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ABSTRACT: The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.
Journal of Neurochemistry 08/2008; 106(5):2056-67. · 4.06 Impact Factor
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ABSTRACT: Scaffolding proteins including kinase suppressor of Ras-1 (KSR1) determine specificity of signaling by extracellular signal-regulated kinase 1/2 (ERK1/2), enabling it to couple diverse extracellular stimuli to various cellular responses. The scaffolding protein(s) that contributes to ERK1/2-mediated neuronal survival has not yet been identified. In cultured rat cortical neurons, BDNF activates ERK1/2 to enhance neuronal survival by suppressing DNA damage- or trophic deprivation-induced apoptosis. Here we report that in this system, BDNF increased KSR1 association with activated ERK1/2, whereas KSR1 knockdown with a short hairpin (sh) RNA reduced BDNF-mediated activation of ERK1/2 and protection against a DNA-damaging drug, camptothecin (CPT). In contrast, BDNF suppression of trophic deprivation-induced apoptosis was unaffected by shKSR1 although blocked by shERK1/2. Also, overexpression of KSR1 enhanced BDNF protection against CPT. Therefore, KSR1 is specifically involved in antigenotoxic activation of ERK1/2 by BDNF. To test whether KSR1 contributes to ERK1/2 activation by other neuroprotective stimuli, we used a cAMP-elevating drug, forskolin. In cortical neurons, ERK1/2 activation by forskolin was protein kinase A (PKA) dependent but TrkB (receptor tyrosine kinase B) independent and was accompanied by the increased association between KSR1 and active ERK1/2. Forskolin suppressed CPT-induced apoptosis in a KSR1 and ERK1/2-dependent manner. Inhibition of PKA abolished forskolin protection, whereas selective PKA activation resulted in an ERK1/2- and KSR1-mediated decrease in apoptosis. Hence, KSR1 is critical for the antiapoptotic activation of ERK1/2 by BDNF or cAMP/PKA signaling. In addition, these novel data indicate that stimulation of cAMP signaling is a candidate neuroprotective strategy to intervene against neurotoxicity of DNA-damaging agents.
Journal of Neuroscience 11/2007; 27(42):11389-400. · 7.11 Impact Factor
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ABSTRACT: To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.
Journal of Neurochemistry 02/2006; 96(2):335-48. · 4.06 Impact Factor
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ABSTRACT: N-methyl-D-aspartate receptors (NMDARs) are critical for neuronal plasticity and survival, whereas their excessive activation produces excitotoxicity and may accelerate neurodegeneration. Here, we report that stimulation of NMDARs in cultured rat hippocampal or cortical neurons and in the adult mouse brain in vivo disinhibited glycogen synthase kinase 3beta (GSK3beta) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3beta at the serine 9 residue. NMDA-triggered GSK3beta activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3beta inhibition reduced inhibitory phosphorylation of the PP1 inhibitor 2 (I2) and attenuated serine 9 dephosphorylation by PP1. These data suggest existence of a feedback loop between GSK3beta and PP1 that results in amplification of PP1 activation by GSK3beta. In addition, GSK3beta inhibition decreased PP1-mediated dephosphorylation of the cAMP-response element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3beta abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3beta and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3beta-PP1 circuitry may contribute to the deficits of CREB-dependent neuronal plasticity in neurodegenerative diseases.
Journal of Biological Chemistry 12/2005; 280(45):37526-35. · 4.77 Impact Factor
