Abstract
The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity.
Highlights
Rho GTPases are monomeric, small GTP-binding proteins belonging to the Ras superfamily
Small GTP-binding proteins, the regulatory cycle of RhoA is controlled by three distinct families of proteins: guanine nucleotide exchange factors (GEFs) that activate RhoA by promoting uptake of free nucleotide, GTPase-activating proteins (GAPs) that negatively regulate RhoA by stimulating its intrinsic GTPase activity leading to an inactive GDP-bound state, and guanine nucleotide dissociation inhibitors (GDIs) that inhibit the dissociation of GDP from RhoA and prevent the binding of GDP-RhoA to cell membranes
Our results showed that GST-RhoAΔD pulled down substantially less p-extracellular signal-regulated kinases (ERK) and total ERK compared with wild type GST-RhoA, indicating that the docking site (D-site) is required for the interaction between RhoA and ERK
Summary
Rho GTPases are monomeric, small GTP-binding proteins belonging to the Ras superfamily. Phosphorylation of RhoA by ERK small GTP-binding proteins, the regulatory cycle of RhoA is controlled by three distinct families of proteins: guanine nucleotide exchange factors (GEFs) that activate RhoA by promoting uptake of free nucleotide, GTPase-activating proteins (GAPs) that negatively regulate RhoA by stimulating its intrinsic GTPase activity leading to an inactive GDP-bound state, and guanine nucleotide dissociation inhibitors (GDIs) that inhibit the dissociation of GDP from RhoA and prevent the binding of GDP-RhoA to cell membranes. The GTPase cycle is essential for the biological functions of Rho GTPases, leading to its interaction with downstream effectors [5,6]. It has become evident, that a simple GTPase cycle cannot solely explain the variety of functions and signaling initiated by Rho proteins. Evidence is accumulating that phosphorylation is playing an increasingly important role in the regulation of Rho GTPase functions
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