DHCR24 Knockdown Lead to Hyperphosphorylation of Tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites by Membrane Lipid-Raft Dependent PP2A Signaling in SH-SY5Y Cells

Neurochemical Research - Accumulating data suggest that the downregulation of DHCR24 is linked to the pathological risk factors of AD, denoting a potential role of DHCR24 in AD pathogenesis....     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

HeLa、HEK293、SH-SY5Y细胞中的Tau蛋白

通过间接免疫荧光测定了HeLa、HEK-293、SH-SY5Y细胞内Tau蛋白的分布,观察到在细胞间期单克隆抗体Tau-1的荧光信号分布于细胞质和胞核中．特别是HeLa细胞,其胞核内具有相对较高的Tau蛋白免疫荧光信号．通过分离SH-SY5Y的细胞核,更为清楚地显示了Tau蛋白在细胞核中的分布,并且免疫荧光信号与DNA的Hoechst33258染色信号相重合．Western blotting的测定结果进一步证明了SH-SY5Y细胞的胞质和胞核中均含有Tau蛋白的不同异构体．以上结果提示,Tau蛋白不仅存在于神经、肌肉等细胞内,也存在于肿瘤细胞系,并且分布于间期的胞核中．     

The Co-chaperone BAG2 Mediates Cold-Induced Accumulation of Phosphorylated Tau in SH-SY5Y Cells

Inclusions of phosphorylated tau (p-tau) are a hallmark of many neurodegenerative disorders classified as “tauopathy,” of which Alzheimer’s disease is the most prevalent form. Dysregulation of tau phosphorylation disrupts neuron structure and function, and hyperphosphorylated tau aggregates to form neurotoxic inclusions. The abundance of ubiquitin in tau inclusions suggests a defect in ubiquitin-mediated tau protein degradation by the proteasome. Under the temperature of 37 °C, the co-chaperone BAG2 protein targets phosphorylated tau for degradation via by a more-efficient, ubiquitin-independent pathway. In both in vivo and in vitro studies, cold exposure induces the accumulation of phosphorylated tau protein. The SH-SY5Y cell line differentiates into neuron-like cells on treatment with retinoic acid and is an established model for research on the effects of cold on tau phosphorylation. The aim of the present study was to investigate whether BAG2 mediates the cold-induced accumulation of phosphorylated tau protein. Our findings show that cold exposure causes a decrease in BAG2 expression in undifferentiated cells. Conversely, BAG2 expression is increased in differentiated cells exposed to cold. Further, undifferentiated cells exposed to cold had an increased proportion of p-tau to total tau, suggesting an accumulation of p-tau that is consistent with decreased levels of BAG2. Overexpression of BAG2 in cold-exposed undifferentiated cells restored levels of p-tau to those of 37 °C undifferentiated control. Interestingly, although BAG2 expression increased in differentiated cells, this increase was not accompanied by a decrease in the proportion of p-tau to total tau. Further, overexpression of BAG2 in cold exposed differentiated cells showed no significant difference in p-tau levels compared to 37 °C controls. Taken together, these data show that expression of BAG2 is differently regulated in a differentiation-dependent context. Our results suggest that repression of BAG2 expression or BAG2 activity by cold-sensitive pathways, as modeled in undifferentiated and differentiated cells, respectively, may be a causal factor in the accumulation of cytotoxic hyperphosphorylated tau protein via restriction of BAG2-mediated clearance of cellular p-tau.     

Decreases in valosin-containing protein result in increased levels of tau phosphorylated at Ser262/356

VCP/p97 is a multifunctional AAA+-ATPase involved in vesicle fusion, proteasomal degradation, and autophagy. Reported dysfunctions of these processes in Alzheimer disease (AD), along with the linkage of VCP/p97 to inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD) led us to examine the possible linkage of VCP to the AD-relevant protein, tau. VCP levels were reduced in AD brains, but not in the cerebral cortex of an AD mouse model, suggesting that VCP reduction occurs upstream of tau pathology. Genetic reduction of VCP in a primary neuronal model led to increases in the levels of tau phosphorylated at Ser(262/356), indicating that VCP may be involved in regulating post-translational processing of tau in AD, demonstrating a possible functional linkage between tau and VCP.     

The Neuroprotective Effects of Ginsenosides on Calcineurin Activity and Tau Phosphorylation in SY5Y Cells

Calcineurin (CN) is a Ca²⁺/calmodulin-dependent protein phosphatase expressed at high levels in brain. Many findings have shown that calcineurin plays an important role in tau hyperphosphorylation, which is one of the neuropathologic features in the brains of Alzheimer’s disease (AD). Based on the molecular screening model using p-nitrophenyl phosphate (p-NPP) as a substrate for preliminary screening and ³²P-labeled 19-residue phosphopeptide as a specific substrate for final determination, we found that the total ginsenoside extracts from stems and leaves of Panax ginseng (GSL) could enhance the phosphatase activity of purified CN. In the human neuroblastoma cells SY5Y, inhibition of CN by cyclosporine A (CsA) could induce hyperphosphorylation of tau at multiple sites, accompanied with oxidative stress. Pretreatment of the cells with GSL prior to CsA exposure could alleviate CsA-induced CN inhibition and tau hyperphosphorylation to some degree. Further oxidative parameters demonstrated that GSL caused increased SOD activity and content of SH significantly. It is speculated that GSL weakens CsA-induced CN inhibition through the antioxidant mechanisms. Although our results indicate that GSL may have neuroprotective effects on some characteristic features of AD, the chemical compositions of GSL and their potential for affecting the disease mechanism need to be further studied.     

Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.     

Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH-SY5Y Cells

Abstract: SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a K _D = 0.14 ± 0.06 n M and B _max = 7.3 ± 2.3 p M . Functionality of these receptors was demonstrated by an increased [³H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [³H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF β-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.     

Inhibition of PP-2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356

The regulation of the activity of CaMKII by PP-1 and PP-2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 microM okadaic acid (OA) inhibited approximately 65% of PP-2A and had no significant effect on PP-1 in the 16000xg tissue extract. Calyculin A (CL-A), 0.1 microM under the same conditions, inhibited approximately 50% of PP-1 and approximately 20% of PP-2A activities. In contrast, a mixture of OA and CL-A practically completely inhibited both PP-2A and PP-1 activities. The inhibition of the two phosphatase activities or PP-2A alone resulted in an approximately 2-fold increase in CaMKII activity and an approximately 8-fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN-62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced approximately 60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN-62-treated brain slices when further treated with OA and CL-A did not show any change in CaMKII activity. In vitro, both PP-2A and PP-1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP-2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP-2A/PP-1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity.     

Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells

Chronic exposure to polychlorinated biphenyls (PCBs), ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254) in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 μg/ml) reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase), measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.     

MAPK磷酸酯酶-1通过抑制JNK和p38的磷酸化调节SH-SY5Y神经母细胞瘤的凋亡

目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。     

CDK-5-Mediated Neurofilament Phosphorylation in SHSY5Y Human Neuroblastoma Cells

Cyclin-dependent kinase-5 (CDK-5) has been shown to play important roles in neuronal development and neurogenesis. In vitro studies indicate a role of CDK-5 in phosphorylation of neurofilaments (NFs). In this study, we have chosen the human neuroblastoma cell line SHSY5Y as a model system to study the in vivo phosphorylation of NF proteins by CDK-5. Upon differentiation of SHSY5Y cells with retinoic acid, we found that the phosphorylation of high molecular mass (NF-H) and medium molecular mass (NF-M) NFs increased, whereas the CDK-5 protein level and kinase activity were unaffected. The role of CDK-5 in the phosphorylation of cytoskeletal proteins was studied by using antisense oligonucleotides (ONs) to inhibit the expression of the CDK-5 gene. We found that inhibition of CDK-5 levels by antisense ON treatment resulted in a decrease in phosphorylation of NF-H that correlated with a decline in neurite outgrowth. These results demonstrate that CDK-5 is a major proline-directed kinase phosphorylating the human NF-H tail domain.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

K-252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells

Abstract: The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.     

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin.     

八氯腺苷诱导SH-SY5Y和SK-N-SH细胞G2/M期阻滞的分子机制不同

为探索八氯腺苷的抗肿瘤作用机制,以神经母细胞瘤SH-SY5Y和SK-N-SH细胞为对象,采用四唑盐比色实验（MTT法）证明,八氯腺苷具有明显的抑制肿瘤细胞增殖的作用,这种抑制作用呈剂量-时间依赖性.流式细胞分析显示,10 μmol/L八氯腺苷作用48 h后可导致靶细胞生长停滞于G ^₂/M期;SH-SY5Y细胞发生明显细胞凋亡,但SK-N-SH细胞却未见凋亡.Hoechst 33342染色显示,SK-N-SH细胞发生了核分裂异常.蛋白质免疫印迹分析证明,10 μmol/L 八氯腺苷处理SH SY5Y 48～72 h后,G^₂检验点调节蛋白ATM、Chk1、Cdc25C和Cdc2磷酸化形式明显上调,同时伴有caspase-3的激活,提示SH-SY5Y细胞发生了G^₂检验点通路和细胞凋亡途径的激活.与SH-SY5Y细胞不同,在SK-N-SH细胞中,八氯腺苷处理24～96 h时,磷酸化ATM、磷酸化Chk1/Chk2、磷酸化Cdc25C以及磷酸化Cdc2的水平呈现逐渐降低的趋势.结果提示,SK-N-SH细胞在八氯腺苷处理后发生了G^₂检验点失败.蛋白质免疫印迹分析还显示,八氯腺苷可诱导p53在SH-SY5Y细胞的表达,但却不能影响SK—N-SH细胞的p53组成性表达水平.p21在SK-N-SH的组成性表达随八氯腺苷处理时间延长而逐渐减少,但在处理前后的SH-SY5Y细胞均未检测到p21蛋白的表达.上述实验结果提示,八氯腺苷抑制两种细胞增殖的机制不同：在SH-SY5Y细胞,八氯腺苷可激活ATM-Chk-Cdc25C-Cdc2/cyclin途径和凋亡通路,使细胞发生G_²/M期阻滞和细胞凋亡;在SK-N-SH细胞,八氯腺苷诱导G^₂检验点失败,导致细胞阻滞在有丝分裂期,并发生有丝分裂异常.2种不同的细胞命运可能还与p53和p21表达不同有关.     

Enhanced Toxicity to the Catecholamine Tyramine in Polyglutamine Transfected SH-SY5Y Cells

Huntington’s disease (HD) is a progressive neurodegenerative disorder, of which the pathogenesis is not completely understood. In patients with Huntington’s disease, there is a mutation in the gene encoding the protein huntingtin, which results in an expanded polyglutamine sequence leading to degeneration of the basal ganglia. There is mounting evidence that metabolism of the transmitter dopamine by the enzyme monoamine oxidase may contribute to striatal damage in mitochondrial toxin-induced models of HD. In this study, we have examined the role of the catecholamine tyramine in neural SH-SY5Y cells transfected with normal and expanded polyglutamine repeat numbers. Our findings demonstrate that cells containing a pathological number of polyglutamines are more sensitive to tyramine than cells with a non-pathological number. Tyramine-induced cell death was attenuated by MAO inhibitors as well as with catalase and the iron chelator deferoxamine, suggesting that H₂O₂ might mediate the observed toxicity. These observations support the notion that the metabolism of dopamine plays a role in neuron death in Huntington’s disease.     

Carbachol Inhibits Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity in SH-SY5Y Neuroblastoma Cells

Abstract: SH-SY5Y human neuroblastoma cells express muscarinic M₃ receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross-talk following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3′ phosphate or its 4′ phosphate, respectively. In this study, the effect of carbachol on insulin-stimulated PI 3-kinase (PI3K) activity was examined in SH-SY5Y cells. Insulin addition to the cell medium induced a 10–26-fold increase in anti-phosphotyrosine-immunoprecipitable PI3K activity. Preincubation with 1 mM carbachol inhibited the insulin-stimulated PI3K activity in a time-dependent manner, with half-maximal and maximal inhibition times of 4 and 15 min, respectively. Atropine blocked the inhibitory effect of carbachol. Although carbachol did not change the amount of 85-kDa subunit protein regulatory unit associated with tyrosine-phosphorylated proteins, either in control or in insulin-stimulated cells, it appears to decrease the amount of associated 110-kDa catalytic subunit protein in the latter instance. Because PI3K activity from SH-SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP-DAG) or phosphatidate (PA), we examined the presence of these lipids in SH-SY5Y cells that had been treated with carbachol. Formation of both lipids was increased in a time-dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell-permeable homologue didecanoyl-CDP-DAG to intact cells inhibited insulin-stimulated PI3K activity up to 75%, with an IC₅₀ of 0.5 µM, a result that further supports a proposed lipid-mediated inhibition of PI3K. Exogenously added didecanoyl-PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4-kinase-mediated signal transduction pathway leads to down-regulation of the PI3K-mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP-DAG or by other consequences of phosphoinositidase C-linked receptor activation, is discussed.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.