Pragmin, a Novel Effector of Rnd2 GTPase, Stimulates RhoA Activity

Hiroko Tanaka,Hironori Katoh,Manabu Negishi

doi:10.1074/jbc.m511314200

Abstract

The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.

Highlights

The growth cone and stimulation of neurite outgrowth
To examine whether Pragmin interacts with Rnd2 among the Rho family GTPases, HA-tagged Rnd subfamily GTPases and well characterized Rho family GTPases were expressed and used in a pull-down assay with the glutathione S-transferase (GST)-fused COOH-terminal region of Pragmin (Pragmin-CT*), a fragment originally isolated from the yeast two-hybrid screening
Knockdown of Pragmin Expression Promotes Neurite Elongation—Since overexpression of Pragmin inhibited neurite outgrowth in PC12 cells, we further examined if knockdown of endogenous Pragmin with small interfering RNAs (siRNAs) induces neurite extension

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructions and Antibodies—Wild-type and mutant forms of Rnd, wild-type Rnd and Rnd, mutant forms of RhoA, Rac, and Cdc were obtained as described previously [14, 20, 27, 28]. For expression in mammalian cells, cDNAs of RhoAV14, Rac1V12, Cdc42V12, Rnd, Rnd, Rnd2V16, Rnd2N21, and Rnd were subcloned into pcDNA3 encoding an initiating Met followed by the hemagglutinin (HA) epitope tag sequence at the NH2 terminus. The small interfering RNAs (siRNAs) for Pragmin were designed to target 19 nucleotides of rat Pragmin (Pragmin siRNA-A, nucleotides 224 –242, 5Ј-CCACCATGATGACTTCTGA3Ј; Pragmin siRNA-B, nucleotides 1339 –1357, 5Ј-GCAACCACTATTACTGTTA-3Ј), and the siRNA for Rapostlin was designed to target 19 nucleotides of rat Rapostlin (nucleotides 132–150, 5Ј-ACAGCTCAGGAATCTTTCA-3Ј). They were subcloned into an siRNA expression vector pSilencer (Ambion) to express a 21-nucleotides hairpin siRNA

A Novel Rnd2 Effector Stimulating RhoA Activity

RESULTS

DISCUSSION