Proteomic Analysis of Protein Phosphorylation in Pc12 Cells Induced Bypituitary Adenylate Cyclase Activating Polypeptide 38

This dissertation, "Proteomic Analysis of Protein Phosphorylation in PC12 Cells Induced Bypituitary Adenylate Cyclase Activating Polypeptide 38" by Wai-him, Lee, 李偉謙, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled PROTEOMIC ANALYSIS OF PROTEIN PHOSPHORYLATION IN PC12 CELLS INDUCED BY PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE 38 Submitted by LEE WAI HIM for the degree of Master of Philosophy at The University of Hong Kong in August 2002 Protein phosphorylation is one of the post-translational modifications of proteins. It plays a central role in signal transduction in cells. Pituitary adenylate cyclase activating polypeptide (PACAP) was first discovered in ovine hypothalamus in 1989. It is a member of the Secretin/Glucagons/VIP superfamily. PACAP and its receptors are widely distributed, and affect a wide range of physiological functions. PACAP can act as a neurohormone, neurotransmitter or neurotrophic factor in tissues. Rat phenochromocytoma PC12 cells were used in this study. PC12 cells have been used as a model for investigation of neural differentiation and neurodegenerative diseases. In order to analyze the protein phosphorylation in PC12 cells by PACAP, a proteomic approach was used. PC12 cells were treated -7 with 10 M of PACAP38 for 10 and 20 minutes, respectively. Protein extracts from the cells were separated by two-dimensional electrophoresis (2-DE) at a pI range from 4 to 8. Immunoblotting was done using anti-phosphotyrosine, anti-phosphothreonine and anti-phosphoserine antibodies and the immunoactive spots were developed by the Enhanced Chemiluminescence (ECL) system. The intensities of the immunoactive spots were estimated by densitometric scanning. Attempts were made to identify protein spots by either N-terminal amino-acid sequencing or internal amino-acid sequencing. The results showed that PACAP38 can increase or decrease the phosphorylation on tyrosine, threonine and serine residues. Two clusters of protein spots and six distinct protein spots that were tyrosine-phosphorylated were analyzed in phosphotyrosine immunoblots. All of them showed an increase in tyrosine phosphorylation after the 10 minute and 20 minute-PACAP38 treatments. In phosphothreonine 2-DE western blots, there were only a few immunoactive protein spots. A cluster of these protein spots and one distinct protein spot were analyzed. It was found that PACAP38 decreased threonine phosphorylation of these proteins. It was also found that more threonine-phosphorylated proteins were separated by SDS-PAGE. In phosphoserine western blots, one cluster of protein spots and four distinct protein spots were analyzed. All of them showed an increase in serine phosphorylation after the 20 minute-PACAP38 treatment, but spots 7, 9 and 10 showed a decrease in serine phosphorylation in the 10 minute-PACAP38 induction. Attempts were made to identify the protein spots. Two tyrosine-phosphorylated protein spots were subjected to N-terminal protein sequencing, but no sequence data were obtained. The N-terminals of the two proteins might have been modified and sequencing was not possible, or the amount of proteins was too small to be sequenced. In-gel cyanogen bromide digestion was performed for internal protein sequencing, but no digested peptides were obtained. In order to detect low-abundance proteins and to separate the clustered protein spots, large 2-DE was performed. However, the low-abundance proteins could not be seen in the large 2-DE gels and the separation of the clustered protein spots were not improved.