Acetylation of Tat Defines a CyclinT1-Independent Step in HIV Transactivation

Kaehlcke, Katrin; Dorr, Alexander; Hetzer-Egger, Claudia; Kiermer, Véronique; Henklein, Peter; Schnoelzer, Martina; Loret, Erwann; Cole, Philip A.; Verdin, Eric; Ott, Melanie

doi:10.1016/s1097-2765(03)00245-4

Cited by 117 publications

(128 citation statements)

References 49 publications

Supporting

Mentioning

125

Contrasting

Order By: Relevance

“…Recombinant acetyltransferase domains were prepared as described before (39,40). Incubation was performed at 30°C for 30 min in a total volume of 20 l of reaction buffer containing 50 mM HEPES, pH 8.0, 10% glycerol, 1 mM DTT, and 10 mM sodium butyrate.…”

Section: Methodsmentioning

confidence: 99%

Probing Lysine Acetylation in Proteins

Dormeyer

Ott

Schnölzer

2005

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in falsepositive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/ CBP-associated factor (PCAF). The acetylation of proteins by acetyltransferases is increasingly considered a biologically relevant regulatory modification like phosphorylation (1). Acetyltransferases transfer acetyl groups from acetyl-coenzyme A (AcCoA) 1 either to the ␣-amino group of the amino-terminal residue (N-acetyltransferases) or to the ⑀-amino group of specific lysine residues (histone/factor acetyltransferases) of substrate proteins. The reverse reaction is catalyzed by deacetylases that remove acetyl groups from specific acetyllysine residues in their substrates. The reversible lysine acetylation of histones and nonhistone proteins plays a vital role in the regulation of many cellular processes including chromatin dynamics and transcription (2-5), gene silencing (6, 7), cell cycle progression (8 -11), apoptosis (12-14), differentiation (15-19), DNA replication (20, 21), DNA repair (22-27), nuclear import (28 -30), and neuronal repression (31-33). More than 20 acetyltransferases and 18 deacetylases have been identified so far, but the mechanistic details of substrate selection and site specificity of these enzymes remain unclear. Over 40 transcription factors and 30 other nuclear, cytoplasmic, bacterial, and viral proteins have been shown to be acetylated in vivo (34,35), and the investigation of protein acetylation continues.In recent years, the analysis of protein acetylation by MS has become increasingly popular because MALDI-TOF MS and ESI MS represent fast and sensitive methods for the characterization of co-and posttranslational protein modifications (36 -38). In this study, we demonstrate the advantages of various MS techniques for the characterizat...

show abstract

Section: Methodsmentioning

confidence: 99%

Probing Lysine Acetylation in Proteins

Dormeyer

Ott

Schnölzer

2005

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…[32][33][34][35][36][37][38][39] In particular, D'Orso and Frankel have found that acetylation of Tat Lys28 by PCAF modulates the assembly of Tat•TAR•P-TEFb complex. 33 They also found that Asn257 in the TRM of Cyclin T1 is essential for the recognition of acetylated Tat.…”

Section: Acetylation Of Tat Lys28 and Implications For Tar Bindingmentioning

confidence: 99%

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4

Babayeva

Suwa

et al. 2014

Cell Cycle

View full text Add to dashboard Cite

“…The mechanism by which Tat/TAR-RNA complexes regulate transcription from the HIV-1 LTR involves the concerted recruitment of a plethora of cellular factors, including p300/CREB-binding protein (p300/CBP) (18 -25), PCAF/hGCN5 (20 -22, 26 -30), P-TEFb (30 -33), SET7/SET9 methyltransferases (34), SIRT1 (35), the Brm component of the SWI/SNF chromatin-remodeling com-plex (36), and the splicing factor, SKIP (37). The Tat protein is acetylated on Lys 28 , Lys 50 , and Lys 51 by the transcriptional coactivators/acetyltransferases, p300/CBP and PCAF/hGCN5 (20 -22, 24, 27-30, 38), which has been shown to modulate Tat interactions with P-TEFb and Brm, as well as the ability of Tat for binding TAR-RNA (20,30,35,36,39). The formation of Tat/TAR-RNA/P-TEFb/PCAF complexes on the HIV-1 LTR stimulates Ser 2 -Ser 5 -phosphorylation of the RNA pol II carboxyl-terminal domain associated with increased transcriptional elongation (30,37,40).…”

mentioning

confidence: 99%

The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

Sharma

Awasthi

Harrod

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

(1) have demonstrated that WRN contributes to general RNA pol II 4 -dependent transcription, although its mechanism remains unclear. Interestingly, these authors found that a 27-amino acid direct-repeat sequence strongly activated transcription in yeast two-hybrid experiments, independent of WRN 3Ј 3 5Ј DNA helicase activity (1) (Fig. 1A) suggesting that WRN interacts with cellular factors to modulate RNA pol II-dependent transcription. The WRN protein localizes to nucleoli and the nucleoplasm of transcriptionally active cells (1, 2). Moreover, Laine et al. (3) have shown that WRN stimulates topoisomerase I DNA-unwinding activity that could influence cellular transcription. The yeast WRN homologue, SGS1, also participates in DNA replication and RNA pol I-dependent transcription (4), and the WRN helicase enhances RNA pol I-dependent transcription of ribosomal RNA (5).In the present study, we have investigated whether WRN contributes to HIV-1 LTR transactivation and retroviral replication. The HIV-1 LTR contains upstream enhancer elements (e.g. NF-B and SP1) that synergize with the transactivator protein, Tat, bound to TAR-RNA, to promote retroviral gene expression in HIV-1-infected tissues, macrophages/monocytes, and CD4 ϩ T-lymphocytes (6 -17). The mechanism by which Tat/TAR-RNA complexes regulate transcription from the HIV-1 LTR involves the concerted recruitment of a plethora of cellular factors, including p300/CREB-binding protein (p300/CBP) (18 -25), [26][27][28][29][30], P-TEFb (30 -33), SET7/SET9 methyltransferases (34), SIRT1 (35), the Brm component of the SWI/SNF chromatin-remodeling com-

show abstract

Acetylation of Tat Defines a CyclinT1-Independent Step in HIV Transactivation

Cited by 117 publications

References 49 publications

Probing Lysine Acetylation in Proteins

Probing Lysine Acetylation in Proteins

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4

The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

Contact Info

Product

Resources

About