Lysine acetylation is a reversible posttranslational modification that occurs at thousands of sites on human proteins. However, the stoichiometry of acetylation remains poorly characterized, and is important for understanding acetylation-dependent mechanisms of protein regulation. Here we provide accurate, validated measurements of acetylation stoichiometry at 6829 sites on 2535 proteins in human cervical cancer (HeLa) cells. Most acetylation occurs at very low stoichiometry (median 0.02%), whereas high stoichiometry acetylation (>1%) occurs on nuclear proteins involved in gene transcription and on acetyltransferases. Analysis of acetylation copy numbers show that histones harbor the majority of acetylated lysine residues in human cells. Class I deacetylases target a greater proportion of high stoichiometry acetylation compared to SIRT1 and HDAC6. The acetyltransferases CBP and p300 catalyze a majority (65%) of high stoichiometry acetylation. This resource dataset provides valuable information for evaluating the impact of individual acetylation sites on protein function and for building accurate mechanistic models.

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin‐homology domains 1 and 2. Diaphanous‐related formins (Drfs) are regulated by a RhoGTPase‐binding domain situated in the amino‐terminal (N‐terminal) region and a carboxy‐terminal Diaphanous‐autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N‐terminus of mDia1 (mDiaN) and its release by Rho•GTP. We have defined the elements required for tight binding and solved the three‐dimensional structure of a complex between an mDiaN construct and DAD by X‐ray crystallography. The core DAD region is an α‐helical peptide, which binds in the most highly conserved region of mDiaN using mainly hydrophobic interactions. The structure suggests a two‐step mechanism for release of autoinhibition whereby Rho•GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho•GTP accelerates the dissociation of DAD from the mDiaN•DAD complex.

Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDia(N), including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDia(N)-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42.Gpp(NH)p and mDia(N)-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.