Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.     

Suppression of native defense mechanisms,SIRT1 and PPARγ, by dietary glycoxidants precedes disease in adult humans; relevance to lifestyle-engendered chronic diseases

SIRT1 and PPARγ, host defenses regulating inflammation and metabolic functions, are suppressed under chronic high oxidant stress and inflammation (OS/Infl) conditions. In diabetes, dietary advanced glycation end products (dAGEs) cause OS/Infl and suppress SIRT1. Herein, we ask whether dAGEs also suppress host defense in adults without diabetes. The relationships between dAGEs and basal SIRT1 mRNA, PPARγ protein levels in mononuclear cells (MNC) and circulating inflammatory/metabolic markers were examined in 67 healthy adults aged >60 years and in 18 subjects, before and after random assignment to either a standard diet (regular >15 AGE Eq/day) or an isocaloric AGE-restricted diet (<10 AGE Eq/day) for 4 months. Also, the interactions of AGEs and anti-AGE receptor-1 (AGER1) with SIRT1 and PPARγ were assessed in wild type (WT) and AGER1-transduced (AGER1⁺) MNC-like THP-1 cells. We found that dAGE, but not caloric intake, correlated negatively with MNC SIRT1 mRNA levels and positively with circulating AGEs (sAGEs), OS/infl, MNC TNFα and RAGE. Basal MNC PPARγ protein was also lower in consumers of regular vs. AGE-restricted diet. AGE restriction restored MNC SIRT1 and PPARγ, and significantly decreased sAGEs, 8-isoprostanes, VCAM-1, MNC TNFα and RAGE. Model AGEs suppressed SIRT1 protein and activity, and PPARγ protein in WT, but not in AGER1⁺ cells in vitro. In conclusion, chronic consumption of high-AGE diets depletes defenses such as SIRT1 and PPARγ, independent of calories, predisposing to OS/Infl and chronic metabolic disease. Restricted entry of oral AGEs may offer a disease-prevention alternative for healthy adults.     

p66shc negatively regulates insulin-like growth factor I signal transduction via inhibition of p52shc binding to Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 leading to impaired growth factor receptor-bound protein-2 membrane recruitment

Our previous studies have indicated an essential role of p52shc in mediating IGF-I activation of MAPK in smooth muscle cells (SMC). However, the role of the p66 isoform of shc in IGF-I signal transduction is unclear. In the current study, two approaches were employed to investigate the role of p66shc in mediating IGF-I signaling. Knockdown p66shc by small interfering RNA enhanced IGF-I-stimulated p52shc tyrosine phosphorylation and growth factor receptor-bound protein-2 (Grb2) association, resulting in increased IGF-I-dependent MAPK activation. This was associated with enhanced IGF-I-stimulated cell proliferation. In contrast, knockdown of p66shc did not affect IGF-I-stimulated IGF-I receptor tyrosine phosphorylation. Overexpression of p66shc impaired IGF-I-stimulated p52shc tyrosine phosphorylation and p52shc-Grb2 association. In addition, IGF-I-dependent MAPK activation was also impaired, and SMC proliferation in response to IGF-I was inhibited. IGF-I-dependent cell migration was enhanced by p66shc knockdown and attenuated by p66shc overexpression. Mechanistic studies indicated that p66shc inhibited IGF-I signal transduction via competitively inhibiting the binding of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to SHP substrate-1 (SHPS-1), leading to the disruption of SHPS-1/SHP-2/Src/p52shc complex formation, an event that has been shown previously to be essential for p52shc phosphorylation and Grb2 recruitment. These findings indicate that p66shc functions to negatively regulate the formation of a signaling complex that is required for p52shc activation in response to IGF-I, thus leading to attenuation of IGF-I-stimulated cell proliferation and migration.     

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs.     

Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.     

Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C β (PKCβ) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.     

Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

Antagonism of p66shc by melanoma inhibitory activity

The p66shc protein governs oxidant stress and mammalian lifespan. Here, we identify melanoma inhibitory activity (MIA), a protein secreted by melanoma cells, as a novel binding partner and antagonist of p66shc. The N-terminal collagen homology-2 (CH2) domain of p66shc binds to the Src Homology-3 (SH3)-like domain of MIA in vitro. In cells, ectopically expressed MIA and p66shc colocalize and co-precipitate. MIA also co-precipitates with the CH2 domain of p66shc in vivo. MIA expression in vivo suppresses p66shc-stimulated increase in endogenous hydrogen peroxide (H(2)O(2)), and inhibits basal and H(2)O(2)-induced phosphorylation of p66shc on serine 36 and H(2)O(2)-induced death. In human melanoma cells expressing MIA, endogenous MIA and p66shc co-precipitate. Downregulation of MIA in melanoma cells increases basal and ultraviolet radiation (UVR)-induced phosphorylation of p66shc on serine 36, augments endogenous H(2)O(2) levels, and increases their susceptibility to UVR-induced death. These findings show that MIA binds to p66shc, and suggest that this interaction antagonizes phosphorylation and function of p66shc.     

Distinct mechanisms of taxol-induced serine phosphorylation of the 66-kDa Shc isoform in A549 and RAW 264.7 cells

Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.     

Tau phosphorylation and μ-calpain activation mediate the dexamethasone-induced inhibition on the insulin-stimulated Akt phosphorylation

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.     

Amelioration of oxidant stress by the defensin lysozyme

Reactive oxidant species (ROS), products of normal metabolism, cause oxidant injury if they accumulate in pathological amounts. Lysozyme (LZ) contains an 18-amino acid domain that binds agents such as advanced glycation end products (AGE) that generate ROS. We examined whether endogenous LZ affected physiological, or baseline, antioxidant balance and provided protection against both acute and chronic oxidant injury, using paraquat and H2O2 as agents of acute injury and AGE for chronic injury. Hen egg LZ-Tg mice had threefold higher serum LZ levels and decreased baseline AGE levels in serum and liver. These findings were linked to an enhanced baseline systemic GSH-to-GSSG ratio. Baseline levels of stress response genes p66(Shc) and c-Jun were also lower in liver tissue of LZ-Tg mice. Survival from severe oxidant injury induced by paraquat was twofold greater in LZ-Tg mice. In addition, LZ-Tg mice were resistant to chronic exogenous oxidant stress (OS) induced by AGE administration. Preincubation of hepatocytes (Hep G2) with LZ suppressed redox balance at baseline, as well as OS after added paraquat, AGE, or H2O2. LZ also ameliorated paraquat-enhanced cell apoptosis in a dose-dependent manner and suppressed AGE-induced p66(Shc) expression and c-Jun phosphorylation in Hep G2 cells. Thus LZ provides protection against acute and chronic oxidant injury by mechanisms involving suppression of ROS generation and of OS response genes.     

Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.