Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors

Abstract

The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.

Keywords: CDC42; GTPase; Rac; Rho; actin; mutation; phosphorylation; signal transduction; ubiquitin.

Figure 1.

Filamentous actin structures. MDA MB 231 human breast cancer cells were fixed and stained with fluorescently labeled phalloidin to visualize filamentous actin structures. The image shows a maximum-projection assembled from 22 Z-plane images acquired with a Zeiss LSM 880 Airyscan microscope.

Figure 2.

Rho GTPases are molecular switches. When associated GEF molecules promote GDP release, GTP is bound which results in conformational changes in the Switch 1 and Switch 2 regions that enable interactions with downstream effector proteins and consequent signal transduction. Upon association with GAPs, GTP is hydrolysed to GDP and Pi is released to inactivate the Rho protein.

Figure 3.

The Rho GTPase family. (A) An amino acid identity matrix was generated by comparing the primary sequence of 20 human Rho proteins. Grouping into Rac (red), Rho (purple), RhoH (brown) and RhoBTB (green) groups was by hierarchical clustering using the Multalin multiple sequence alignment program. (B) A rooted phylogenetic tree of the 20 human Rho GTPases was generated by hierarchical clusing using the Multalin multiple sequence alignment program. Distances between proteins are proportional to their PAM (point accepted mutation) score of molecular evolution.

Figure 4.

Amino acid conservation in the Rho GTPases. (A) The core conserved segments, with important features numbered by their positions within amino acids 4–177 of Rac1, of the 18 most related Rho GTPases are depicted with percentages of amino acid identity ranging from 100% (red) to less than 50% (white). Guanine nucleotide binding regions are shown as G1 to G5 boxes (green). Switch 1 (yellow) and Switch 2 (magenta) regions are also depicted. (B) Rac2-GDP (PDB ID: 2W2T) structure was rendered with Chimera, with G boxes (green), Switch 1 (yellow) and Switch 2 (magenta) regions indicated. (C) Amino acid identity for 18 Rho GTPases was determined with Clustal Omega and mapped onto Rac2-GDP with Chimera. Colors depicting amino acid identity from 100% (red) to 0% (blue) as indicated.

Figure 5.

Phosphorylation and ubiquitylation of Rho GTPases. (A) Phosphorylation (purple) and ubiquitylation (brown) events reported in the literature or PhosphoSite (www.phosphosite.org) were mapped onto the amino acid identity grid depicting the core conserved segments, analogous to amino acids 4–177 of Rac1, of the 18 most related Rho GTPases, ranging from 100% identity (salmon) to less than 50% (white). (B) The phosphorylations (purple) and ubiquitylations (brown) of Rac2 were mapped onto the 3-dimensional structure, with G boxes (green), Switch 1 (yellow) and Switch 2 (magenta) regions depicted. (C) View rotated 180° of Rac2 phosphorylations and ubiquitylations.