Inhibition of insulin-like growth factor-1 receptor and IRS-2 signaling by ethanol in SH-SY5Y neuroblastoma cells

The effect of ethanol on insulin-like growth factor-1 (IGF-I)-mediated signal transduction and functional activation in neuronal cells was examined. In human SH-SY5Y neuroblastoma cells, ethanol inhibited tyrosine autophosphorylation of the IGF-I receptor. This corresponded to the inhibition of IGF-I-induced phosphorylation of p42/p44 mitogen-activated/extracellular signal-regulated protein kinase (MAPK) by ethanol. Insulin-related substrate-2 (IRS-2) and focal adhesion kinase phosphorylation were reduced in the presence of ethanol, which corresponded to the prevention of lamellipodia formation (30 min). By contrast, ethanol had no effect on Shc phosphorylation when measured up to 1 h, and did not affect the association of Grb-2 with Shc. Neurite formation at 24 h was similarly unaffected by ethanol. The data indicate that the IGF-I receptor is a target for ethanol in SH-SY5Y cells However, there is diversity in the sensitivity of signaling elements within the IGF-I receptor tyrosine kinase signaling cascades to ethanol, which can be related to the inhibition of specific functional events in neuronal activation.     

Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.     

Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice

Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.     

NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1

Protein synthesis is essential for the stabilization of glutamate receptor-dependent forms of long-lasting hippocampal synaptic plasticity and for the consolidation of memory, but the signal transduction mechanisms that regulate translation factors during these processes are not well understood. As a first step towards understanding how translation is activated during synaptic plasticity, we investigated how the eukaryotic initiation factor 4E (eIF4E), a rate-limiting mRNA cap-binding protein, and its kinase, Mnk1, are regulated by protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and N-methyl-D-aspartate (NMDA) receptor activation in hippocampal area CA1. We found that treatment of mouse hippocampal slices with either phorbol ester, to activate PKC, or forskolin, to activate PKA, resulted in activation of Mnk1 and increased eIF4E phosphorylation that was dependent on extracellular signal-regulated kinase (ERK). Similarly, brief treatment of hippocampal slices with NMDA resulted in activation of Mnk1 and increased phosphorylation of eIF4E. The NMDA-induced activation of Mnk1 and increased phosphorylation of eIF4E were dependent on PKA and ERK, but not PKC, and were present in synaptoneurosome preparations. Immunohistochemical analysis revealed that the PKA- and ERK-dependent increases in Mnk1 activation induced by NMDA also occurred in dendrites. These findings identify a specific regulatory pathway that can couple NMDA receptor activation to translation initiation factors in the hippocampus, and may represent a mechanism for triggering dendritic protein synthesis during long-term potentiation and long-term memory formation.     

Identification of a novel signaling pathway and its relevance for GluA1 recycling

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.     

Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons.

The molecular effects of use-dependent changes in synaptic transmission were studied in individual CA1 pyramidal neurons from rat hippocampal slices. Potentiation of excitatory postsynaptic currents was associated with coordinate changes in the relative abundance of several mRNAs 30 min to 3 hr after stimulation. There was a 300% increase in calcium/calmodulin-dependent protein kinase II mRNA levels concordant with a 50% decrease in protein kinase C beta 1 isoform mRNA. A 2-fold increase in zif-268 mRNA was seen, while increases in c-fos and c-jun mRNA levels were inconsistent, gamma-Aminobutyric acid A receptor beta 1 subunit mRNA levels increased 3-fold. Potentiation-induced changes were prevented by N-methyl-D-aspartate receptor blockade. Changes in mRNA abundance in individual cells, with synaptic and glial interactions intact, combine to produce a molecular fingerprint of a potentiated CA1 neuron.     

Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

Learning-induced glutamate receptor phosphorylation resembles that induced by long term potentiation

Long term potentiation and long term depression of synaptic responses in the hippocampus are thought to be critical for certain forms of learning and memory, although until recently it has been difficult to demonstrate that long term potentiation or long term depression occurs during hippocampus-dependent learning. Induction of long term potentiation or long term depression in hippocampal slices in vitro modulates phosphorylation of the alpha-amino-3-hydrozy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor subunit GluR1 at distinct phosphorylation sites. In long term potentiation, GluR1 phosphorylation is increased at the Ca2+/calmodulin-dependent protein kinase and protein kinase C site serine 831, whereas in long term depression, phosphorylation of the protein kinase A site serine 845 is decreased. Indeed, phosphorylation of one or both of these sites is required for long term synaptic plasticity and for certain forms of learning and memory. Here we demonstrate that training in a hippocampus-dependent learning task, contextual fear conditioning is associated with increased phosphorylation of GluR1 at serine 831 in the hippocampal formation. This increased phosphorylation is specific to learning, has a similar time course to that in long term potentiation, and like memory and long term potentiation, is dependent on N-methyl-D-aspartate receptor activation during training. Furthermore, the learning-induced increase in serine 831 phosphorylation is present at synapses and is in heteromeric complexes with the glutamate receptor subunit GluR2. These data indicate that a biochemical correlate of long term potentiation occurs at synapses in receptor complexes in a final, downstream, postsynaptic effector of long term potentiation during learning in vivo, further strengthening the link between long term potentiation and memory.     

Epidermal growth factor receptor phosphorylates protein kinase C {delta} at Tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H(2)O(2)-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCdelta at Tyr332 in vitro. PKCdelta was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H(2)O(2)-induced tyrosine phosphorylation of PKCdelta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKCdelta at Tyr332 in the H(2)O(2)-stimulated but not in the growth-factor treated cells, and suggest that PKCdelta in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.     

CaM kinase II and protein kinase C activations mediate enhancement of long-term potentiation by nefiracetam in the rat hippocampal CA1 region

Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as cognitive-enhancing effect. We previously showed that nefiracetam potentiates NMDA-induced currents in cultured rat cortical neurons. To address questions whether nefiracetam affects NMDA receptor-dependent synaptic plasticity in the hippocampus, we assessed effects of nefiracetam on NMDA receptor-dependent long-term potentiation (LTP) by electrophysiology and LTP-induced phosphorylation of synaptic proteins by immunoblotting analysis. Nefiracetam treatment at 1-1000 nM increased the slope of fEPSPs in a dose-dependent manner. The enhancement was associated with increased phosphorylation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) without affecting synapsin I phosphorylation. In addition, nefiracetam treatment increased PKCalpha activity in a bell-shaped dose-response curve which peaked at 10 nM, thereby increasing phosphorylation of myristoylated alanine-rich protein kinase C substrate and NMDA receptor. Nefiracetam treatment did not affect protein kinase A activity. Consistent with the bell-shaped PKCalpha activation, nefiracetam treatment enhanced LTP in the rat hippocampal CA1 region with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with CaMKII and PKCalpha activation with concomitant increases in phosphorylation of their endogenous substrates except for synapsin I. These results suggest that nefiracetam potentiates AMPA receptor-mediated fEPSPs through CaMKII activation and enhances NMDA receptor-dependent LTP through potentiation of the post-synaptic CaMKII and protein kinase C activities. Together with potentiation of nicotinic acetylcholine receptor function, nefiracetam-enhanced AMPA and NMDA receptor functions likely contribute to improvement of cognitive function.     

Hyperosmotic stress activates the insulin receptor in CHO cells

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.     

行为训练与小鼠海马突触蛋白磷酸化水平的相关性

本文对梭箱回避行为训练是否能改变小鼠海马突触蛋白磷酸化水平进行了检定。在小鼠训练结束的不同时程制备突触体,以γ-~(32)P-ATP作为标记进行离体测定。结果如下:(1)正常小鼠各脑区突触蛋白磷酸化水平无显著差异;(2)学习训练刚结束时,海马突触蛋白磷酸化水平与学习训练成绩的优劣有相关性;(3)学习训练结束后24h,上述相关性更为明显,且成绩良好组右侧海马突触蛋白磷酸化水平显著高于左侧,而成绩低劣组相反。海马突触蛋白磷酸化的这种不对称现象的成因尚不清楚。这些结果表明,海马突触蛋白磷酸化反应可能是习得行为的分子机制。     

Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc,Raf-1, and ERK pathways in lung epithelial cells

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)α/c-Src and Gβγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/β (IKKα/β) activation, NF-κB transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKKα/β phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gβγ, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through Gβγ, c-Src, and Shc complex formation to induce IKKα/β phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.     

Polyomavirus Large T Antigen Induces Alterations in Cytoplasmic Signalling Pathways Involving Shc Activation

It has been extensively demonstrated that growth factors play a key role in the regulation of proliferation. Several lines of evidence support the hypothesis that for the induction of cell cycle progression in the absence of exogenous growth factors, oncogenes must either induce autocrine growth factor secretion or, alternatively, activate their receptors or their receptor substrates. Cells expressing polyomavirus large T antigen (PyLT) display reduced growth factor requirements, but the mechanisms underlying this phenomenon have yet to be explored. We conducted tests to see whether the reduction in growth factor requirements induced by PyLT was related to alterations of growth factor-dependent signals. To this end, we analyzed the phosphorylation status of a universal tyrosine kinase substrate, the transforming Shc adapter protein, in fibroblasts expressing the viral oncogene. We report that the level of Shc phosphorylation does not decrease in PyLT-expressing fibroblasts after growth factor withdrawal and that this PyLT-mediated effect does not require interaction with protein encoded by the retinoblastoma susceptibility gene. We also found that the chronic activation of the adapter protein is correlated with the binding of Shc to Grb-2 and with defects in the downregulation of mitogen-activated protein kinases. In fibroblasts expressing the nuclear oncoprotein, we also observed the formation of a PyLT-Shc complex that might be involved in constitutive phosphorylation of the adapter protein. Viewed comprehensively, these results suggest that the cell cycle progression induced by PyLT may depend not only on the direct inactivation of nuclear antioncogene products but also on the indirect induction, through the alteration of cytoplasmic pathways, of growth factor-dependent nuclear signals.     

Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.