Oncogenic Activity of Ect2 Is Regulated through Protein Kinase Cι-mediated Phosphorylation

Verline Justilien,Lee Jameison,Channing J Der,Kent L Rossman,Alan P Fields

doi:10.1074/jbc.m110.196113

Abstract

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells. PKCι directly phosphorylates Ect2 at Thr-328 in vitro, and RNAi-mediated knockdown of either PKCι or Par6 leads to a decrease in phospho-Thr-328 Ect2, indicating that PKCι regulates Thr-328 Ect2 phosphorylation in NSCLC cells. Both wild-type Ect2 and a phosphomimetic T328D Ect2 mutant bind the PKCι-Par6 complex, activate Rac1, and restore transformed growth and invasion when expressed in NSCLC cells made deficient in endogenous Ect2 by RNAi-mediated knockdown. In contrast, a phosphorylation-deficient T328A Ect2 mutant fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation. Our data support a model in which PKCι-mediated phosphorylation regulates Ect2 binding to the oncogenic PKCι-Par6 complex thereby activating Rac1 activity and driving transformed growth and invasion.

Highlights

Epithelial cell transforming sequence 2 (Ect2) is a guanine nucleotide exchange factor (GEF)[3] for Rho family small GTPases (RhoA, Rac[1], and Cdc42), and Ect[2] function is essential in the regulation of cytokinesis (1–5)
We recently demonstrated that Ect[2] is a potential oncogene in nonsmall cell lung cancer (NSCLC) (6)
We find that Ect[2] isolated from NSCLC cells is highly phosphorylated at the novel and previously uncharacterized site Thr-328 within its hinge-like domain

Summary

Introduction

Epithelial cell transforming sequence 2 (Ect2) is a guanine nucleotide exchange factor (GEF)[3] for Rho family small GTPases (RhoA, Rac[1], and Cdc42), and Ect[2] function is essential in the regulation of cytokinesis (1–5). Ect[2] is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKC␫-Par[6] complex, leading to activation of the Rac[1] small GTPase. We identify a previously uncharacterized phosphorylation site on Ect[2], threonine 328, that serves to regulate the oncogenic activity of Ect[2] in NSCLC cells.

Results

Conclusion