Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6

Abstract

The binding of integrin adhesion receptors to their extracellular matrix ligands controls cell morphology, movement, survival, and differentiation in various developmental, homeostatic, and disease processes. Here, we report a methodology to isolate complexes associated with integrin adhesion receptors, which, like other receptor-associated signaling complexes, have been refractory to proteomic analysis. Quantitative, comparative analyses of the proteomes of two receptor-ligand pairs, α 4 β 1 -VCAM-1 and α 5 β 1 -fibronectin, defined both core and receptorspecific components. Regulator of chromosome condensation-2 (RCC2) was detected in the α 5 β 1 -fibronectin signaling network at an intersection between the Rac1 and Arf6 sub-networks. RCC2 knockdown enhanced fibronectin-induced activation of both Rac1 and Arf6 and accelerated cell spreading, suggesting that RCC2 limits the signaling required for membrane protrusion and delivery. Dysregulation of Rac1 and Arf6 function by RCC2 knockdown also abolished persistent migration along fibronectin fibers, indicating a functional role for RCC2 in directional cell movement. This proteomics workflow now opens the way to further dissection and systems-level analyses of adhesion signaling.

Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells

“…Therefore, to address the mechanisms for how and to what extent the kindlins compensate for each other requires development of new molecular tools and approaches, such as quantitative proteomics to elucidate the kinetics of kindlin-containing protein complexes in keratinocytes. 23 However, it became obvious that kindlin-1 and -2 also have distinct dominant functions. Although both kindlins had similar effects on cell morphology and size, kindlin-2 deficiency seemed to affect formation of cell-cell junctions and FAs in a more drastic manner, as demonstrated by diffuse cytoplasmic distribution of ␤ 1 integrin, ␤-catenin, desmoplakin, and talin in K2 Ϫ and K1 Ϫ K2 Ϫ cells, instead of a membrane-associated localization.…”

Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

“…Searches of CID MS2 and MS3 data both specified matches to y and b ions. Results then were analyzed using the program Scaffold (V3_00_08, Proteome Software, Portland, OR) (45,46) with minimum peptide and protein probabilities of 95 and 99% being used. IGFBP-5 peptides from MS3 results were sorted by scan number, and cysteine containing peptides were identified from groups of MS3 scans produced from the 5 most intense ions observed in ETD MS2 scans.…”

Selenoprotein K Is a Novel Target of m-Calpain, and Cleavage Is Regulated by Toll-like Receptor-induced Calpastatin in Macrophages

“…HEK293 cells were transfected with this plasmid or empty control plasmid using Lipofectamine 2000 (Invitrogen) for 24 h before lysis and analysis for protein expression. Associations between calpastatin, m-calpain and SelK were detected by a ligand affinity purification assay method (18,19). Anti-m-calpain antibody (10 g) was cross-linked to Dynabeads using 5 mM Bis (sulfosuccinimidyl) suberate (Thermo Fisher), and beads were incubated with BMDM at 70 rpm for 30 min at 37°C.…”