O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.     

Akt1 is dynamically modified with O-GlcNAc following treatments with PUGNAc and insulin-like growth factor-1

The Ser/Thr kinase Akt1 is activated by growth factors subsequent to its phosphorylation on Thr308 and Ser473. In the present study, Akt1 was found to be constitutively modified with O-GlcNAc. Treatment of SH-SY5Y cells with O(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which inhibits the enzymatic removal of O-GlcNAc from proteins, increased cytosolic O-GlcNAc-Akt1 levels. Treatment of cells with insulin-like growth factor-1 (IGF-1) also increased O-GlcNAc-Akt1 levels and increased Akt1 phosphorylation. PUGNAc treatment did not attenuate IGF-1 induced Akt1 phosphorylation. These results indicate that Akt1 can be simultaneously modified with O-GlcNAc and phosphorylated. However, PUGNAc induced the nuclear accumulation of Akt1 suggesting that the O-GlcNAc-modification on Akt1 may play a role in Akt1 nuclear localization.     

Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells

LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO(-))-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO(-) (5 microM) significantly increased the phosphorylation of LKB1 at Ser(428) and protein kinase Czeta (PKCzeta) at Thr(410). These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser(473)) of Akt, and induction of apoptosis. ONOO(-) enhanced Akt-Ser(473) phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO(-) inhibited Akt Ser(473) phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKCzeta directly phosphorylated LKB1 at Ser(428) in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser(380)/Thr(382/383). Finally, ONOO(-) enhanced PKCzeta nuclear import and LKB1 nuclear export. We conclude that PKCzeta mediates LKB1-dependent Akt inhibition in response to ONOO(-), resulting in endothelial apoptosis.     

Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Nontargeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.     

In silico modulation of apoptotic Bcl-2 proteins by mistletoe lectin-1: functional consequences of protein modifications

The mistletoe lectin-1 (ML-1) modulates tumor cell apoptosis by triggering signaling cascades through the complex interplay of phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modification in pro- and anti-apoptotic proteins. In particular, ML-1 is predicted to induce dephosphorylation of Bcl-2-family proteins and their alternative O-GlcNAc modification at specific, conserved Ser/Thr residues. The sites for phosphorylation and glycosylation were predicted and analyzed using Netphos 2.0 and YinOYang 1.2. The involvement of modified Ser/Thr, and among them the potential Yin Yang sites that may undergo both types of posttranslational modification, is proposed to mediate apoptosis modulation by ML-1.     

Glycosylation of the murine estrogen receptor-alpha

O-linked N-acetylglucosamine (O-GlcNAc) is a highly dynamic and abundant modification found on nuclear and cytoplasmic proteins of nearly all eukaryotes. O-GlcNAc addition is required for life at the single cell level and is analogous to protein phosphorylation in most respects. In a previous study (M.S. Jiang, G.W. Hart, A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J. Biol. Chem. 270 (1997) 2421-2428), we demonstrated that a subpopulation of the murine estrogen receptor-alpha (mER-alpha) is modified by O-GlcNAc at Thr(575). Here we mutated mER-alpha to convert Thr(575) and Ser(576) to Val and Ala, respectively. Surprisingly, this glycosylation-site mutant is still extensively modified by O-GlcNAc. Analyses of glycopeptides identified two additional sites of modification on mER-alpha, at Ser(10) and Thr(50) near the N-terminus. The major glycosylation sites are within or near PEST regions, suggesting that O-GlcNAc may regulate mER-alpha turnover.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

Oct-2 DNA binding transcription factor: functional consequences of phosphorylation and glycosylation

Phosphorylation and O-GlcNAc modification often induce conformational changes and allow the protein to specifically interact with other proteins. Interplay of phosphorylation and O-GlcNAc modification at the same conserved site may result in the protein undergoing functional switches. We describe that at conserved Ser/Thr residues of human Oct-2, alternative phosphorylation and O-GlcNAc modification (Yin Yang sites) can be predicted by the YinOYang1.2 method. We propose here that alternative phosphorylation and O-GlcNAc modification at Ser191 in the N-terminal region, Ser271 and 274 in the linker region of two POU sub-domains and Thr301 and Ser323 in the POUh subdomain are involved in the differential binding behavior of Oct-2 to the octamer DNA motif. This implies that phosphorylation or O-GlcNAc modification of the same amino acid may result in a different binding capacity of the modified protein. In the C-terminal domain, Ser371, 389 and 394 are additional Yin Yang sites that could be involved in the modulation of Oct-2 binding properties.     

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).     

Developmental Regulation of Protein O-GlcNAcylation, O-GlcNAc Transferase, and O-GlcNAcase in Mammalian Brain

O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins by β-N-acetylglucosamine (GlcNAc). The dynamic addition and removal of O-GlcNAc groups to and from proteins are catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA), respectively. O-GlcNAcylation often modulates protein phosphorylation and regulates several cellular signaling and functions, especially in the brain. However, its developmental regulation is not well known. Here, we studied protein O-GlcNAcylation, OGT, and OGA in the rat brain at various ages from embryonic day 15 to the age of 2 years. We found a gradual decline of global protein O-GlcNAcylation during developmental stages and adulthood. This decline correlated positively to the total protein phosphorylation at serine residues, but not at threonine residues. The expression of OGT and OGA isoforms was regulated differently at various ages. Immunohistochemical studies revealed ubiquitous distribution of O-GlcNAcylation at all ages. Strong immunostaining of O-GlcNAc, OGT, and OGA was observed mostly in neuronal cell bodies and processes, further suggesting the role of O-GlcNAc modification of neuronal proteins in the brain. These studies provide fundamental knowledge of age-dependent protein modification by O-GlcNAc and will help guide future studies on the role of O-GlcNAcylation in the mammalian brain.     

Autophosphorylation of Akt at threonine 72 and serine 246. A potential mechanism of regulation of Akt kinase activity

Activation of the serine/threonine protein kinase Akt is a multistep process. We here propose that the kinase activity of Akt is regulated via autophosphorylation in trans at two putative sites (threonine 72 and serine 246) that lie in the characteristic Akt substrate motif (RXRXX(S/T)). Incubation of Akt immunoprecipitated from transfected cells with a pre-activated Akt recombinant protein and gamma-32P-labeled ATP led to marked incorporation of radioactivity in wild-type Akt but not Akt/T72A/S246A mutant. Western blot analysis using a phosphorylated Akt substrate-specific antibody of Akt immunoprecipitated from transfected cells confirmed the autophosphorylation of wild-type Akt but not Akt/T72A/S246A mutant in insulin-like growth factor-1 (IGF-1)-stimulated cells. Autophosphorylation of Akt on Thr-72 and Ser-246 appeared to require prior phosphorylation of Akt on Thr-308 and Ser-473. Compared with wild-type Akt, Akt/T72A/S246A mutant exhibited markedly reduced basal Akt kinase activity and response to cellular stimulation by insulin-like growth factor-1, and also conferred less cellular resistance to doxorubicin-induced apoptosis. The findings from these pilot studies suggest that Akt regulates its kinase activity through autophosphorylation. Further investigation of this potential novel regulatory mechanism by which Akt performs its cellular functions is warranted.     

Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.     

Hsp90 regulates O-linked β-N-acetylglucosamine transferase: a novel mechanism of modulation of protein O-linked β-N-acetylglucosamine modification in endothelial cells

O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is involved in many important cellular processes. Increased O-GlcNAc has been implicated in major diseases, such as diabetes and its complications and cardiovascular and neurodegenerative diseases. Recently, we reported that O-GlcNAc modification occurs in the proteasome and serves to inhibit proteasome function by blocking the ATPase activity in the 19S regulatory cap, explaining, at least in part, the adverse effects of O-GlcNAc modification and suggesting that downregulating O-GlcNAc might be important in the treatment of human diseases. In this study, we report on a novel mechanism to modulate cellular O-GlcNAc modification, namely through heat shock protein 90 (Hsp90) inhibition. We observed that O-linked β-N-acetylglucosamine transferase (OGT) interacts with the tetratricopeptide repeat binding site of Hsp90. Inhibition of Hsp90 by its specific inhibitors, radicicol or 17-N-allylamino-17-demethoxygeldanamycin, destabilized OGT in primary endothelial cell cultures and enhanced its degradation by the proteasome. Furthermore, Hsp90 inhibition downregulated O-GlcNAc protein modifications and attenuated the high glucose-induced increase in O-GlcNAc protein modification, including high glucose-induced increase in endothelial or type 3 isoform of nitric oxide synthase (eNOS) O-GlcNAcylation. These results suggest that Hsp90 is involved in the regulation of OGT and O-GlcNAc modification and that Hsp90 inhibitors might be used to modulate O-GlcNAc modification and reverse its adverse effects in human diseases.     

Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308). We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.