). Although ERK2 is clearly important in human cells, our results show that it does not seem to have a role in S2 cells. This suggests that in S2 cells, inhibiting secretion when serum and/or amino acids are missing is not only a passive mechanism of not stimulating ERK2, but an active mechanism involving ERK7. Furthermore, HeLa cells also exhibit this active mechanism (our results). In short, ERK2 has an opposite effect on Sec16 from our proposed function for ERK7/MAPK15 (Supplementary Figure S8): (1) Growth factors stimulate Ras and ERK2 that directly phosphorylates Sec16 on T415. (2) This results in an increased mobility of Sec16 (Sec16 recruitment to ERES is increased, either from the cytosol or from the general ER membrane). (3) The number of ERES as well as the secretory capacity increase (Farhan et al, 2010). Conversely, (1) Amino-acid starvation stabilizes ERK7 in a TORC1-independent pathway. This induces Sec16 phosphorylation in a starvation responsive domain’-dependent manner. (2) This results in Sec16 release from the tER sites leading to (3) tER site disassembly and ERCplasma membrane transport inhibition, thus negatively regulating cell growth. Taken together, our results point towards ERK7 as a novel mediator of nutrient availability in the control of secretion DL-Methionine and provide a framework for a better mechanistic understanding of the regulation of protein secretion and cell growth as DL-Methionine a response to environmental stimuli. Materials and methods Cell lines and culture conditions, primary and validation screen, antibodies, imaging data analysis and quantitation of cell proliferation, cell-cycle distribution by flow cytometry and statistical analysis The reagents, cell lines and RNAi screen design, data acquisition and analysis are described in detail in Supplementary data and Kondylis et al, 2011. dsRNA designing for the characterization of selected hits The dsRNAs used for the characterization of selected screen hits were independently designed and each probe was evaluated for its efficiency and potential off-target effects on the website http://e-rnai.dkfz.de. Only probes with 100% specificity for the targeted gene were used. The primers and dsRNA sizes of each targeted gene are mentioned DL-Methionine in Supplementary Table S3. Cloning/Sec16 truncations To test the subcellular localization of selected hits, the full-length coding sequences were amplified by PCR and cloned into pMT/V5-HisA, B, C vectors (Invitrogen). The expressed proteins were C-terminally tagged. The primers and restriction sites used to clone each gene are mentioned in Supplementary Table S4. Wallenda K188A, CG32703/ERK7 K54R and T195A/Y197F mutants were created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and confirmed by sequencing (Supplementary Table S4). For expression in HeLa cells, Wallenda-V5 was subcloned into pcDNA3.1 vector (Invitrogen). Sec16 truncations were cloned in pMT/V5-HisB or pRmeGFP (Farkas et al, 2004) using the primers and restriction sites mentioned in Supplementary Table S4. Transient transfections S2 cells were transiently transfected for 2 days in 35 mm dish as previously described (Kondylis et al, 2005). The expression DL-Methionine of each tagged protein was induced for 2 h with CuSO4 followed by a 2-h chase, sometimes in the presence of cycloheximide. HeLa cells were transfected with Effectene (Qiagen) for 24 h as described by the manufacture then fixed in 4% PFA for immunofluorescence. Immunoelectron microscopy S2 cells were fixed and.