Anti-RAPGEF2 (aa 1398-1487) polyclonal antibody

Integrin activation by Rap1-GTP is pivotal for lymphocyte trafficking. In this study, we show the phosphatidic acid (PA)-dependent membrane distribution of RA-GEF-1 and -2 (also known as Rapgef2 and 6), which are guanine nucleotide exchange factors for Rap1, plays important roles in lymphocyte migration. RA-GEF-1 associates with PA through 919-967 aa within CDC25 homology domain, and the deletion of this region of RA-GEF-1 inhibits chemokine-dependent migration. Chemokine stimulation induces temporal production of PA on the plasma membrane, which is not necessary for Rap1 activation, but the translocation of RA-GEFs. Thus, chemokine-dependent generation of PA is critical for lymphocyte migration through membrane localization of RA-GEFs. (C) 2020 Elsevier Inc. All rights reserved.

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study beta TrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCFFBXW11 E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11 bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.