The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl-phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose, and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. Since glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl-phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl-phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.

The CpxAR two-component signal transduction system in Escherichia coli and other pathogens senses diverse envelope stresses and promotes the transcription of a variety of genes that remedy these stresses. An important member of the CpxAR regulon is cpxP. The CpxA-dependent transcription of cpxP has been linked to stresses such as misfolded proteins and alkaline pH. It also has been proposed that acetyl phosphate, the intermediate of the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, can activate the transcription of cpxP in a CpxA-independent manner by donating its phosphoryl group to CpxR. We tested this hypothesis by measuring the transcription of cpxP using mutants with mutations in the CpxAR pathway, mutants with mutations in the Pta-AckA pathway, and mutants with a combination of both types of mutations. From this epistasis analysis, we learned that CpxR integrates diverse stimuli. The stimuli that originate in the envelope depend on CpxA, while those associated with growth and central metabolism depend on the Pta-AckA pathway. While CpxR could receive a phosphoryl group from acetyl phosphate, this global signal was not the primary trigger for CpxR activation associated with the Pta-AckA pathway. On the strength of these results, we contend that the interactions between central metabolism and signal transduction can be quite complex and that successful investigations of such interactions must include a complete epistatic analysis.

Atypical antipsychotics such as olanzapine have high affinity for multiple monoamine neurotransmitter receptors and are the mainstay of pharmacological therapy for treatment of schizophrenia. In addition to blocking monoamine receptors, these drugs also affect intracellular signaling cascades. We now report that 24-h treatment with 300 nM olanzapine causes desensitization of serotonin (5-HT) 2A receptors in A1A1v cells, a rat cortical cell line, as indicated by a reduction in inositol phosphate accumulation following stimulation with a 5-HT 2A/2C receptor agonist (Ϫ)-1-(2,5-dimethoxy-4-lodophenyl)-2-aminopropane HCl. Olanzapine treatment for 24 h increased the levels of 5-HT 2A receptors in both cytosol (234 Ϯ 34% of control level) and membrane fractions (206 Ϯ 14% of control levels) and RGS7 proteins in both cytosol (193 Ϯ 32% of control levels) and membrane fractions (160 Ϯ 18% of control levels) as measured on Western blots. Increased phosphorylation of Janus tyrosine kinase (JAK) 2 and increased phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT) 3 with 24-h olanzapine treatment demonstrate activation of the JAK-STAT signaling cascade. Pretreatment with a JAK inhibitor, AG490 [␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide], prevented the olanzapine-induced increase in membrane RGS7 protein levels; AG490 alone had no effect on RGS7 protein levels. We verified that treatment with AG490 reduced phosphorylation of JAK2 and inhibited the nuclear localization of phospho-STAT3. Interestingly, treatment with the JAK inhibitor had no effect on 5-HT 2A receptor protein levels. These data suggest that olanzapine-induced activation of the JAK-STAT signaling cascade causes increased expression of RGS7 protein, which in turn could mediate desensitization of 5-HT 2A receptor signaling caused by olanzapine because RGS7 binds to G␣ q protein and accelerates GTP hydrolysis.

Agonist treatment causes desensitization of many G proteincoupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of G␣ q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT 2A ) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (Ϫ)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT 2A/2C receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of G␣ q/11 proteins, as demonstrated by immunoprecipitation of G␣ q/11 and both incorporation of 32 P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of G␣ q/11 proteins, suggesting that different mechanisms are involved in desensitization after short-and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in G␣ 11 significantly reduced DOI-stimulated phosphorylation of G␣ 11 and DOI-induced desensitization of 5-HT 2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of G␣ q/11 proteins and DOI-induced desensitization of 5-HT 2A receptors. Expression of G␣ 11 S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of G␣ 11 protein by PKC and CaMK contributes to agonistinduced homologous desensitization of 5-HT 2A receptor signaling as well as heterologous desensitization. The phosphorylation of G␣ protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.