Epac-induced alterations in the proteome of human SH-SY5Y neuroblastoma cells

Abstract

The hitherto proteome of the human neuroblastoma cell line SH-SY5Y consists of 1029 unique proteins. By using subcellular fractionation, SDS PAGE and nanoLC-Q-TOF-MS/MS we enlarged the proteome with additionally 72.2%, resulting in 3707 unique proteins. The stage of neuroblastoma tumor cell differentiation is known to influence patient outcome, with a high differentiation stage correlating to favourable prognosis. To elucidate the effects of cAMP in SH-SY5Y neuroblastoma differentiation we used selective cAMP analogs to activate Epac and PKA. We found that activation of Epac induced actin and tubulin polymerization and neurite outgrowth, whereas PKA activation did not. The effects of the Epac stimulation were abolished by knock down of Epac1 with ShRNA. Stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry were used to disclose the long-time effects of Epac activation on the SH-SY5Y proteome. We found 101 expressed proteins upregulated and 74 downregulated. Upregulated proteins were in general associated with neuronal cell differentiation and adhesion, whereas downregulated proteins typically were involved in RNA processing. We conclude that cAMP-induced morphological and biochemical neuronal differentiation of human neuroblastoma SH-SY5Y cells are mediated by Epac. © 2009 Birkeland E, et al.