14 research outputs found
Shoc2/Sur8 protein regulates neurite outgrowth
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Creative Commons Attribution License.-- et al.The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.GL, TG and LMD were recipients of fellowships from the Ministerio de Educación y Ciencia (MEC) (to GL, TG), and Fondo de Investigaciones Sanitarias (FIS) (to LMD). LSR
held a postdoctoral research contract from CIBERNED. This work was supported by FIS grant
(PI10/00815) to JLO; CIBERNED to MC; SAF2008-01951, Comunidad Autónoma de Madrid (CAM) SSAL-0202-2006-01 and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) to TI; FIS grant PI12/00775 and ISCIII-RETIC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0027 from the Instituto de Salud Carlos III to PSG; and FIS grants (PI09/0562 and PI13/00703), ISCIIIRETIC
(RD06/0020/0003 and RD12/0036/0021), and the Spanish Association Against Cancer
(AECC) to JMR.Peer Reviewe
Shoc2/Sur8 Protein Regulates Neurite Outgrowth
The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.This work was supported by FIS grant (PI10/00815) to JLO; CIBERNED to MC; SAF2008-01951, Comunidad Autónoma de Madrid (CAM) S-SAL-0202-2006-01 and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) to TI; FIS grant PI12/00775 and ISCIII-RETIC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0027 from the Instituto de Salud Carlos III to PSG; and FIS grants (PI09/0562 and PI13/00703), ISCIII-RETIC (RD06/0020/0003 and RD12/0036/0021), and the Spanish Association Against Cancer (AECC) to JMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S
SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues
Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to
genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility
and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component.
Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci
(eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene),
including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform
genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer
SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the
diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types
Shoc2 depletion reduces phospho-ERK and phospho-MEK levels induced by RTK activation.
<p>HEK293T cells were transiently transfected (100 nM) with either control siRNA (siRNA-Control) or human Shoc2-specific siRNA (siRNA-Shoc2). At 48 h post-transfection, cells were serum-starved for 18 h and then incubated with vehicle (0), or 5-25 ng/ml FGF for 10 min. (<b>A</b>), endogenous Shoc2 expression was assessed, from the cell lysates, by immunoblot using anti-Shoc2 rabbit polyclonal antibody and normalized to tubulin levels, expressed as a percentage (corresponding to the mean of five separate assays, in all cases with a SD ≤10% of the mean). The specific effect of the siRNA-Shoc2 was tested throughout detection of Grb2, Ras, C-Raf, ERK and MEK protein levels, by immunoblot of the cell lysates with the corresponding antibodies. Cell lysates were also analyzed by immunoblot using anti-p-ERK/ERK, and anti-p-MEK/MEK antibodies (bottom panels); in each case, fold increase denotes the ratio p-ERK/ERK, and p-MEK/MEK levels estimated as the mean of five separate assays (in all cases with a SD ≤10% of the mean). (<b>B</b>), the expression of Shoc2 was assessed by qRT-PCR. Data represents the mean ± SD of four independent experiments. The detection of GAPDH (normalization control) was performed using specific primers. Results were calculated by the 2-<sup>ΔΔCT</sup> method. *P≤0.001 compared with siRNA-control; bars show SD.</p
Shoc2 knockdown reduces NGF-elicited of PC12 cell differentiation.
<p>A), PC12 cells were nucleofected with shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP plasmids (ShRNA Shoc2 #1-3). At 48 h post-transfection, cell lysates were analyzed by immunoblot. B), Shoc2 levels determined using anti-Shoc2 polyclonal antibody were normalized to total ERK levels, and expressed as a percentage (right). Results were similar in two additional experiments. *P≤0.001 and n.s. (not significant) compared with siRNA-control; bars show SD. C), PC12 cells nucleofected with either shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP plasmids (ShRNA Shoc2 (2) and (3)) were stimulated with 100 ng/ml NGF for 72 h. After fixing, GFP-positive cells were detected by fluorescence microscopy (right: arrows) and total cells were visualized with transmitted light (left). Extending processes equal in length or greater than two cell bodies were considered neurites; bars, 10 µm. D), histograms represent the percentage of transfected PC12 cells (GFP-positive) with neurites after NGF treatment; values are the mean of three separate assays, in which at least 50 GFP-positive cells/assay were analyzed (*ShRNA Shoc2 (2 or 3) vs ShRNA Control; P<0.01), bars show SD.</p
Shoc2 knockdown reduces NGF-induced ERK activation in PC12 cells.
<p>PC12 cells nucleofected with shRNA-control-GFP plasmid (ShRNA Control) or specific rat shRNA-Shoc2-GFP #3 plasmid (ShRNA Shoc2) were stimulated with 100 ng/ml NGF for the times indicated. In each assay, 2 µg PC12 cell extracts were analyzed by xMAP technology in a Luminex-200 platform for quantitative and simultaneous detection of phospho-ERK (pTpY183/185) and total ERK protein (Panel <b>A</b>), phospho-AKT and total AKT (Panel <b>B</b>) or phospho-GSK3 and total GSK3 protein (Panel <b>C</b>). Histograms show the ratio of p-ERK/ERK (Panel <b>A</b>), p-AKT/AKT (Panel <b>B</b>) or p-GSK3/GSK3 levels (Panel <b>C</b>) after the indicated time of NGF stimulation; values are the mean of three separate assays performed in duplicate (*P≤0.001), bars show SD.</p
Shoc2 overexpression increases EGF-induced PC12 differentiation.
<p><b>A</b>), PC12 cells were co-transfected with plasmids pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5 (0.7 µg) (top: GFP + EGF) or pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5-hShoc2 (0.7 µg) (bottom: Shoc2-GFP + EGF). Transfected PC12 cells were stimulated with EGF (100 ng/ml) for 72 h, fixed, and the presence of extending neurites was analyzed in GFP-positive cells by fluorescence microscopy (right: white arrows). Total cells were visualized with transmitted-light (left). A process equal in length or greater than one cell body was considered a neurite; bars, 10 µm. PC12 cells co-transfected with pCEFL-KZ-GFP and pCEFL-KZ-AU5-hShoc2 but not stimulated with EGF or NGF did not have neurites (not shown). <b>B</b>), PC12 transfected cells (as in A) were stimulated with EGF (100 ng/ml) for 72 h, fixed. Cells were incubated with anti-AU5 monoclonal antibody for 1h at room temperature and, after several washes, were incubated with Alexa 488-conjugated secondary antibodies. The presence of extending neurites was analyzed with transmitted-light (left-picture). PC12 cells overexpressing exogenous Shoc2 were detected as AU5-positive cells by fluorescence microscopy (right-picture and white arrows). A process equal to or greater than one cell body in length was considered a neurite; bars, 10 µm. <b>C</b>), histograms represent the percentage of transfected PC12 cells (GFP-positive: GFP vs Shoc2-GFP) with neurites after EGF treatment of panel A; values are the mean of three separate assays, in which at least 50 GFP-positive cells/assay were analyzed (*P<0.01); bars show SD.</p
Shoc2 overexpression enhances the intensity and/or duration of RTK-elicited ERK activation.
<p>HEK293T cells transiently co-transfected with 1 µg pCEFL-KZ-HA-ERK1 and 2 µg pCEFL-KZ-AU5-hShoc2 (S) or 2 µg pCEFL-KZ-AU5 (-) were serum-starved for 18 h and then incubated with vehicle (0), 25 ng/ml FGF (<b>A</b>), or 10 ng/ml EGF (<b>B</b>), for 5 to 240 min. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by immunoblot using anti-p-ERK and -HA antibodies, to determine p-ERK1 and total HA-ERK1. Fold increase denotes the ratio p-ERK/ERK levels estimated as the mean of three separate assays (in all cases with a SD ≤10% of the mean). Expression levels of transfected AU5-hShoc2 constructs were detected by immunoblotting whole cell extracts (WCL) with the appropriate anti-AU5 antibody, and total Shoc2 expression was assessed by re-blotting of the same filters against anti-Shoc2 rabbit polyclonal antibody and using tubulin detection as control. In each point-time, the protein levels of Shoc2 were normalized versus tubulin amount and vector sample (-). Results were similar in three independent experiments.</p