Amyloid β peptide specifically promotes phosphorylation and nuclear translocation of the extracellular signal-regulated kinase in cultured rat cortical astrocytes

Abstract

To explore cellular signaling molecules that respond to amyloid β protein (Aβ), we investigated the effect of Aβ on tyrosine phosphorylation of cellular proteins in cultured rat cortical astrocytes. Western blotting with the phosphotyrosine-specific monoclonal antibody 4G10 demonstrated that exposure of cultured rat cortical astrocytes to 20 μM Aβ1-40 or Aβ25 - 35 for 24 h resulted in a prominent increase in the phosphotyrosine content of 44-kDa protein. The Aβ-induced increase in tyrosine phosphorylation of 44-kDa protein was blocked by U0126, a specific inhibitor of the extracellular signal-regulated kinase (ERK) kinase MEK. Western blotting with anti-phospho-ERK1/2 antibody and anti-ERK1/2 antibody demonstrated that Aβ1 - 40 or Aβ25 - 35 induced an increase in the dually (tyrosine and threonine) phosphorylated form of ERK1 and ERK2, with no change in total ERK1/2 level. In addition, immunofluorescent staining with anti-ERK1/2 antibody revealed that Aβ induced a significant increase in the number of cells expressing ERK1/2 mainly in the nucleus. These results suggest that Aβ specifically promotes tyrosine phosphorylation and nuclear translocation of ERK in astrocytes.