Structural and Mechanistic Insights into the Regulation of the Fundamental Rho Regulator RhoGDIα by Lysine Acetylation

Abstract

Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor ␣ (RhoGDI␣). RhoGDI␣ has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDI␣ is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDI␣ function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDI␣ acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDI␣ is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDI␣-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.

The use of unnatural amino acids to study and engineer protein function

“…Acetylation of critical lysine residues in Ran modulates its activities related to the regulation of nuclear transport processes [19 ]. Similarly, acetylation of the guanine nucleotide dissociation inhibitor a impairs its binding to RhoA, thereby indirectly enhancing Rho-mediated alteration of actin filaments [20].…”

“…For example, fluorinated UAAs (20) have been used to study the interaction of phosphorylated GPCR tail-peptides with b-arrestin-1 and how this modulates the conformation and downstream signalling of the latter (Figure 4b) [67 ]. The tert-butyl group of (21) can be used in 1 H-NMR spectroscopy of proteins without any isotope labelling.…”

Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins

“…Therefore, another promising strategy is to design mechanistic inhibitors by combining substrate-based peptides with acetyl-lysine analogues such as trifluoroacetyl-lysine and thioacetyl-lysine, which show a markedly reduced rate of deacetylation (32, 66 -68). We and others show that some sirtuins present a remarkable level of substrate specificity for certain acetylation sites (14,15,69). However, whether structural features or the primary sequence is the main determinant of specificity remains an unresolved question.…”

Regulation of RhoA activation and cytoskeletal organization by acetylation

“…These findings are distinct from recently published effects on Rho guanine nucleotide dissociation inhibitor a (RhoGDIa), where it was observed that its acetylation can be stimulated by overexpression of PCAF, p300 and CBP and inhibited by overexpression of HDAC6 or SIRT2. 22 It is noteworthy that we did not observe significant relocalization of the Net1 isoform following acetylation. This may be due to differences in the number of NLS sequences between isoforms, as Net1 contains 4 NLS sequences while Net1A only has 2.…”

“…24,25 In addition, the ability of RhoGDIa to bind RhoA is inhibited by lysine acetylation, leading to activation of RhoA and F-actin accumulation. 22 It is perhaps not surprising that the activity of Rho regulatory proteins is controlled by acetylation, as acetylation is an important regulatory mechanism for other cytoskeletal elements (Fig. 2).…”