Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-specific Guanine Nucleotide Exchange Factor, by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells

Katsuya Sato,Tsuyoshi Sugiyama,Takahiro Nagase,Yukio Kitade,Hiroshi Ueda

doi:10.1074/jbc.m113.521880

Abstract

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.

Highlights

Rho GTPase-specific guanine nucleotide exchange factors (RhoGEFs) play important roles in regulation of the actin cytoskeleton
FLJ00018 Is Activated by ␤1-Adrenergic Receptor Stimulation through a G␤␥-dependent and -independent Pathway—We previously reported that FLJ00018 was activated by G␤␥ and m2 muscarinic acetylcholine receptor stimulation (7)
To examine whether FLJ00018 is activated by stimulation of other G-protein-coupled receptor (GPCR), we used a ␤-adrenergic receptor, ␤1-AR, that is known to couple with multiple signaling pathways via Gs protein

Summary

Background

RhoGEFs play important roles in regulation of the actin cytoskeleton. Results: The FLJ00018/PLEKHG2 was phosphorylated, and activated by EGF signaling might be interpreted as a direct effect on its GEF activity (which is not established). The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein G␤␥ subunits or cytosolic actin. In the present study we show that FLJ00018 is phosphorylated and activated by ␤1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to G␤␥ signaling. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Phosphorylation of FLJ00018 by EGF Receptor Signaling phosphoinositide through direct interaction to activate the GDP/GTP exchange activity for Rac. We previously reported that one novel RhoGEF, FLJ00018/PLEKHG2, was activated by direct interaction with G␤␥ subunits and regulated cell spreading through the activation of Rac and Cdc (7). We found that phosphorylation of FLJ00018 by EGFR-activated Ras/MAPK signaling caused the activation of FLJ00018 and controlled the cell morphological change in Neuro-2a cells independently of interaction with G␤␥ subunits

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION