Phosphorylation of neuroglycan C, a brain-specific transmembrane chondroitin sulfate proteoglycan, and its localization in the lipid rafts

Neuroglycan C (NGC) is a brain-specific transmembrane chondroitin sulfate proteoglycan. In the present study, we examined whether NGC could be phosphorylated in neural cells. On metabolic labeling of cultured cerebral cortical cells from the rat fetus with (32)P(i), serine residues in NGC were radiolabeled. Some NGC became detectable in the raft fraction from the rat cerebrum, a signaling microdomain of the plasma membrane, with cerebral development. NGC from the non-raft fraction, not the raft fraction, could be phosphorylated by an in vitro kinase reaction. The phosphorylation of NGC was inhibited by adding to the reaction mixture a recombinant peptide representing the ectodomain of NGC, but not by adding a peptide representing its cytoplasmic domain. NGC could be labeled by an in vitro kinase reaction using [gamma-(32)P]GTP as well as [gamma-(32)P]ATP, and this kinase activity was partially inhibited by 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a selective inhibitor of casein kinase II. In addition to the intracellular phosphorylation, NGC was also phosphorylated at the cell surface by an ectoprotein kinase. This is the first report to demonstrate that NGC can be phosphorylated both intracellularly and pericellularly, and our findings suggest that a kinase with a specificity similar to that of casein kinase II is responsible for the NGC ectodomain phosphorylation.     

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.     

Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan

Neuroglycan C (NGC), a brain-specific transmembrane proteoglycan, is thought to bear not only chondroitin sulfate but also N- and O-linked oligosaccharides on its core protein. In this study, we isolated and purified NGC from rat brains at various developmental stages by immunoaffinity column chromatography or by immunoprecipitation, and examined the structural characters of its carbohydrate moiety. The chondroitin sulfate disaccharide composition of NGC at postnatal day 10 was significantly different from those of two secreted chondroitin sulfate proteoglycans, neurocan and phosphacan, purified from the brain at the same developmental stage; higher levels of 4-sulfate unit and E unit, a disulfated disaccharide unit, and a lower level of 6-sulfate unit. The levels of both 6-sulfate and E units decreased with a compensatory increase of 4-sulfate unit with postnatal development of the brain. Lectin-blot analysis of the NGC core glycoprotein prepared by chondroitinase digestion confirmed that NGC actually bore both N- and O-linked carbohydrates, and also revealed that lectin-species reactive with NGC did not always recognize other brain-specific proteoglycans, neurocan and phosphacan, and vice versa, even though they were isolated from the brain at the same stage. The reactivity of NGC with lectins and with the HNK-1 antibody markedly changed as the brain matured. These findings indicate that the structure of the carbohydrate moiety of NGC is developmentally regulated, and differs from those of neurocan and phosphacan. The developmentally-regulated structural change of the carbohydrates on NGC may be partly implicated in the modulation of neuronal cell recognition during brain development.     

Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan

Neuroglycan C (NGC), a brain-specific transmembrane proteoglycan, is thought to bear not only chondroitin sulfate but also N- and O-linked oligosaccharides on its core protein. In this study, we isolated and purified NGC from rat brains at various developmental stages by immunoaffinity column chromatography or by immunoprecipitation, and examined the structural characters of its carbohydrate moiety. The chondroitin sulfate disaccharide composition of NGC at postnatal day 10 was significantly different from those of two secreted chondroitin sulfate proteoglycans, neurocan and phosphacan, purified from the brain at the same developmental stage; higher levels of 4-sulfate unit and E unit, a disulfated disaccharide unit, and a lower level of 6-sulfate unit. The levels of both 6-sulfate and E units decreased with a compensatory increase of 4-sulfate unit with postnatal development of the brain. Lectin-blot analysis of the NGC core glycoprotein prepared by chondroitinase digestion confirmed that NGC actually bore both N- and O-linked carbohydrates, and also revealed that lectin-species reactive with NGC did not always recognize other brain-specific proteoglycans, neurocan and phosphacan, and vice versa, even though they were isolated from the brain at the same stage. The reactivity of NGC with lectins and with the HNK-1 antibody markedly changed as the brain matured. These findings indicate that the structure of the carbohydrate moiety of NGC is developmentally regulated, and differs from those of neurocan and phosphacan. The developmentally-regulated structural change of the carbohydrates on NGC may be partly implicated in the modulation of neuronal cell recognition during brain development. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

B cell receptor-mediated Syk-independent activation of phosphatidylinositol 3-kinase,Ras, and mitogen-activated protein kinase pathways

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.     

Atypical protein kinase C (PKCzeta/lambda) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways

Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCzeta/lambda) to plasma membrane lipid raft microdomains. This redistribution of PKCzeta/lambda was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCzeta/lambda to the plasma membrane through an indirect association with the Par6-Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCzeta. Although PI 3-kinase activation also resulted in PKCzeta/lambda phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3beta phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways. Together, these data demonstrate that PKCzeta/lambda can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.     

The role of phosphatidylinositol 3-kinase, Src kinase, and protein kinase A signaling pathways in insulin and glucagon regulation of CYP2E1 expression

The signaling pathways involved in insulin and glucagon regulation of CYP2E1 expression were examined in primary cultured rat hepatocytes. Insulin addition to primary cultured rat hepatocytes for 24 h resulted in an approximately 80% and >90% decrease in CYP2E1 mRNA levels at 1 and 10 nM insulin, respectively, relative to untreated cells. Addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, or the Src kinase inhibitor geldanamycin, prior to insulin addition, inhibited the insulin-mediated decline in CYP2E1 mRNA. In contrast, treatment of cells with glucagon (100 nM), or the cAMP analogue dibutyryl-cAMP (50 microM), for 24 h increased CYP2E1 mRNA levels by approximately 7-fold. Addition of the protein kinase A inhibitor H89 prior to glucagon treatment attenuated the glucagon-mediated increase in CYP2E1 mRNA by approximately 70%. Glucagon (100 nM) opposed the effects of insulin (1 nM) on CYP2E1 mRNA expression and conversely, insulin blocked the effects of glucagon. These data provide compelling evidence for the regulation of CYP2E1 expression via mutually antagonistic signaling pathways involving insulin and glucagon.     

Identification from cDNA of the precursor form of a chondroitin sulfate proteoglycan core protein

The yolk sac carcinoma cell line L2 secretes a chondroitin/dermatan sulfate proteoglycan that has an Mr 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1321-1325). From additional cDNA sequences described in this report we have identified the prepro core protein precursor of the yolk sac carcinoma chondroitin/dermatan sulfate proteoglycan. From the amino acid sequence of the core protein precursor can be deduced the protein processing events in the biosynthesis of the proteoglycan. The amino acid sequence shows that the 104-amino acid mature core protein is processed from a 179-amino acid prepro core protein precursor which, in addition to the mature core protein, contains a 26-amino acid signal peptide as well as a 49-amino acid propeptide. The molecular weight of the prepro core protein predicted from the cDNA sequence (Mr = 18,600) was in good agreement with the molecular weight of the in vitro translation product (Mr = 19,000) of hybrid-selected mRNA. Accordingly, we have designated the proteoglycan core protein PG19. Further analysis of the PG19 mRNA by RNA sequencing confirmed the identification of the core protein translation initiation codon by revealing stop codons in all three reading frames of the upstream mRNA sequence. Primer extension analyses demonstrated that the 5' untranslated sequence of the proteoglycan mRNA is approximately 220 nucleotides in length, which, combined with the length of cDNA clones, accounts for the entire length of the coding sequence of PG19 mRNA from L2 cells. The cDNA sequences presented here establish the complete protein sequence of PG19 and provide evidence of polypeptide processing during the biosynthesis of the proteoglycan core protein.     

Akt2, a novel functional link between p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in myogenesis

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentiation, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2.     

Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase,extracellular signal-regulated kinase and protein kinase C signaling pathways

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.     

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase,phosphatidylinositol 3-kinase,Akt and mitogen-activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.     

Phosphoinositide fatty acids regulate phosphatidylinositol 5-kinase, phospholipase C and protein kinase C activities.

PtdIns(4,5)P(2) generally results from phosphorylation of PtdIns(4)P by the phosphatidylinositol 5-kinase (PtdIns5-K). Its hydrolysis by phospholipase C (PLC) yields inositol 1,4,5-trisphosphate and diacylglycerol, which stimulates protein kinase C (PKC). We show that epithelial cells of the cockroach rectum contain three different inositol lipids: PtdIns(4,5)P(2), PtdIns(4)P, and PtdIns. They are composed of six major fatty acids: palmitic (16:0) stearic (18:0), oleic (18:1n--9), linoleic (18:2n--6), linolenic (18:3n--3), and arachidonic (20:4n-6) acids. The fatty acid preference of each of the above enzymes was evaluated by incorporating different fatty acids in pairs into membrane lipids. Incorporation of 16:0 plus 18:1n--9 provoked an increase in PtdIns(4,5)P2-PLC activity and a decrease in PtdIns5-K activity. In contrast, incorporation of 16:0 plus 18:3n--3 led to a potentiation of PtdIns5-K activity and a decrease in PtdIns(4,5)P(2)-PLC activity. Furthermore, PLC and PtdIns5-K acted preferentially on substrates containing 18:3n--3, and 18:3n--3-containing diacylglycerol specifically potentiated PKC activity. Thus, we propose that the fatty acids that make up the phosphoinositides function as intracellular modulators of the activity of certain enzymes.     

Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase

Although protein kinase C (PKC) and phosphatidylinositol 3 (PI3)-kinase are implicated in cardioprotective signal transduction mediated by ischemic preconditioning, their role in pharmacological preconditioning (PPC) has not been determined. Cultured neonatal rat cardiomyocytes (CMCs) were subjected to simulated ischemia for 2 h followed by 15 min of reoxygenation. PPC of CMCs consisted of administration of 50 microM adenosine, 50 microM diazoxide, and 50 microM S-nitroso-N-acetylpenicillamine (SNAP), each alone or in combination, for 15 min followed by 30 min of washout before simulated ischemia. Although PKC-epsilon and PI3-kinase were significantly activated during treatment with adenosine, activation of these kinases dissipated after washout. In contrast, PPC combined with adenosine, diazoxide, and SNAP elicited sustained activation of PKC-epsilon and PI-3 kinase after washout. The combined-PPC, but not the single-PPC, protocol conferred antiapoptotic and antinecrotic effects after reoxygenation. The PKC inhibitor chelerythrine (5 microM) or the PI3-kinase inhibitor LY-294002 (10 microM) given during the washout period partially blocked the activation of PKC-epsilon and PI3-kinase mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely inhibited activation of PKC-epsilon and PI3-kinase. Chelerythrine or LY-294002 partially blocked antiapoptotic and antinecrotic effects mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely abrogated antiapoptotic and antinecrotic effects. These results suggest that the combined-PPC protocol confers cardioprotective memory through sustained and interdependent activation of PKC and PI3-kinase.     

Macrophage's proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase

Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca(2+) flux is an important step in its capacity to halt phagosome maturation. This affect on Ca(2+) release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca(2+) in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca(2+)-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF-alpha production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-alpha, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.     

Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.     

Differential regulation of phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase, and AMP-activated protein kinase pathways during menadione-induced oxidative stress in the kidney of young and old rats

We investigated regulation of various signal transduction pathways during oxidative stresses in the kidney of young and aged rats. Menadione-induced regulation of molecules in PI 3-kinase, MAPK, and AMPK pathways was determined in the young (2 months) and old (24 months) groups. PI 3-kinase activity and Akt phosphorylation were significantly reduced in the old compared with the young. PTEN tumor suppressor was also lower in its expression and phosphorylation levels in the old. Response of the molecules in PI 3-kinase pathway to menadione was minimized. In contrast, over 5-fold induction of ERK1/2 phosphorylation by menadione was observed in both groups. On the other hand, basal activities as well as menadione-induced activities of JNK1 and AMPK were higher in the old than in the young. While p27(Kip1), p53, and p21(Waf1) were slightly increased by menadione in both groups, the basal induction level in the old was considerably higher. In conclusion, the results suggest that the age-related down-regulation of PI 3-kinase/Akt pathway and up-regulation of JNK1, AMPK, and p53 pathways may be responsible for the increased susceptibility to oxidative stress.