Supervillin binds the Rac/Rho‐GEF Trio and increases Trio‐mediated Rac1 activation

Abstract

We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4–7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276 – 669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4–7, amino acids 531 – 1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.

Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway

“…4a , and Additional file 1 : Figure S4A and B). As has been described previously in HeLa cervical carcinoma cells, supervillin knockdown with RNAi #2 or #3 decreased Rac1 loading in normoxia [ 41 ]. During hypoxia, knockdown of supervillin with RNAi #2 or RNAi #3 was accompanied by a significant reduction in the amount of GTP-loaded (active) RhoA, as assessed by pulldown assays with GST-tagged Rho-binding domain (RBD).…”

“…GTP loading assays were carried out as described [ 41 ]. Briefly, cells were extracted in lysis buffer at 4 °C and centrifuged for 10 min at 14000 g. Supernatants were incubated with GST-RBD or GST-PBD pre-bound to glutathione-Sepharose beads for 1 h at 4 °C.…”

“…Cancer cells contain at least three supervillin isoforms (SV1, SV4, and SV5) [ 32 – 36 ]. These proteins are involved in actin filament assembly, cell spreading and lamellipodia extension, and focal adhesion maturation and/or disassembly [ 37 – 41 ]. Supervillin is also a component of invadosomes, where it regulates the invadosome half-life and matrix degradation, and enhances the secretion of matrix metalloproteinases (MMPs) [ 29 – 31 ].…”

Trio family proteins as regulators of cell migration and morphogenesis in development and disease – mechanisms and cellular contexts

“…One interesting candidate is Supervillin (SVIL) isoform 4 (Supervillin4), which links the actin cytoskeleton to the plasma membrane. Trio employs its SR6 and SR7 to bind Supervillin4 directly (Son et al, 2015). Supervillin4 expression in HeLa cells induces Rac1 activation, and depletion of Trio prevents this, suggesting Supervillin4 signals through Trio to activate Rac1.…”

Generation and characterization of monoclonal antibodies that recognize human and murine supervillin protein isoforms

“…Actin was used as a loading control to calculate percent knockdown [ 11 ]. Detection was by ECL [SuperSignal West Pico or Femto reagents (Thermo Scientific, Rockford, IL)], according to the manufacturer’s instructions, on a Chemidoc MP Imaging System, using ImageLab software, version 4.1 (Bio-Rad Life Science Research, Hercules, CA) [ 20 ].…”