The RhoA-binding protein, Rhophilin-2, Regulates Actin Cytoskeleton Organization

Abstract

The Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP-and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rhoinduced serum response element 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 Rho mutant reversed the disassembly phenotype, in which the 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. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity.

Normal Thyroid Structure and Function in Rhophilin 2-Deficient Mice

“…To our surprise, the comparison of our exon 7-containing genomic DNA fragment of the mouse Rhpn2 gene with data available in the NCBI and Ensembl databases revealed a discrepancy in the number of exons in the gene as well as in the length of the coding region of its cDNA. Cloning and sequencing of the coding region of the Rhpn2 cDNA in mouse confirmed that it is 2,058 bp long, as in dog and human, and as more recently described by Peck et al (10).…”

“…Second, the most widely cited explanation for the absence of major alterations in knockout mice is functional redundancy. Rhpn2 is highly similar to rhophilin 1 (Rhpn1): comparison between mouse Rhpn2 and Rhpn1 revealed that they are similar in size and have three domains in common: the Rho-binding motif at the amino terminus, a central Bro1-like domain of ϳ200 amino acids, and a carboxy-terminal PDZ domain of limited homology (8,10). Rhpn1 is expressed in all tissues examined, and seems to be present in the thyroid, as is Rhpn2 (9,14).…”

“…Among these, rhophilin 2 (Rhpn2, previously reported as clone 45, p76 RBE , or Rho binding protein 2) was particularly well analyzed in our laboratory. Rhpn2, whose mRNA and protein expression is enhanced in thyrocytes following TSH stimulation of the cAMP transduction cascade in vitro (8), presents important similarities with rhophilin and contains several protein-protein interaction motifs, like HR1 and PDZ motifs, as well as a potential PDZ binding motif (8,10). Yeast two-hybrid screening and coimmunoprecipitation experiments have recently revealed that rhophilin 2 binds specifically to the GTPbound form of the small GTPase RhoB and to components of the cytoskeleton (8).…”

Nanoliter scale microbioreactor array for quantitative cell biology

“…Cancer cell serum response was one of the earliest and most studied signaling pathways in systems biology (Jones and Kazlauskas, 2000;Schoeberl et al, 2002). Various growth factors isolated from serum have been found to activate the MAP kinase proliferation pathway (Srsen et al, 1999), influence cell attachment, and spreading (Barnes et al, 1980;Sato et al, 1995), and promote the formation of actin fibers (Peck et al, 2002). In the microbioreactor array, 64 cell cultures were simultaneously observed at 8 serum concentrations between 0% and 10%.…”

Genes involved in cell adhesion and signaling: A new repertoire of Retinoic Acid Receptors target genes in mouse embryonic fibroblasts

“…Indeed, Rab20 encodes a GTPase, which associates with and dissociates from membranes and which like all the GTPases of the Rab family is a key regulator of all transport steps between intracellular compartments (Pylypenko and Goud, 2012). Rhpn2 encodes rhophilin-2, which is a RhoA-GTPase-binding protein and which regulates actin cytoskeleton organization (Peck et al, 2002). Tnfrsf1b belongs to the tumor necrosis factor receptor super family and is linked to pro-and anti-apoptotic pathways (Aggarwal, 2003).…”