performed experiments and discussed data. == Acknowledgments == We thank Dr . prerequisites to constitute abona fideGDI displacement factor. RhoGDI acetylation interferes with Rho signaling, resulting in modification 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 Entacapone significance of this post-translational modification. Keywords: acetylation; acetyltransferase; histone deacetylase (HDAC); post-translational modification (PTM); Ras homolog gene family, member A (RhoA); Rho (Rho GTPase); sirtuin; guanine-nucleotide-dissociation inhibitor alpha; lysine acetylation; lysine acetyltransferase Entacapone == Introduction == Rho proteins are guanine nucleotide-binding proteins (GNBPs)3predominantly regulating the actin and microtubule cytoskeleton (1, 2). They are molecular switches and cycle between a GDP-bound inactive and a GTP-bound active conformation. In the GTP-bound state, Rho proteins bind to effector proteins regulating Entacapone essential cellular processes: maintenance of cell architecture, intracellular transport, cell migration, cell movement, cytokinesis, and signal transduction. Rho protein dysfunction results in severe cellular disorders, such as neurodegenerative diseases, metastasis, and tumor invasion (3). Rho proteins show a low intrinsic GTP hydrolysis and nucleotide exchange rate, which is strongly accelerated by RhoGTPase-activating proteins and Rho guanine nucleotide exchange factors, respectively (4). In the GTP-bound state, they are mostly bound to the plasma membrane via a polybasic region and a prenyl group (farnesyl or geranylgeranyl) forming a thioether with the C-terminal CaaX box cysteine side chain. About 80 different RhoGTPase-activating proteins and 80 Rho guanine nucleotide exchange factors have been described in humans to date (5, 6). Another key regulator of Rho function is the Rho guanine nucleotide dissociation inhibitor (RhoGDI) that couples the GTP/GDP cycle to a membrane/cytosol cycle. Only three RhoGDIs have been found in mammals. RhoGDI is ubiquitously expressed, RhoGDI is mainly expressed in hematopoietic cells, and RhoGDI is present in the brain, lung, kidney, and testis (7). This led to the hypothesis that RhoGDIs are housekeeping regulators of Rho proteins. However , recently, it has been found that RhoGDIs play more complex roles than originally expected. They are highly regulated by phosphorylation, can bind cytosolic GDP- and GTP-loaded Rho guanine nucleotide-binding protein (RhoGNBPs), are Entacapone capable of transporting Rho proteins specifically to different cellular membranes, and regulate their turnover (810). The interaction of Rho proteins and GDIs has been studied functionally and structurally. The crystal structures of full-length RhoGDI alone and in complex with RhoA, Cdc42, and Rac1 have been solved by NMR and by x-ray crystallography (1114). These studies revealed a modular structure of RhoGDI, a C-terminal immunoglobulin (Ig) domain forming a hydrophobic pocket accommodating the prenyl group of the RhoGNBPs and an N-terminal intrinsically unfolded domain. This domain adopts a helix-turn-helix conformation upon binding the lipidated RhoGNBP contacting the switch I and II regions essential for effector binding. For membrane extraction and membrane relocation of RhoGNBPs by RhoGDI, a two-step reaction mechanism has been postulated, supported by its modular structure (7). In the first step of delivery, positively charged patches in the Ig domain of RhoGDI are electrostatically attracted to the negatively charged membrane phospholipids. In the second step, the RhoGNBP inserts its lipid moiety into the membrane. An electrostatic network encompassing the negatively charged RhoGDI N terminus competes with the membrane phospholipids for binding to the positively charged C terminus of the RhoGNBP (polybasic region) (15). It is still unclear how the tight RhoGDPRhoGDI complexes are dissociated for RhoGNBPs to be reactivated by GEF-catalyzed GTP loading (14, 16). It was shown that RhoGDI is targeted by phosphorylation and lysine acetylation (10, 1721). Some phosphorylation sites are in the direct vicinity of the identified lysine acetylation sites. Phosphorylation of RhoGDI Ser-174 and Ser-101 by PAK1 upon stimulation by PDGF or EGF releases Rac1 but not RhoA and Cdc42 from its complex with RhoGDI (17). RhoA phosphorylation at Ser-188 and Cdc42 at Ser-185 by PKA/PKG leads to stabilization of its complexes with RhoGDI, translocation to the cytosol, and its protection from proteasomal degradation (2224). Recently, RhoGDI has been found to be SUMOylated at Lys-138, leading to a stabilization of RhoARhoGDI, resulting in decreased cancer cell motility (25). Several RhoGDI lysine acetylation sites have been found in various quantitative proteomic screens performed in diverse cell and tissue types (20, 21, 2628). For one site, RhoGDI Lys-141, it Entacapone has been shown by site-directed mutagenesis (K141Q as an acetylation mimic) that it leads to formation of thickened actin stress fibers and filopodia in HeLa cells (21). Functionally, the Tead4 acetylation sites in RhoGDI identified by quantitative mass spectrometry have only marginally been characterized so far. Here we present the first comprehensive study using a combined synthetic biological, biophysical, and cell biological approach to unravel how lysine acetylation regulates.