Screening of factors that affect GSK-3beta expression and study of tau phosphorylation by GSK-3beta in cultured cell lines

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that phosphorylates on the proline-rich sequence. It has been implicated in different physiological roles including energy metabolism, development and neuronal function.;The first objective of this study is to explore the possible pathways that may regulate the transcription of GSK-3beta. By transfecting cell lines using various factors and p2090-CAT, a 2.5kb GSK-3beta promoter fragment, p2090, fused with the promoterless chloramphenicol acetyltransferase (CAT) reporter gene vector, I attempted to find candidates that can alter the promoter activity of p2090. Factors tested include insulin, aspirin, dexamethasone, beta-amyloid peptidel-42 (Abeta 42), cAMP elevators---dibutyryl cAMP (dcAMP), forskolin and isobutyl methaxanthine (IBMX) and PKA inhibitors---Rp-cAMP, KT5720, 6-22 amide and H89. Results from screening showed that aspirin, Abeta42 and cAMP elevators can upregulate the promoter activity of GSK-3beta.;Further study on the dose-response effects of dcAMP and forskolin showed that the p2090 promoter activity was induced up to 225% and 336% by 2 mM dcAMP and 100 muM forskolin, respectively. However the same treatment exerted no significant effect in the endogenous mRNA and protein level of GSK-3beta, whereas the kinase activity of endogenous GSK-3beta was inhibited to approximately 50% of control at 4 mM dcAMP and 400 muM forskolin treatments.;The effect of Fe65, X11alpha and X11beta on the promoter activity of p2090 was also examined by the co-transfection of the corresponding cDNA and the p2090-CAT construct. After normalization with transfection efficiency, only X11alpha was found to inhibit the promoter activity of p2090 by 30%.;The second objective is the study on the tau phosphorylation induced by GSK-3beta. It was performed by co-transfection of GSK-3beta and tau0N3R cDNA containing plasmids into the CHO-K1, COS-7 and SH-SY5Y cell lines. Tau phosphorylation at different epitopes was assessed by immunoblotting with phosphorylation-dependent antibodies. The AT-270 and AT-8 epitopes were consistently phosphorylated by GSK-3beta among the three cell lines. Phosphorylation on AT-180 and PHF-1 epitopes by GSK-3beta was significant only in CHO-K1 and SH-SY5Y cells respectively.