The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The mass spectrometry data reported in this paper have be submitted to the Massive database under project ID MSV000084656. This short article contains Figs. cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation. (21). Thus, the association of IQGAP1 with CDC42 and RAC1 has biological relevance. Post-translational modifications increase the functional diversity of the proteome by the covalent addition of functional groups to proteins. Reported modifications on IQGAP1 include phosphorylation (22), acetylation (23), ISG (interferon-stimulated gene 15)-ylation (24, 25), SUMOylation (26), and ubiquitination (27,C30). Many of the studies that have recognized these post-translational modifications, used high throughput MS methods to identify proteome-wide modifications, with little subsequent functional analysis. Ubiquitination results from enzymatic linkage of the polypeptide Hesperidin ubiquitin to a lysine residue on target proteins. Following monoubiquitination, ubiquitin can itself be modified on any of its seven lysine residues or at its N terminus, leading to formation EBR2A of polymeric ubiquitin chains (31). The role of ubiquitination has been extensively Hesperidin analyzed in the ubiquitin proteasome system where substrate-linked ubiquitin provides a signal for proteasomal degradation of target proteins (32). In addition, ubiquitination regulates nonproteolytic processes, including protein activity, localization, and conversation with other proteins (33, 34). Ubiquitinated IQGAP1 peptides have been recognized in large-scale MS analyses of several cellular proteomes (27,C30). However, the sites found globally in these studies often reflect ubiquitination of newly synthetized proteins that are misfolded and therefore targeted for immediate degradation (27). We are not aware of any studies where a detailed characterization of IQGAP1 ubiquitination was carried out or where the functional effects of IQGAP1 ubiquitination was decided. In this study, we investigated ubiquitination of IQGAP1 and its functional effects. Using biochemical and MS analysis we demonstrate that IQGAP1 is usually ubiquitinated in HEK293 cells and identify the ubiquitination sites. Replacement of ubiquitinated lysine residues with arginine reduces ubiquitination of IQGAP1 in cells. In addition, mutation of the ubiquitinated lysines in the GRD increases the conversation of IQGAP1 with CDC42 and RAC1, and increases the amount of active CDC42 in cells. Results IQGAP1 is usually ubiquitinated in cells Ubiquitination of IQGAP1 was assessed in HEK293 cells. Cells were transfected with Myc-IQGAP1 and/or His-ubiquitin. Proteins were resolved by SDS-PAGE and the amount of ubiquitination was determined by Western blotting with anti-ubiquitin antibodies. Incubation with the proteasome inhibitor MG132 increases ubiquitination of numerous cellular proteins (Fig. 1and HEK293 cells were transfected with (+) or without (?) Myc-IQGAP1 and/or His-ubiquitin (HEK293 cells were transfected with (+) or without (?) His-ubiquitin and incubated with MG132 (+) or DMSO (?). His-ubiquitin was pulled-down (and and and HEK293 cells were transfected with (+) or without (?) His-ubiquitin (LC-MS/MS analysis of the in-gel tryptic digested samples allowed the identification of ubiquitinated lysine residues. A representative MS/MS spectrum of a ubiquitinated peptide is usually shown for peptide IQGAP1 1517C1532 made up of ubiquitinated Lys-1528. schematic representation of IQGAP1 showing the six ubiquitination sites recognized by LC-MS/MS and the tryptic peptides in which each is located. and and schematic representation of IQGAP1 mutant constructs. Individual lysine residues are replaced with arginine for each construct: CC-1K (Lys-556 is usually replaced with Arg), GRD-2K (K1155R; K1230R), RGCT-3K (K1465R; K1475R; K1528R), and 6K (K556R; K1155R; K1230R; K1465R; K1475R; K1528R). All plasmids contain a Myc-tag. equal amounts of protein lysate from HEK293 control (+/+) and IQGAP1-knockdown (?/?) cells generated with the CRISPR/Cas9 system were resolved by Western blotting. PVDF membranes were probed with anti-IQGAP1 and anti-actin (loading control) antibodies. A representative blot is usually shown. WT Myc-IQGAP1 was expressed in IQGAP1-knockdown HEK293 cells (?/?). Equivalent amounts of protein lysate from control (+/+) and knockdown cells were resolved by Western blotting using Hesperidin anti-IQGAP1, anti-Myc,.