Title Extracellular Signal-Regulated Kinase Regulates RhoA Activation and Tumor Cell Plasticity by Inhibiting Guanine Exchange Factor H 1 Activity

27 In certain Ras mutant cell lines the inhibition of ERK signalling increases RhoA 28 activity and inhibits cell motility, which was attributed to a decrease in Fra-1 29 levels. Here, we report a Fra-1 independent augmentation of RhoA signalling 30 during short-term inhibition of ERK signalling. Using mass spectrometry based 31 proteomics we identified the Rho exchange factor GEF-H1 as mediating this 32 effect. ERK binds to GEF-H1 and phosphorylates it on S959 causing inhibition 33 of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown 34 experiments and expression of a non-phosphorylatable S959A GEF-H1 mutant 35 showed that this site is crucial in regulating cell motility and invasiveness. Thus, 36 we identified GEF-H1 as a critical ERK effector that regulates motility, cell 37 morphology and invasiveness. 38