PRL-1 Protein Promotes ERK1/2 and RhoA Protein Activation through a Non-canonical Interaction with the Src Homology 3 Domain of p115 Rho GTPase-activating Protein

Yunpeng Bai,Yong Luo,Sijiu Liu,Lujuan Zhang,Kui Shen,Yuanshu Dong,Chad D Walls,Lawrence A Quilliam,Clark D Wells,Youjia Cao,Zhong-Yin Zhang

doi:10.1074/jbc.m111.286302

Abstract

Phosphatases of the regenerating liver (PRL) play oncogenic roles in cancer development and metastasis. Although previous studies indicate that PRL-1 promotes cell growth and migration by activating both the ERK1/2 and RhoA pathways, the mechanism by which it activates these signaling events remains unclear. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). p115 RhoGAP directly binds PRL-1 in vitro and in cells via its SH3 domain. Structural analyses of the PRL-1·Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. Furthermore, PRL-1 binding activates RhoA signaling by inhibiting the catalytic activity of p115 RhoGAP. The results demonstrate that PRL-1 binding to p115 RhoGAP provides a coordinated mechanism underlying ERK1/2 and RhoA activation.

Highlights

The mechanism for the oncogenic phosphatase Phosphatases of the regenerating liver (PRL)-1 remains undefined
Identification and Characterization of a PRL-1-binding Peptide—We hypothesized that the discovery of PRL-1-binding proteins may yield new insight into the mechanism that accounts for PRL-1-mediated ERK1/2 and RhoA activation
From HEK293 cell lysate (Fig. 1C), and GST-Peptide 1 readily co-immunoprecipitated with HA-PRL-1 when both were expressed in HEK293 cells (Fig. 1D)

Summary

Background

The mechanism for the oncogenic phosphatase PRL-1 remains undefined. Results: We identified and characterized a novel PRL-1-binding protein, p115 RhoGAP. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). Structural analyses of the PRL11⁄7Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. We provide evidence that PRL-1 activates RhoA by directly inhibiting the catalytic activity of p115 RhoGAP

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Space group Cell dimensions