Altered Rho-Dependent Protein Trafficking in Breast Cancer Cells
Abstract
Rho, a member of the Rho GThase family, regulates stress fiber formation, cell motility, cytokinesis, and kinase signaling pathways. Rho signaling pathways are important for cancer progression since they are involved in controlling cell adhesion, anchorage-independent growth, and cell cycle progression. While no mutations in Rho GTPases have been found in breast or other cancers, numerous studies indicate that these OThases are often either overexpressed or hyperactive in breast cancer tissue (1). A number of downstream effector proteins of Rho have been identified that are involved in Rho-mediated effects (2). We identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2 (3). Rhophiiin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Brol-like, and a C-terminal PDZ domain. GST-capture experiments revealed that Rhophilin- 1 and Rhophilin-2 interacted with both GDP- and 0Th-bound RhoA in vitro. Despite the ability of Rhophilin- 1 and Rhophilin- 2 to interact with RhoA in a nucleotide independent fashion, Rho-induced serum response element (SRE) transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2- expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active RhoA mutant reversed the disassembly phenotype, in which coexpressing cells were more spread and less contracted than Rho alone expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. Our results with a minor splice variant of Rhophilin-2 suggested that it was involved in protein trafficking and cytokinesis.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2004
- Accession Number
- ADA429541
Entities
People
- Adam E. Kisailus
Organizations
- George Washington University