A Novel Strategy for Specifically Down-regulating Individual Rho GTPase Activity in Tumor Cells

Abstract

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorageindependent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.

Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

“…The relative levels of GTP-bound RhoA, Rac1, or Cdc42 were determined by an effector pulldown assay as described previously (Wang et al, 2003). Briefly, the log phase growing WT, knockout, or reconstituted cells were lysed in a buffer containing 1% Triton X-100 and 10 mM MgCl 2 , and the lysates were probed with glutathione-agarose immobilized GST-Rhotekin (for RhoA) or GST-PAK1 (for Cdc42 or Rac1) effector domain.…”

Genetic deletion of Cdc42GAP reveals a role of Cdc42 in erythropoiesis and hematopoietic stem/progenitor cell survival, adhesion, and engraftment

“…24 In brief, low-density fetal liver cells were lysed in a buffer containing 1% Triton X-100 and incubated with the glutathione beadbound GST-Rhotekin (for RhoA activity) or GST-PAK1 (for Cdc42, Rac1, and Rac2 activities). The bead-immobilized, GTP-bound Rho GTPases and the Rho GTPases in the lysates were probed by immunoblotting with respective antibodies, and the relative amount of proteins bound to the beads were quantified by densitometry measurements.…”

Cdc42GAP regulates c-Jun N-terminal kinase (JNK)-mediated apoptosis and cell number during mammalian perinatal growth

“…Current knowledge of Cdc42 function in mammalian cells came mostly from studies using overexpression of dominant negative or constitutively active mutants of Cdc42 in clonal cell lines. Although the dominant mutant approach has provided useful insights into the functions of Cdc42 in mammalian cells, it also has significant drawbacks (10,11). For example, the dominant negative form of Cdc42 can be nonspecific because of its ability to sequester the upstream guanine nucleotide exchange factors that are needed for multiple Rho GTPase functions (12).…”

“…Relative levels of GTP-bound RhoA, Rac1, or Cdc42 were determined by an effector pull-down assay, as described (11). Briefly, the asynchronously growing WT, heterozygous, or knockout cells or tissues were lysed, and the lysates were probed with the glutathione-agaroseimmobilized GST-Rhotekin (for RhoA) or GST-PAK1 (for Cdc42 or Rac1) effector domain.…”