Insulin-like growth factor 1 inhibits extracellular signal-regulated kinase to promote neuronal survival via the phosphatidylinositol 3-kinase/protein kinase A/c-Raf pathway

J Neurosci. 2005 Mar 16;25(11):2838-52. doi: 10.1523/JNEUROSCI.5060-04.2005.

Abstract

Extracellular signal-regulated kinase (ERK) activation has been shown to promote neuronal death in various paradigms. We demonstrated previously that the late and sustained ERK activation in cerebellar granule neurons (CGNs) cultured in low potassium predominantly promotes plasma membrane (PM) damage. Here, we examined the effects of a well established neuronal survival factor, insulin-like growth factor 1 (IGF-1), on the ERK cell death pathway. Stimulation of CGNs with IGF-1 induced an early and transient ERK activation but abrogated the appearance of late and sustained ERK. Withdrawal or readdition of IGF-1 after 4 h in low potassium failed to prevent sustained ERK activation and cell death. IGF-1 activated the protein kinase A (PKA) to mediate ERK inhibition via c-Raf phosphorylation at an inhibitory site (Ser259). Phosphatidylinositol 3-kinase (PI3K) or PKA inhibitors, but not a specific Akt inhibitor, abrogated PKA signaling. This suggests that the PI3K/PKA/c-Raf-Ser259 pathway mediates ERK inhibition by IGF-1 independent of Akt. In addition, adenoviral-mediated expression of constitutively active MEK (mitogen-activated protein kinase kinase) or Sindbis viral-mediated expression of mutant Raf Ser259Ala both attenuated IGF-1-mediated prevention of PM damage. Activation of caspase-3 promoted DNA damage. Its inhibition by IGF-1 was both PI3K and Akt dependent but PKA independent. 8-Br-cAMP, an activator of PKA, induced phosphorylation of c-Raf-Ser259 and inhibited ERK activation without affecting caspase-3. This indicates a selective role for PKA in ERK inhibition through c-Raf-Ser259 phosphorylation. Together, these data demonstrate that IGF-1 can positively and negatively regulate the ERK pathway in the same neuronal cell, and provide new insights into the PI3K/Akt/PKA signaling pathways in IGF-1-mediated neuronal survival.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Animals
  • Animals, Newborn
  • Blotting, Western / methods
  • Cell Survival / drug effects
  • Cells, Cultured
  • Cerebellum / cytology
  • Cyclic AMP-Dependent Protein Kinases / physiology*
  • Dose-Response Relationship, Drug
  • Drug Interactions
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay / methods
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Gene Expression Regulation, Enzymologic / drug effects
  • Green Fluorescent Proteins / metabolism
  • Immunohistochemistry / methods
  • In Situ Nick-End Labeling / methods
  • Indoles
  • Insulin-Like Growth Factor I / pharmacology*
  • Neurons / drug effects*
  • Neurons / physiology
  • Phosphatidylinositol 3-Kinases / physiology*
  • Potassium Chloride / pharmacology
  • Proto-Oncogene Proteins c-raf / physiology*
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Serine / metabolism
  • Signal Transduction / drug effects*
  • Time Factors
  • Transfection / methods

Substances

  • Enzyme Inhibitors
  • Indoles
  • RNA, Messenger
  • Green Fluorescent Proteins
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Serine
  • DAPI
  • Potassium Chloride
  • Insulin-Like Growth Factor I
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-raf
  • Cyclic AMP-Dependent Protein Kinases
  • Extracellular Signal-Regulated MAP Kinases