Identification of proteins regulated by PACAP in PC12 cells by 2D gel electrophoresis coupled to mass spectrometry.
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 apoptosis. Under such conditions, treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) promotes cell survival and induces neurite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable information 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 MALDI-ToF 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 protein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin beta-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.
Lebon
Alexis
A
INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France.
Seyer
Damien
D
Cosette
Pascal
P
Coquet
Laurent
L
Jouenne
Thierry
T
Chan
Philippe
P
Leprince
Jerome
J
Fournier
Alain
A
Vaudry
Hubert
H
Gonzalez
Bruno J
BJ
Vaudry
David
D
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011506:Proteins