Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: EGFR activation leads to phosphorylation of LARG at S1288 through RSK . A , schematic representation of of the LARG protein showing the functional domains: PDZ, RH, DH, PH, and CC. These domains coordinate LARG activation, RhoA interaction and localization. B , posttranslational modifications determined by Mass Spec are shown as curated by PhosphoSitePlus ( www.phosphosite.com ). The height of the lines relates to the number of reports in which that modification was identified. C , Western blot analysis of LARG phosphorylation from U87 MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV for 48h. Cells were starved for 24h, then treated with EGF (100 ng/ml) for 10 min. Cell lysates were immunoprecipitated with a Myc antibody, and LARG phosphorylation was detected using a phospho-AKT substrate antibody. LARG phosphorylation was quantified relative to Myc tag expression ( right ). D , starved cells were subjected to RSK inhibitors B-ID1780 or PMD-026 for 24h or 4h before treatment with 100 ng/ml EGF for 10 min. LARG, RSK and YB1 phosphorylation levels were quantified as fold changes ( right ). Results are representative of three independent experiments, each of which was done in triplicate and presented as the mean ± standard deviation. ∗ p ˂ 0.05.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Functional Assay, Mass Spectrometry, Modification, Western Blot, Transfection, Immunoprecipitation, Expressing, Standard Deviation