Rac1-induced cell migration requires membrane recruitment of the nuclear oncogene SET

Abstract

The Rho GTPase Rac1 controls cell adhesion and motility. The effector loop of Rac1 mediates interactions with downstream effectors, whereas its C-terminus binds the exchange factor beta-Pix, which mediates Rac1 targeting and activation. Here, we report that Rac1, through its C-terminus, also binds the nuclear oncogene SET/I2PP2A, an inhibitor of the serine/threonine phosphatase PP2A. We found that SET translocates to the plasma membrane in cells that express active Rac1 as well as in migrating cells. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner. Conversely, reduction of SET expression inhibits Rac1-induced migration, indicating that efficient Rac1 signalling requires membrane recruitment of SET. The recruitment of the SET oncogene to the plasma membrane represents a new feature of Rac1 signalling. Our results suggest a model in which Rac1-stimulated cell motility requires both effector loop-based downstream signalling and recruitment of a signalling amplifier, that is, SET, through the hypervariable C-terminus.

Figure 1

Rac1 interacts with the nuclear oncogene SET. (A) Identification of the Rac1-interacting protein SET. Streptavidin-based pull-down assays (PD) were performed with biotinylated peptides encoding the hypervariable domain of Rac1 and Rac2. Total cell lysates (Lys) and Rac-interacting proteins were detected by Western blotting followed by Coomassie staining. The protein band indicated by the dotted square was identified by peptide sequencing as SET. (B, C) SET interacts specifically with the C-terminus of Rac1. Streptavidin-based pull-down assays were performed with a control peptide (Ctrl) or the C-terminal peptides derived from Rac1, Rac2 and Cdc42 in lysates from HeLa cells (B) or from Cos7 cells, expressing HA-tagged SET (C). Samples were blotted and immunostained for endogenous (B) or HA-tagged (C) SET. Lys, cell lysates blotted for SET (B) or HA (C). (D) Purified, full-length Rac1 interacts directly with full-length SET. Rac1, GST and GST SET were purified from bacteria. Pull-down assays were performed with GST and GST-SET in the presence of Rac1. Isolated proteins were subsequently blotted and immunostained for Rac1 and GST. Input represents the Rac1 protein, added to the assay. (E) Endogenous Rac1 binds to endogenous SET. Rac1 was isolated from HeLa cells with GST RhoGDI or the Crib domain of PAK1 and subsequently immunostained for endogenous SET and Rac1. Controls represent GST alone or a control peptide (Ctrl). (F) The SET–Rac1 interaction is nucleotide independent. Cos7 lysates expressing HA-tagged SET were incubated with GST or GST-Rac1 loaded with either GDP or GTPγS. Isolated proteins were subsequently blotted and immunostained for PAK, HA and GST. Input represents SET or PAK protein, added to the assay. Detection of two bands for PAK is due to PAK isoforms. (G) The C-terminus of Rac1 interacts with the NAP domain of SET. Streptavidin-based pull-down assays were performed with the C-terminal Rac1 peptide in Cos7-derived cell lysates expressing different MYC-tagged SET mutants, indicated in the left panel, and subsequently immunostained with anti-MYC.

Figure 2

Active Rac1 induces translocation of SET from the nucleus to the plasma membrane. (A) HeLa cells expressing constitutively active Rac1 (red) and SET (green). Note the difference in the localisation of SET in the single SET-transfected cell (left) and the double transfected cell (right, cell body indicated by the dashed line). Dashed box in the merged image indicates the enlarged area, which is shown in the lower panels. Asterisks indicate nuclei; arrow indicates SET localisation at the plasma membrane. (B) HeLa cells expressing β-Pix (red) and SET (green). (C) HeLa cells expressing dominant-negative N17Rac1 (red) and SET (green). The merge also contains the corresponding phase-contrast image to show the outline of the cell. Scale bars, 10 μm.

Figure 3

SET translocates to the plasma membrane in migrating cells. (A) SET-transfected HeLa cells, either growing in a monolayer (stationary, upper panels) or induced to migrate into a wound (migrating, lower panels), were immunostained for SET (green) and counterstained for F-actin (red). The direction of migration is indicated by the arrow. Scale bar, 10 μm. (B) HeLa cells were cotransfected with SET and the pSUPER vector with either control siRNA (Ctrl) or Rac1 siRNA, of which two examples are shown. SET is indicated in green and actin in red. Arrows indicate the direction of migration. Scale bar, 10 μm.

Figure 4

Serine phosphorylation of SET regulates its interaction with Rac1 and PP2A. (A) The phospho-mimetic SET E9 mutant binds Rac1. GST-SET A9 and GST-SET E9 were purified from bacteria and subsequently incubated with Cos7 cell lysates expressing Rac1 (Input). GST pull-downs were analysed by Western blotting for Rac1. (B) The phospho-mimetic SET E9 mutant does not dimerise. HA-tagged WT-SET was coexpressed with MYC-tagged SET A9 and E9 in Cos7 cells. Immunoprecipitations (IPs) with anti-MYC were performed and lysates (Lys) and IPs were immunostained with both anti-MYC and anti-HA. (C) The SET E9 mutant interacts with PP2A. GST-SET A9 and -E9 mutant proteins were purified from bacteria and incubated with Cos7-derived cell lysates (input). Endogenous PP2A was detected following GST pull-down assay and Western blotting by anti-PP2A staining. (D) The hypervariable domain of Rac1 associates with PP2A. Streptavidin-based pull-down assays with a biotinylated control peptide (Ctrl) or the C-terminal domain of Rac1 were performed in HeLa cell lysates (input). Endogenous PP2A was detected following Western blotting of the isolated proteins by anti-PP2A. (E) Phospho-deficient SET A9 mutant does not translocate in the presence of activated Rac1. Merged images show SET A9, E9 and WT SET (green) in HeLa cells, coexpressed with empty vector (left panels) or activated L61Rac1 (in red, right panels). F-actin is shown in red (left panels only). Scale bar, 10 μm. (F) Quantification of SET localisation was determined by immunostaining in combination with confocal analysis (E) and divided in three categories: nuclear staining (black bars), nuclear and cytosol (white bars) and plasma membrane (grey bars). Quantification of the SET distribution is based on counting at least 50 cells per group.

Figure 5

Rac1 and SET cooperate in cell migration. (A) Images show transfected HEK293 cells immunostained for SET or MYR-SET (green) and counterstained for F-actin (red). Scale bars, 10 μm. (B) Western blot analysis of HEK293-derived cell lysates expressing SET or MYR-SET. (C) Membrane-targeted SET enhances migration. HeLa cells expressing either the empty retroviral vector (EV), SET or MYR-SET were allowed to migrate across a fibronectin-coated Transwell filter towards 10% FCS for 4 h. During migration, cells were incubated with a cell-permeable control peptide (Ctrl) or the C-terminal peptide of Rac1 (0.2 mg/ml). Migration is indicated as fold increase relative to the migration of the empty vector-expressing cells. Western blot analysis shows expression of SET and MYR-SET in HeLa cells. (D) Reduction of SET expression does not impair cell adhesion/spreading. HeLa cells, transduced with control siRNA (Ctrl) or siSET, were seeded on fibronectin-coated gold electrodes and analysed by ECIS for 12 h. Western blot analysis shows expression of endogenous SET in retrovirally transduced HeLa cells expressing control siRNA (Ctrl) or siSET. (E) Rac1-induced migration is SET-dependent. HeLa cells expressing either the empty retroviral vector (EV), siSET, V12Rac1 or V12Rac1 in combination with the SET siRNA were allowed to migrate in a Transwell system towards 10% FCS for 4 h. Migration is indicated as fold increase relative to the migration of the empty vector-expressing cells. Western blot analysis shows expression of endogenous SET and V12Rac1 in HeLa cells without or with expression of the siRNA.

Figure 6

Model for SET acting as a signalling amplifier for Rac1. In its unphosphorylated form, SET is primarily nuclear and forms a dimer (1). Phosphorylation of SET at serine 9 dissociates the dimer and allows SET redistribution to the nucleus as well as the cytoplasm (2). This may allow interaction with Rac1 in the cytoplasm. Upon activation of Rac1, for example, by the Rac-GEF β-Pix, SET translocates to the plasma membrane (3) where active Rac1 can stimulate kinase-mediated signalling (4). This requires SET-mediated inhibition of the serine/threonine phosphatase PP2A (5). Kinase-driven signalling is subsequently required for efficient cell migration, which is initiated by Rac1, in a SET-dependent fashion (6).