Shalini Pathak

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

Structural insights into mechanism and specificity of O ‐GlcNAc transferase

Post‐translational modification of protein serines/threonines with N‐acetylglucosamine (O‐GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O‐GlcNAcylation is regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O‐GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X‐ray crystallography and mutagenesis, that OGT adopts the (metal‐independent) GT‐B fold and binds a UDP‐GlcNAc analogue at the bottom of a highly conserved putative peptide‐binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide‐binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.

Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy

Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.

O ‐GlcNAcylation of TAB1 modulates TAK1‐mediated cytokine release

Transforming growth factor (TGF)‐β‐activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro‐inflammatory cytokines such as transforming growth factor‐β, tumour necrosis factor (TNF), interleukin‐1 (IL‐1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1–3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N‐acetylglucosamine (O‐GlcNAc) on a single site, Ser395. With the help of a novel O‐GlcNAc site‐specific antibody, we demonstrate that O‐GlcNAcylation of TAB1 is induced by IL‐1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild‐type or an O‐GlcNAc‐deficient mutant TAB1 (S395A) into Tab1−/− mouse embryonic fibroblasts, we determined that O‐GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL‐1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL‐6 and TNFα. This is one of the first examples of a single O‐GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.

A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription

In mammalian RNA polymerase I transcription, SL1, an assembly of TBP and associated factors (TAFs), is essential for preinitiation complex formation at ribosomal RNA gene promoters in vitro. We provide evidence for a novel component of SL1, TAFI41 (MGC5306), which functions in Pol I transcription. TAFI41 resides at the rDNA promoter in the nucleolus and co‐purifies and co‐immunoprecipitates with SL1. TAFI41 immunodepletion from nuclear extracts dramatically reduces Pol I transcription; addition of SL1 restores the ability of these extracts to support Pol I transcription. In cells, siRNA‐mediated decreased expression of TAFI41 leads to loss of SL1 from the rDNA promoter in vivo, with concomitant loss of Pol I from the rDNA and reduced synthesis of the pre‐rRNA. Extracts from these cells support reduced levels of Pol I transcription; addition of SL1 to the extracts raises the level of Pol I transcription. These data suggest that TAFI41 is integral to transcriptionally active SL1 and imply a role for SL1, including the TAFI41 subunit, in Pol I recruitment and, therefore, preinitiation complex formation in vivo.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence.

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

Substrate and Product Analogues as Human O-Glcnac Transferase Inhibitors.

Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

A sweet TET-à-tête-synergy of TET proteins and O-GlcNAc transferase in transcription

5-hydroxy methyl cytosine (5hmC) is a modification identified in vertebrates several decades ago. More recently, a possible role of 5hmC as an epigenetic modifier and/or transcriptional regulator has started to emerge, with altered levels in early embryonic development, embryonic stem (ES) cell differentiation and tumours (Tahiliani et al, 2009; Yang et al, 2012). The balance between 5hmC and 5-methyl cytosine (5mC) at gene promoters and CpG islands in the genome appears to be linked to pluripotency and lineage commitment of a cell (Ito et al, 2010). However, proteins with 5hmC binding capability have not yet been identified, and it has been proposed that 5hmC may only be a reaction intermediate in the process of demethylation (He et al, 2011; Ito et al, 2011). Over the last few years, ten-eleven translocation (Tet) family proteins have been shown to be responsible for the conversion of 5mC to 5hmC (Iyer et al, 2009; Loenarz and Schofield, 2009; Tahiliani et al, 2009). However, how Tet family proteins and 5hmC are linked to transcriptional regulation is currently not clear.