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

  • Author Footnotes
    1 Both authors contributed equally to this work.
    Nora Kuhlmann
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Sarah Wroblowski
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Philipp Knyphausen
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Susanne de Boor
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Julian Brenig
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Anke Y. Zienert
    Affiliations
    the Institute for Genetics, Zülpicher Strasse 47a, University of Cologne, 50674 Cologne, Germany,
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  • Katrin Meyer-Teschendorf
    Affiliations
    the Institute for Genetics, Zülpicher Strasse 47a, University of Cologne, 50674 Cologne, Germany,
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  • Gerrit J.K. Praefcke
    Affiliations
    the Institute for Genetics, Zülpicher Strasse 47a, University of Cologne, 50674 Cologne, Germany,

    the Paul-Ehrlich-Institute, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany, and
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  • Hendrik Nolte
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Marcus Krüger
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Magdalena Schacherl
    Affiliations
    the Institute for Biochemistry, Zülpicher Strasse 47, University of Cologne, 50674 Cologne, Germany,
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  • Ulrich Baumann
    Affiliations
    the Institute for Biochemistry, Zülpicher Strasse 47, University of Cologne, 50674 Cologne, Germany,
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  • Leo C. James
    Affiliations
    the Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom
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  • Jason W. Chin
    Affiliations
    the Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom
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  • Michael Lammers
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne, Germany,
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  • Author Footnotes
    * This work was supported by Emmy Noether Grant LA2984-1/1, Sonderforschungsbereich 635 (SFB635; Post-translational Control of Protein Function), and Priority Programs SPP1365 and SPP1580, all from Deutsche Forschungsgemeinschaft. This work was also supported by the European BIOSTRUCTX_5870. The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
Open AccessPublished:December 30, 2015DOI:https://doi.org/10.1074/jbc.M115.707091
      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 α (RhoGDIα). RhoGDIα has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDIα is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDIα 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. RhoGDIα acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDIα is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDIα-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.

      Introduction

      Rho proteins are guanine nucleotide-binding proteins (GNBPs)
      The abbreviations used are: GNBP, guanine nucleotide-binding protein; RhoGDI, Rho guanine-nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; mantGDP, 2/3′-O-(N-methyl-anthraniloyl)-guanosine-5′-diphosphate; PDB, Protein Data Bank; PTM, post-translational modification; NTA, nitrilotriacetic acid; aa, amino acid(s); BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; KAT, lysine acetyltransferase; ITC, isothermal titration calorimetry; KDAC, lysine deacetylase; ESI, electrospray ionization; RhoA-G, geranylgeranylated RhoA; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; EGFP, enhanced GFP.
      predominantly regulating the actin and microtubule cytoskeleton (
      • Hall A.
      Rho GTPases and the actin cytoskeleton.
      ,
      • Gundersen G.G.
      • Wen Y.
      • Eng C.H.
      • Schmoranzer J.
      • Cabrera-Poch N.
      • Morris E.J.
      • Chen M.
      • Gomes E.R.
      Regulation of microtubules by Rho GTPases in migrating cells.
      ). 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 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 (
      • Jaffe A.B.
      • Hall A.
      Rho GTPases: biochemistry and biology.
      ).
      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 (
      • Vetter I.R.
      • Wittinghofer A.
      The guanine nucleotide-binding switch in three dimensions.
      ). 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 (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ,
      • Geyer M.
      • Wittinghofer A.
      GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins.
      ).
      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. RhoGDIα is ubiquitously expressed, RhoGDIβ is mainly expressed in hematopoietic cells, and RhoGDIγ is present in the brain, lung, kidney, and testis (
      • Dovas A.
      • Couchman J.R.
      RhoGDI: multiple functions in the regulation of Rho family GTPase activities.
      ). 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 capable of transporting Rho proteins specifically to different cellular membranes, and regulate their turnover (
      • de Toledo M.
      • Senic-Matuglia F.
      • Salamero J.
      • Uze G.
      • Comunale F.
      • Fort P.
      • Blangy A.
      The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway.
      ,
      • Brunet N.
      • Morin A.
      • Olofsson B.
      RhoGDI-3 regulates RhoG and targets this protein to the Golgi complex through its unique N-terminal domain.
      • DerMardirossian C.
      • Rocklin G.
      • Seo J.Y.
      • Bokoch G.M.
      Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling.
      ).
      The interaction of Rho proteins and GDIs has been studied functionally and structurally. The crystal structures of full-length RhoGDIα alone and in complex with RhoA, Cdc42, and Rac1 have been solved by NMR and by x-ray crystallography (
      • Scheffzek K.
      • Stephan I.
      • Jensen O.N.
      • Illenberger D.
      • Gierschik P.
      The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.
      • Grizot S.
      • Fauré J.
      • Fieschi F.
      • Vignais P.V.
      • Dagher M.C.
      • Pebay-Peyroula E.
      Crystal structure of the Rac1-RhoGDI complex involved in NADPH oxidase activation.
      ,
      • Hoffman G.R.
      • Nassar N.
      • Cerione R.A.
      Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI.
      • Tnimov Z.
      • Guo Z.
      • Gambin Y.
      • Nguyen U.T.
      • Wu Y.W.
      • Abankwa D.
      • Stigter A.
      • Collins B.M.
      • Waldmann H.
      • Goody R.S.
      • Alexandrov K.
      Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.
      ). These studies revealed a modular structure of RhoGDIα, 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 RhoGDIα, a two-step reaction mechanism has been postulated, supported by its modular structure (
      • Dovas A.
      • Couchman J.R.
      RhoGDI: multiple functions in the regulation of Rho family GTPase activities.
      ). In the first step of delivery, positively charged patches in the Ig domain of RhoGDIα 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 RhoGDIα N terminus competes with the membrane phospholipids for binding to the positively charged C terminus of the RhoGNBP (polybasic region) (
      • Dransart E.
      • Morin A.
      • Cherfils J.
      • Olofsson B.
      RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs.
      ). It is still unclear how the tight Rho·GDP·RhoGDIα complexes are dissociated for RhoGNBPs to be reactivated by GEF-catalyzed GTP loading (
      • Tnimov Z.
      • Guo Z.
      • Gambin Y.
      • Nguyen U.T.
      • Wu Y.W.
      • Abankwa D.
      • Stigter A.
      • Collins B.M.
      • Waldmann H.
      • Goody R.S.
      • Alexandrov K.
      Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.
      ,
      • Takahashi K.
      • Sasaki T.
      • Mammoto A.
      • Takaishi K.
      • Kameyama T.
      • Tsukita S.
      • Takai Y.
      Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.
      ).
      It was shown that RhoGDIα is targeted by phosphorylation and lysine acetylation (
      • DerMardirossian C.
      • Rocklin G.
      • Seo J.Y.
      • Bokoch G.M.
      Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling.
      ,
      • DerMardirossian C.
      • Schnelzer A.
      • Bokoch G.M.
      Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase.
      • DerMardirossian C.M.
      • Bokoch G.M.
      Phosphorylation of RhoGDI by p21-activated kinase 1.
      ,
      • Gorvel J.P.
      • Chang T.C.
      • Boretto J.
      • Azuma T.
      • Chavrier P.
      Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity.
      ,
      • Choudhary C.
      • Kumar C.
      • Gnad F.
      • Nielsen M.L.
      • Rehman M.
      • Walther T.C.
      • Olsen J.V.
      • Mann M.
      Lysine acetylation targets protein complexes and co-regulates major cellular functions.
      • Kim S.C.
      • Sprung R.
      • Chen Y.
      • Xu Y.
      • Ball H.
      • Pei J.
      • Cheng T.
      • Kho Y.
      • Xiao H.
      • Xiao L.
      • Grishin N.V.
      • White M.
      • Yang X.J.
      • Zhao Y.
      Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.
      ). Some phosphorylation sites are in the direct vicinity of the identified lysine acetylation sites. Phosphorylation of RhoGDIα Ser-174 and Ser-101 by PAK1 upon stimulation by PDGF or EGF releases Rac1 but not RhoA and Cdc42 from its complex with RhoGDIα (
      • DerMardirossian C.
      • Schnelzer A.
      • Bokoch G.M.
      Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase.
      ). RhoA phosphorylation at Ser-188 and Cdc42 at Ser-185 by PKA/PKG leads to stabilization of its complexes with RhoGDIα, translocation to the cytosol, and its protection from proteasomal degradation (
      • Boulter E.
      • Garcia-Mata R.
      RhoGDI: A rheostat for the Rho switch.
      ,
      • Sawada N.
      • Itoh H.
      • Miyashita K.
      • Tsujimoto H.
      • Sone M.
      • Yamahara K.
      • Arany Z.P.
      • Hofmann F.
      • Nakao K.
      Cyclic GMP kinase and RhoA Ser188 phosphorylation integrate pro- and antifibrotic signals in blood vessels.
      • Rolli-Derkinderen M.
      • Sauzeau V.
      • Boyer L.
      • Lemichez E.
      • Baron C.
      • Henrion D.
      • Loirand G.
      • Pacaud P.
      Phosphorylation of serine 188 protects RhoA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells.
      ).
      Recently, RhoGDIα has been found to be SUMOylated at Lys-138, leading to a stabilization of RhoA·RhoGDIα, resulting in decreased cancer cell motility (
      • Yu J.
      • Zhang D.
      • Liu J.
      • Li J.
      • Yu Y.
      • Wu X.R.
      • Huang C.
      RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility.
      ). Several RhoGDIα lysine acetylation sites have been found in various quantitative proteomic screens performed in diverse cell and tissue types (
      • Choudhary C.
      • Kumar C.
      • Gnad F.
      • Nielsen M.L.
      • Rehman M.
      • Walther T.C.
      • Olsen J.V.
      • Mann M.
      Lysine acetylation targets protein complexes and co-regulates major cellular functions.
      ,
      • Kim S.C.
      • Sprung R.
      • Chen Y.
      • Xu Y.
      • Ball H.
      • Pei J.
      • Cheng T.
      • Kho Y.
      • Xiao H.
      • Xiao L.
      • Grishin N.V.
      • White M.
      • Yang X.J.
      • Zhao Y.
      Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.
      ,
      • Park J.
      • Chen Y.
      • Tishkoff D.X.
      • Peng C.
      • Tan M.
      • Dai L.
      • Xie Z.
      • Zhang Y.
      • Zwaans B.M.
      • Skinner M.E.
      • Lombard D.B.
      • Zhao Y.
      SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.
      ,
      • Lundby A.
      • Lage K.
      • Weinert B.T.
      • Bekker-Jensen D.B.
      • Secher A.
      • Skovgaard T.
      • Kelstrup C.D.
      • Dmytriyev A.
      • Choudhary C.
      • Lundby C.
      • Olsen J.V.
      Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns.
      • Chen Y.
      • Zhao W.
      • Yang J.S.
      • Cheng Z.
      • Luo H.
      • Lu Z.
      • Tan M.
      • Gu W.
      • Zhao Y.
      Quantitative acetylome analysis reveals the roles of SIRT1 in regulating diverse substrates and cellular pathways.
      ). For one site, RhoGDIα Lys-141, it 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 (
      • Kim S.C.
      • Sprung R.
      • Chen Y.
      • Xu Y.
      • Ball H.
      • Pei J.
      • Cheng T.
      • Kho Y.
      • Xiao H.
      • Xiao L.
      • Grishin N.V.
      • White M.
      • Yang X.J.
      • Zhao Y.
      Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.
      ).
      Functionally, the acetylation sites in RhoGDIα 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 RhoGDIα function. Our results reveal general mechanisms of how lysine acetylation regulates protein function and might open up new therapeutic strategies.

      Discussion

      RhoGDIα is a major regulator of RhoGNBP function modulating its subcellular distribution and turnover, thereby creating a cytosolic pool of inactive Rho easily activatable for a fast cellular response. Thus, RhoGDIα is essential for the precise spatial and temporal regulation of RhoGNBPs and affects their downstream signaling. How RhoGDIα is regulated to create a high specificity of action is only marginally understood. Here, we present the first functional and structural study describing how RhoGDIα function is controlled by post-translational lysine acetylation (Fig. 7F). Using the powerful genetic code expansion concept to produce site-specifically lysine-acetylated proteins allows us to study its real impact on protein function.
      As we show here, RhoGDIα acetylation influences the membrane/cytosol and GTP/GDP cycle of Rho proteins, phenotypically observable by affecting the cellular actin cytoskeleton (Lys-52G, Lys-99G, Lys-141G, and Lys-178G). It furthermore interferes with binding to prenylated (Ac-Lys-52G, Ac-Lys-138G, and Ac-Lys-178G) RhoGNBPs. Lysine acetylation per se leads to an electrostatic quenching of the lysine side chain's positive charge and an increase of the hydrophobicity. Additionally, we observe that Ac-Lys-178G structurally couples β5, β9, and β10 of the Ig domain, most likely interfering with the inherent Ig flexibility (structural coupling). Moreover, we show structurally that the volume of the hydrophobic cavity of the Ig domain is increased, adopting the farnesyl group differently compared with the geranylgeranyl group (PDB entry 4F38). These mechanisms might contribute to the observed reduced RhoA-F binding. Ac-Lys-52G is a loss-of-function modification completely abolishing RhoGDIα function, most likely by interfering with the transition of the intrinsically unfolded N-terminal domain to adopt the helix-loop-helix conformation upon binding to the Rho protein (structural destruction). Thereby, the cell has a system to quickly switch on/off RhoGDIα function. We observed that acetylation at the solvent-exposed Lys-138G also reduced the binding toward RhoA-F. The exact molecular mechanisms need further examination but might also include hydrophobic shielding from the polar solvent as well as structural coupling. RhoGDIα acetylation interferes directly (Ac-Lys-138G) and indirectly (Ac-Lys-141G) with Lys-138G SUMOylation, reported to increase the RhoA affinity (PTM cross-talk). By impairing SUMOylation, Lys-141G acetylation might therefore indirectly affect the observed increase of RhoA at the membrane and cellular F-actin content. Using these mechanisms, RhoGDIα lysine acetylation is a powerful cellular system to tightly regulate the RhoGNBP turnover and residence time at the membrane, finally resulting in a modulation of RhoGNBP signaling.
      We have discovered that acetylation at Lys-178G lowers the affinity toward RhoA-F, resulting in more RhoA located at the plasma membrane. Consistent with an increased Rho signaling, we found more filamentous actin in cells expressing the respective acetylation mimicking mutant.
      RhoGDIα Lys-52 acetylation completely blocks the interaction with RhoA, drastically reduces RhoA-F binding (4 orders of magnitude) and the membrane extraction capability. This is supported by previous results showing that the N-terminal domain of RhoGDIα is essential for membrane extraction as well as delivery of Rho proteins. Consistently, the expression of K52QG in the N-terminal domain resulted in a cellular F-actin content comparable with mock control, indicating that acetylation at Lys-52G leads to a complete loss of function. Notably, Lys-52G acetylation restores the GEF-catalyzed nucleotide exchange rates, nearly approaching values observed for uncomplexed RhoA. This shows that it might act as a bona fide RhoGDIα displacement factor.
      An open question for many proteins identified by quantitative mass spectrometry to be lysine-acetylated is the biological significance of the modification. Many acetylation sites are of low stoichiometry, for which only a gain of function might confer an additional property. Technical progress to determine absolute quantities of acetylation on a whole proteome scale will help to identify biologically important sites (
      • Baeza J.
      • Dowell J.A.
      • Smallegan M.J.
      • Fan J.
      • Amador-Noguez D.
      • Khan Z.
      • Denu J.M.
      Stoichiometry of site-specific lysine acetylation in an entire proteome.
      ,
      • Weinert B.T.
      • Iesmantavicius V.
      • Moustafa T.
      • Schölz C.
      • Wagner S.A.
      • Magnes C.
      • Zechner R.
      • Choudhary C.
      Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.
      ). Notably, in contrast to many reported studies, for RhoGDIα, we were able to find endogenously acetylated protein in several human cell lines, showing that a significant amount is lysine-acetylated and suggesting that acetylation is of biological importance to control RhoGDIα function. However, the exact absolute quantities could vary and might depend on the cellular metabolic, physiologic, and cell cycle state. Additionally, the expression level and activity of KDACs and KATs might play an important role. Here, we show that RhoGDIα is targeted by the KATs CBP, p300, and pCAF. The KATs p300 and pCAF acetylate RhoGDIα at Lys-52, -138, -141, and -178, the sites being important to control RhoGDIα function as shown here. Moreover, RhoGDIα is differentially deacetylated by Sirt2 and HDAC6 at these sites.
      Sirt2 and HDAC6 are the main cytosolic deacetylases known to physically interact, to work synergistically, to colocalize with microtubules, and to deacetylate many proteins involved in cytoskeleton regulation, such as cortactin, mDia2, and α-tubulin (
      • Hubbert C.
      • Guardiola A.
      • Shao R.
      • Kawaguchi Y.
      • Ito A.
      • Nixon A.
      • Yoshida M.
      • Wang X.F.
      • Yao T.P.
      HDAC6 is a microtubule-associated deacetylase.
      ,
      • North B.J.
      • Marshall B.L.
      • Borra M.T.
      • Denu J.M.
      • Verdin E.
      The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.
      • Valenzuela-Fernández A.
      • Cabrero J.R.
      • Serrador J.M.
      • Sánchez-Madrid F.
      HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions.
      ). Down-regulation or inhibition of Sirt2 and HDAC6 were both found to inhibit cell motility as well as cell migration (
      • Zuo Q.
      • Wu W.
      • Li X.
      • Zhao L.
      • Chen W.
      HDAC6 and SIRT2 promote bladder cancer cell migration and invasion by targeting cortactin.
      ). Sirt2 deacetylates and thereby inactivates p300, which in turn is able to inactivate both Sirt2 and HDAC6, constituting a feedback loop (
      • Han Y.
      • Jin Y.H.
      • Kim Y.J.
      • Kang B.Y.
      • Choi H.J.
      • Kim D.W.
      • Yeo C.Y.
      • Lee K.Y.
      Acetylation of Sirt2 by p300 attenuates its deacetylase activity.
      ,
      • Han Y.
      • Jeong H.M.
      • Jin Y.H.
      • Kim Y.J.
      • Jeong H.G.
      • Yeo C.Y.
      • Lee K.Y.
      Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity.
      ). The balance between acetylation/deacetylation of RhoGDIα might interfere with cell adhesion, cell migration, and cell motility by directly affecting Rho signal transduction pathways (
      • Garcia-Mata R.
      • Boulter E.
      • Burridge K.
      The “invisible hand”: regulation of RHO GTPases by RHOGDIs.
      ).
      In summary, the presence of endogenously acetylated RhoGDIα in mammalian cells, site-specific KATs, and KDACs suggests that acetylation is a physiologically important post-translational modification to control RhoGDIα function. Depending on when and where RhoGDIα acetylation takes place in vivo, the cell is able to directly control the Rho protein's lifetime and turnover, its residence time on the membrane, and its subcellular distribution. All of these processes strongly contribute to the initiation and termination of Rho signal transduction pathways, ultimately balancing cell resting and cell motility. A dysfunction in its regulation might therefore lead to severe cellular defects supporting tumorigenesis and neurodegeneration. Because there are just three RhoGDIs in mammalian cells, modification of RhoGDIα by post-translational lysine acetylation can create a sophisticated system to spatially and temporally balance RhoGNBP signal transduction pathways. Tackling the RhoGDIα lysine acetylation machinery might thus be a promising yet underestimated therapeutic approach.

      Author Contributions

      N. K., S. W. L. C. J., J. W. C., M. K., H. N., A. Z., G. P., and M. L. designed the experiments, discussed data, generated the data, and wrote the manuscript. L. S. and L. B. provided technical assistance with the project. S. W., U. B., and M. L. performed structural analysis. M. S., S. W., and M. L. took x-ray data sets. S. d. B., P. K., and J. B. performed experiments and discussed data.

      Acknowledgments

      We thank Dr. Astrid Schauss, Dr. Nikolay Kladt, and Dr. Christian Jüngst (CECAD Imaging Facility) for discussions of the cell biological experiments; Dr. Tobias Lamkemeyer for discussion of the proteomic experiments; and Astrid Wilbrand-Hennes and René Grandjean for technical assistance (CECAD Proteomics Facility). We thank Linda Baldus and Lukas Scislowski for expert technical assistance. We thank Prof. Dr. A. Wittinghofer for discussions and experimental materials. We thank the beamline groups at the SLS Villingen/Switzerland and at the Diamond/Oxford UK, whose outstanding efforts have made these experiments possible.

      References

        • Hall A.
        Rho GTPases and the actin cytoskeleton.
        Science. 1998; 279: 509-514
        • Gundersen G.G.
        • Wen Y.
        • Eng C.H.
        • Schmoranzer J.
        • Cabrera-Poch N.
        • Morris E.J.
        • Chen M.
        • Gomes E.R.
        Regulation of microtubules by Rho GTPases in migrating cells.
        Novartis Found. Symp. 2005; 269 (discussion 116–126, 223–230): 106-116
        • Jaffe A.B.
        • Hall A.
        Rho GTPases: biochemistry and biology.
        Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269
        • Vetter I.R.
        • Wittinghofer A.
        The guanine nucleotide-binding switch in three dimensions.
        Science. 2001; 294: 1299-1304
        • Bos J.L.
        • Rehmann H.
        • Wittinghofer A.
        GEFs and GAPs: critical elements in the control of small G proteins.
        Cell. 2007; 129: 865-877
        • Geyer M.
        • Wittinghofer A.
        GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins.
        Curr. Opin. Struct. Biol. 1997; 7: 786-792
        • Dovas A.
        • Couchman J.R.
        RhoGDI: multiple functions in the regulation of Rho family GTPase activities.
        Biochem. J. 2005; 390: 1-9
        • de Toledo M.
        • Senic-Matuglia F.
        • Salamero J.
        • Uze G.
        • Comunale F.
        • Fort P.
        • Blangy A.
        The GTP/GDP cycling of rho GTPase TCL is an essential regulator of the early endocytic pathway.
        Mol. Biol. Cell. 2003; 14: 4846-4856
        • Brunet N.
        • Morin A.
        • Olofsson B.
        RhoGDI-3 regulates RhoG and targets this protein to the Golgi complex through its unique N-terminal domain.
        Traffic. 2002; 3: 342-357
        • DerMardirossian C.
        • Rocklin G.
        • Seo J.Y.
        • Bokoch G.M.
        Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling.
        Mol. Biol. Cell. 2006; 17: 4760-4768
        • Scheffzek K.
        • Stephan I.
        • Jensen O.N.
        • Illenberger D.
        • Gierschik P.
        The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.
        Nat. Struct. Biol. 2000; 7: 122-126
        • Grizot S.
        • Fauré J.
        • Fieschi F.
        • Vignais P.V.
        • Dagher M.C.
        • Pebay-Peyroula E.
        Crystal structure of the Rac1-RhoGDI complex involved in NADPH oxidase activation.
        Biochemistry. 2001; 40: 10007-10013
        • Hoffman G.R.
        • Nassar N.
        • Cerione R.A.
        Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI.
        Cell. 2000; 100: 345-356
        • Tnimov Z.
        • Guo Z.
        • Gambin Y.
        • Nguyen U.T.
        • Wu Y.W.
        • Abankwa D.
        • Stigter A.
        • Collins B.M.
        • Waldmann H.
        • Goody R.S.
        • Alexandrov K.
        Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.
        J. Biol. Chem. 2012; 287: 26549-26562
        • Dransart E.
        • Morin A.
        • Cherfils J.
        • Olofsson B.
        RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs.
        Biochem. Soc. Trans. 2005; 33: 623-626
        • Takahashi K.
        • Sasaki T.
        • Mammoto A.
        • Takaishi K.
        • Kameyama T.
        • Tsukita S.
        • Takai Y.
        Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.
        J. Biol. Chem. 1997; 272: 23371-23375
        • DerMardirossian C.
        • Schnelzer A.
        • Bokoch G.M.
        Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase.
        Mol. Cell. 2004; 15: 117-127
        • DerMardirossian C.M.
        • Bokoch G.M.
        Phosphorylation of RhoGDI by p21-activated kinase 1.
        Methods Enzymol. 2006; 406: 80-90
        • Gorvel J.P.
        • Chang T.C.
        • Boretto J.
        • Azuma T.
        • Chavrier P.
        Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity.
        FEBS Lett. 1998; 422: 269-273
        • Choudhary C.
        • Kumar C.
        • Gnad F.
        • Nielsen M.L.
        • Rehman M.
        • Walther T.C.
        • Olsen J.V.
        • Mann M.
        Lysine acetylation targets protein complexes and co-regulates major cellular functions.
        Science. 2009; 325: 834-840
        • Kim S.C.
        • Sprung R.
        • Chen Y.
        • Xu Y.
        • Ball H.
        • Pei J.
        • Cheng T.
        • Kho Y.
        • Xiao H.
        • Xiao L.
        • Grishin N.V.
        • White M.
        • Yang X.J.
        • Zhao Y.
        Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.
        Mol. Cell. 2006; 23: 607-618
        • Boulter E.
        • Garcia-Mata R.
        RhoGDI: A rheostat for the Rho switch.
        Small GTPases. 2010; 1: 65-68
        • Sawada N.
        • Itoh H.
        • Miyashita K.
        • Tsujimoto H.
        • Sone M.
        • Yamahara K.
        • Arany Z.P.
        • Hofmann F.
        • Nakao K.
        Cyclic GMP kinase and RhoA Ser188 phosphorylation integrate pro- and antifibrotic signals in blood vessels.
        Mol. Cell Biol. 2009; 29: 6018-6032
        • Rolli-Derkinderen M.
        • Sauzeau V.
        • Boyer L.
        • Lemichez E.
        • Baron C.
        • Henrion D.
        • Loirand G.
        • Pacaud P.
        Phosphorylation of serine 188 protects RhoA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells.
        Circ. Res. 2005; 96: 1152-1160
        • Yu J.
        • Zhang D.
        • Liu J.
        • Li J.
        • Yu Y.
        • Wu X.R.
        • Huang C.
        RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility.
        J. Biol. Chem. 2012; 287: 13752-13760
        • Park J.
        • Chen Y.
        • Tishkoff D.X.
        • Peng C.
        • Tan M.
        • Dai L.
        • Xie Z.
        • Zhang Y.
        • Zwaans B.M.
        • Skinner M.E.
        • Lombard D.B.
        • Zhao Y.
        SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.
        Mol. Cell. 2013; 50: 919-930
        • Lundby A.
        • Lage K.
        • Weinert B.T.
        • Bekker-Jensen D.B.
        • Secher A.
        • Skovgaard T.
        • Kelstrup C.D.
        • Dmytriyev A.
        • Choudhary C.
        • Lundby C.
        • Olsen J.V.
        Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns.
        Cell Rep. 2012; 2: 419-431
        • Chen Y.
        • Zhao W.
        • Yang J.S.
        • Cheng Z.
        • Luo H.
        • Lu Z.
        • Tan M.
        • Gu W.
        • Zhao Y.
        Quantitative acetylome analysis reveals the roles of SIRT1 in regulating diverse substrates and cellular pathways.
        Mol. Cell Proteomics. 2012; 11: 1048-1062
        • Lammers M.
        • Neumann H.
        • Chin J.W.
        • James L.C.
        Acetylation regulates cyclophilin A catalysis, immunosuppression and HIV isomerization.
        Nat. Chem. Biol. 2010; 6: 331-337
        • Weisshaar S.R.
        • Keusekotten K.
        • Krause A.
        • Horst C.
        • Springer H.M.
        • Göttsche K.
        • Dohmen R.J.
        • Praefcke G.J.
        Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML.
        FEBS Lett. 2008; 582: 3174-3178
        • Fres J.M.
        • Müller S.
        • Praefcke G.J.
        Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase.
        J. Lipid Res. 2010; 51: 2454-2459
        • Kabsch W.
        XDS.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132
        • Evans P.R.
        • Murshudov G.N.
        How good are my data and what is the resolution?.
        Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1204-1214
        • Adams P.D.
        • Afonine P.V.
        • Bunkóczi G.
        • Chen V.B.
        • Davis I.W.
        • Echols N.
        • Headd J.J.
        • Hung L.W.
        • Kapral G.J.
        • Grosse-Kunstleve R.W.
        • McCoy A.J.
        • Moriarty N.W.
        • Oeffner R.
        • Read R.J.
        • Richardson D.C.
        • Richardson J.S.
        • Terwilliger T.C.
        • Zwart P.H.
        PHENIX: a comprehensive Python-based system for macromolecular structure solution.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Adams P.D.
        • Winn M.D.
        • Storoni L.C.
        • Read R.J.
        Phaser crystallographic software.
        J. Appl. Crystallogr. 2007; 40: 658-674
        • Afonine P.V.
        • Grosse-Kunstleve R.W.
        • Echols N.
        • Headd J.J.
        • Moriarty N.W.
        • Mustyakimov M.
        • Terwilliger T.C.
        • Urzhumtsev A.
        • Zwart P.H.
        • Adams P.D.
        Towards automated crystallographic structure refinement with phenix.refine.
        Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 352-367
        • Moriarty N.W.
        • Grosse-Kunstleve R.W.
        • Adams P.D.
        Electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation.
        Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 1074-1080
        • Emsley P.
        • Lohkamp B.
        • Scott W.G.
        • Cowtan K.
        Features and development of Coot.
        Acta Crystallogr. D. 2010; 66: 486-501
        • Murshudov G.N.
        • Skubák P.
        • Lebedev A.A.
        • Pannu N.S.
        • Steiner R.A.
        • Nicholls R.A.
        • Winn M.D.
        • Long F.
        • Vagin A.A.
        REFMAC5 for the refinement of macromolecular crystal structures.
        Acta Crystallogr. D. 2011; 67: 355-367
        • Chen V.B.
        • Arendall 3rd, W.B.
        • Headd J.J.
        • Keedy D.A.
        • Immormino R.M.
        • Kapral G.J.
        • Murray L.W.
        • Richardson J.S.
        • Richardson D.C.
        MolProbity: all-atom structure validation for macromolecular crystallography.
        Acta Crystallogr. D. 2010; 66: 12-21
        • Davis I.W.
        • Leaver-Fay A.
        • Chen V.B.
        • Block J.N.
        • Kapral G.J.
        • Wang X.
        • Murray L.W.
        • Arendall 3rd, W.B.
        • Snoeyink J.
        • Richardson J.S.
        • Richardson D.C.
        MolProbity: all-atom contacts and structure validation for proteins and nucleic acids.
        Nucleic Acids Res. 2007; 35: W375-W383
        • DeLano W.L.
        The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002 (version 1.7.2.0)
        • Rappsilber J.
        • Mann M.
        • Ishihama Y.
        Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
        Nat. Protoc. 2007; 2: 1896-1906
        • Cox J.
        • Mann M.
        MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
        Nat. Biotechnol. 2008; 26: 1367-1372
        • Cox J.
        • Neuhauser N.
        • Michalski A.
        • Scheltema R.A.
        • Olsen J.V.
        • Mann M.
        Andromeda: a peptide search engine integrated into the MaxQuant environment.
        J. Proteome Res. 2011; 10: 1794-1805
        • Neumann H.
        • Peak-Chew S.Y.
        • Chin J.W.
        Genetically encoding Nϵ-acetyllysine in recombinant proteins.
        Nat. Chem. Biol. 2008; 4: 232-234
        • de Boor S.
        • Knyphausen P.
        • Kuhlmann N.
        • Wroblowski S.
        • Brenig J.
        • Scislowski L.
        • Baldus L.
        • Nolte H.
        • Krüger M.
        • Lammers M.
        Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112: E3679-E3688
        • Solski P.A.
        • Helms W.
        • Keely P.J.
        • Su L.
        • Der C.J.
        RhoA biological activity is dependent on prenylation but independent of specific isoprenoid modification.
        Cell Growth Differ. 2002; 13: 363-373
        • Moissoglu K.
        • McRoberts K.S.
        • Meier J.A.
        • Theodorescu D.
        • Schwartz M.A.
        Rho GDP dissociation inhibitor 2 suppresses metastasis via unconventional regulation of RhoGTPases.
        Cancer Res. 2009; 69: 2838-2844
        • Platko J.V.
        • Leonard D.A.
        • Adra C.N.
        • Shaw R.J.
        • Cerione R.A.
        • Lim B.
        A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 2974-2978
        • Euden J.
        • Mason S.A.
        • Viero C.
        • Thomas N.L.
        • Williams A.J.
        Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.
        J. Biol. Chem. 2013; 288: 16671-16679
        • Baeza J.
        • Dowell J.A.
        • Smallegan M.J.
        • Fan J.
        • Amador-Noguez D.
        • Khan Z.
        • Denu J.M.
        Stoichiometry of site-specific lysine acetylation in an entire proteome.
        J. Biol. Chem. 2014; 289: 21326-21338
        • Weinert B.T.
        • Iesmantavicius V.
        • Moustafa T.
        • Schölz C.
        • Wagner S.A.
        • Magnes C.
        • Zechner R.
        • Choudhary C.
        Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.
        Mol. Syst. Biol. 2014; 10: 716
        • Hubbert C.
        • Guardiola A.
        • Shao R.
        • Kawaguchi Y.
        • Ito A.
        • Nixon A.
        • Yoshida M.
        • Wang X.F.
        • Yao T.P.
        HDAC6 is a microtubule-associated deacetylase.
        Nature. 2002; 417: 455-458
        • North B.J.
        • Marshall B.L.
        • Borra M.T.
        • Denu J.M.
        • Verdin E.
        The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.
        Mol. Cell. 2003; 11: 437-444
        • Valenzuela-Fernández A.
        • Cabrero J.R.
        • Serrador J.M.
        • Sánchez-Madrid F.
        HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions.
        Trends Cell Biol. 2008; 18: 291-297
        • Zuo Q.
        • Wu W.
        • Li X.
        • Zhao L.
        • Chen W.
        HDAC6 and SIRT2 promote bladder cancer cell migration and invasion by targeting cortactin.
        Oncol. Rep. 2012; 27: 819-824
        • Han Y.
        • Jin Y.H.
        • Kim Y.J.
        • Kang B.Y.
        • Choi H.J.
        • Kim D.W.
        • Yeo C.Y.
        • Lee K.Y.
        Acetylation of Sirt2 by p300 attenuates its deacetylase activity.
        Biochem. Biophys. Res. Commun. 2008; 375: 576-580
        • Han Y.
        • Jeong H.M.
        • Jin Y.H.
        • Kim Y.J.
        • Jeong H.G.
        • Yeo C.Y.
        • Lee K.Y.
        Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity.
        Biochem. Biophys. Res. Commun. 2009; 383: 88-92
        • Garcia-Mata R.
        • Boulter E.
        • Burridge K.
        The “invisible hand”: regulation of RHO GTPases by RHOGDIs.
        Nat. Rev. Mol. Cell Biol. 2011; 12: 493-504
        • Wiseman T.
        • Williston S.
        • Brandts J.F.
        • Lin L.N.
        Rapid measurement of binding constants and heats of binding using a new titration calorimeter.
        Anal. Biochem. 1989; 179: 131-137
        • Diederichs K.
        • Karplus P.A.
        Improved R-factors for diffraction data analysis in macromolecular crystallography.
        Nat. Struct. Biol. 1997; 4 (Erratum (1997) Nat. Struct. Biol. 4, 592): 269-275
        • Diederichs K.
        • Karplus P.A.
        Better models by discarding data?.
        Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1215-1222
        • Brünger A.T.
        Free R value: cross-validation in crystallography.
        Methods Enzymol. 1997; 277: 366-396