Identification of Proteins Regulated by PACAP in PC12 Cells by 2D Gel Electrophoresis Coupled to Mass Spectrometry

摘要

The rat pheochromocytoma PC12 cell line has been widely used as a model to study neuronal differentiation. In particular, after serum depletion, PC12 cells stop to proliferate and undergo apopto-sis. Under such conditions, treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) promotes cell survival and induces neu-rite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable informa-tion concerning the mechanisms controlling neuronal cell survival and differentiation. To this aim, PC12 cells cultured in serum-free medium were treated with PACAP (10~(-7) M), proteins were extracted, separated by two-dimensional gel electrophoresis (2-DE), and identified by MALDIToF mass spectrometry. The comparison between 16 2-DE maps led to the characterization of 110 proteins regulated by PACAP among which 22 have been identified by automatic query of the Mascot, Aldente, and Profound servers with the ProGeR-CDD database. Seventy-six percent of these proteins, including the p17 subunit of caspase-3, the heat shock pro-tein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin (β-5, and calmodulin were overexpressed. Investigation of the putative functions indicated that some of the proteins regulated by PACAP and identified in the present article could control cell survival or differentiation.