The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling

Abstract

The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.

FIG. 1.

TRE17 associates with Cdc42 and Rac1 in a GTP-dependent manner. (A) COS cells were cotransfected with the indicated GST-tagged Cdc42, Rac1, or RhoA mutant and HA-tagged TRE17. The GTPases were pulled down with glutathione-Sepharose, and associated TRE17 was detected by anti-HA immunoblotting. Arrowhead, HA-TRE17. (B) The indicated mutant alleles of Cdc42 or Rac1 were cotransfected with HA-tagged T17(447), which encodes the first 447 amino acids of TRE17, and subjected to the same assay. (C) The indicated GTPases were cotransfected with HA-tagged RN-tre(466), which encodes the first 466 amino acids of RN-tre, or T17(447). Associated RN-tre(466) or T17(447) was detected as described above. PD, glutathione-Sepharose pull-down; WCL, whole-cell lysate.

FIG. 2.

TRE17 displays a complex subcellular distribution that is regulated by the actin cytoskeleton and microtubules. (A) HA-tagged TRE17 was transfected in HeLa cells, and its localization was determined by confocal microscopy with anti-HA antibody (left panel). F-actin in the same cell was visualized with FITC-phalloidin (right panel). (B) HA-TRE17-transfected HeLa cells were treated with 1 μM cytoD for 1 h, and TRE17 localization was analyzed as described in panel A. (C) HA-TRE17 (left panel) and GFP-tubulin (right panel) were cotransfected into HeLa cells, and their localization analyzed as in panel A. (D) HA-TRE17-transfected HeLa cells were treated with nocodazole (10 μg/ml) for 2 h. HA-TRE17 localization was analyzed as described above. Scale bar, 10 μm.

FIG. 3.

Activated Rac1 and Cdc42 colocalize with TRE17 and stimulate its recruitment to the plasma membrane. (A to C) HA-TRE17 was cotransfected with GST-tagged WT Rac1 (A) or Rac1V12 (B and C) into HeLa cells. (C) Cells were treated with 1 μM cytoD for 1 h. TRE17 and Rac1V12 were detected as described above; F-actin was visualized with FITC-phalloidin. (D to F) HA-TRE17 was cotransfected with GST-tagged WT Cdc42 (D) or Cdc42V12 (E and F) and analyzed as described above. (F) Cells were treated with cytoD for 1 h and analyzed as in panel C. Scale bar, 10 μm.

FIG. 4.

EGF stimulates the recruitment of TRE17 to the plasma membrane in an F-actin-dependent manner. (A) HeLa cells were transfected with HA-tagged TRE17, and the following day cells were serum starved for 24 h. Cells were either left untreated (left panels), stimulated with 100 ng of EGF/ml for 10′ (middle panels), or pretreated with cytoD for 1 h prior to EGF stimulation (right panels). HA-TRE17 was visualized with anti-HA antibody (top row); F-actin was visualized with FITC-phalloidin (bottom row). Scale bar, 10 μm. (B) TRE17 localization was scored in either serum-starved cells (solid bars) or EGF-stimulated cells (open bars). The results are the average of three independent experiments, with the standard deviation indicated by the error bars.

FIG. 5.

Rac1 and Cdc42 activity are required for EGF-stimulated recruitment of TRE17 to the plasma membrane. HA-tagged TRE17 was cotransfected with GST (A), GST-Rac1N17 (B), GST-Cdc42N17 (C), or GST-PBD (D), which encodes the p21-binding domain of PAK1. Cells were serum starved for 24 h and then stimulated with EGF (100 ng/ml) for 10 min. TRE17 localization was determined with anti-HA antibody, whereas localization of GST, Rac1N17, Cdc42N17, and PBD was determined with anti-GST antibody. In panel D, the upper panel represents the TRE17 localization, and the lower panel represents PBD localization. The arrow indicates a cell expressing TRE17 alone, whereas the arrowhead indicates a cell coexpressing PBD. Scale bar, 10 μm.

FIG. 6.

Structural determinants of TRE17 localization. (A) Schematic representation of full-length TRE17 and various mutants. PTM refers to the PTM/Gyp/Tbc domain, while Pro refers to the proline-rich region. (B) HeLa cells were transfected with the indicated constructs and then serum-starved for 24 h prior to visualization with anti-HA antibody. Scale bar, 10 μm. (C) TRE17 or the indicated mutants were transfected into HeLa cells. Cells were hypotonically lysed and fractionated as described in Materials and Methods. One twenty-fifth of the soluble fraction and one-fifth of the pellet were loaded into each lane. S, soluble fraction; P, pellet. Fractions were also probed for the presence of ERK (bottom panel), a cytoplasmic protein that should localize to the soluble fraction, to ensure proper fractionation.

FIG. 7.

T17(447) stimulates cortical actin accumulation downstream of Cdc42 and Rac1. (A) HA-T17(447) or GST-Cdc42V12 was transfected into HeLa cells. After serum starvation for 24 h, cells were fixed and T17(447), Cdc42V12, and F-actin were visualized. (B) HeLa cells were transfected with HA-TRE17, HA-T17(447), HA-T17(447/RK), or HA-T17(447/PA) and then quiesced for 24 h. The percentage of cells exhibiting an accumulation of cortical actin relative to the total number of cells expressing the indicated construct is given. The results are the average of seven independent experiments for untransfected cells, HA-TRE17, and HA-T17(447) or of four independent experiments for HA-T17(447/RK) and HA-T17(447/PA), with standard deviations indicated by the error bars. (C) HA-T17(447) was cotransfected with GST-Cdc42N17, -Rac1N17, or -PBD into HeLa cells. After 24 h of serum-starvation, HA-T17(447), F-actin, and the indicated GST-tagged construct were visualized. Scale bar, 10 μm.

FIG. 8.

Model of TRE17 regulation and function. In quiescent cells, TRE17 is localized to filamentous structures in the cytoplasm in an inactive conformation. EGF stimulates the activation of Cdc42 and Rac1, leading to cortical actin accumulation, as well as recruitment of TRE17 to the plasma membrane. This process leads to a conformational change in TRE17, such that it now stimulates further cortical actin accumulation via its N-terminal 447 amino acids. See the text for further details.