SGEF GEF Activity and its Regulation by Scribble and Dlg1

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We discovered a cell polarity complex comprised of SGEF, a guanine nucleotide exchange factor (GEF) for the RhoG GTPase, and the scaffolding proteins Scribble and Dlg1. SGEF binds the Scribble PDZ1 domain and the guanylate kinase (GUK) domain of Dlg1. SGEF-catalyzed nucleotide exchange of RhoG is critical for cell junction polarity. Complications with cell polarity are associated with metastasis in cancer. However, the mechanism(s) for SGEF regulation and the role of Scribble and Dlg1 in this regulation remain unknown. Previous work by Ellerbroek et. al. (2004) suggests that the N-terminus of SGEF is auto-inhibitory to its GEF activity. We hypothesize that the N-terminus of SGEF blocks the GEF catalytic site from RhoG and that SGEF binding to Scribble and/or Dlg1 relieves this auto-inhibition. Here, we begin to test this model using in-vitro GEF exchange assays with SGEF constructs in the presence and absence of the Scribble PDZ1 domain and Dlg1 GUK. The SGEF catalytic domain (DH-PH domain) was expressed, the FL-SGEFΔ5, the Scribble PDZ1 and Dlg1 GUK domains. Next, we conducted GEF-exchange assays that measure the change in fluorescence upon the incorporation of MANT-GDP into RhoG. These assays showed that purified RhoG was active and that the SGEF DH-PH region increased the rate of nucleotide exchange. As a control, we used SGEF DH-PH in the presence of Scribble PDZ1 and Dlg1 GUK. Unexpectedly, the rate of RhoG nucleotide exchange increased further. A preliminary assay of the FL-SGEFΔ5 showed a marked decrease in the rate of nucleotide exchange compared to the DH-PH construct, consistent with auto-inhibition. Future experiments will focus on examining the nucleotide exchange rate of FL-SGEFΔ5 in the presence of Scribble PDZ1 and/or Dlg1 GUK. These studies will help understand the role of SGEF/Scribble/Dlg1-polarity-complex in regulating SGEF GEF activity.