UBC Theses and Dissertations
Investigation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase 1/2 (ERK1/2) activation Chiu, Doris Diane
There have been conflicting studies in the past several years regarding the relationship between PI3K activation and its potential role in ERK1/2 activation. Some studies indicate that PI3K activation is required for ERK1/2 activation, yet other studies suggest that there is no relationship between the two. In this thesis we hypothesize that PI3K activation is not required for ERK1/2 activation. To investigate this hypothesis we used several cell systems, including a human, hemopoietic cell line, TF-1, and murine, hemopoietic cell lines FDC-P1 and BAF-3 cells. Also, we used a human embryonic kidney cell line, HEK 293. With the use of pharmacological inhibitors of PI3K, LY294002 and wortmannin it was concluded that ERK1/2 activation does not require PI3K activation. We also addressed the question of what role PI3K plays in the regulation of protein synthesis. However, in order to test this, we first investigated whether our cell systems contained the important components of the translational protein machinery including, elF-4E and 4E-BPs. Afterwards, we tested the effects of using various inhibitors, including the PI3K inhibitors LY294002, wortmannin, MEK1/2 inhibitors, U0I26 and the FRAP/mTOR inhibitor rapamycin. From our preliminary experiments we determined that 4E-BP1 is present in TF-1 cells and 4E-BP2 is present in FDC-P1, MC-9 and TF-1 cells. Furthermore, elF-4E is present in all three cell systems. With the use of the inhibitors, we observed that LY294002 inhibits the phosphorylation of 4E-BP1, U0126 did not block the phosphorylation of 4E-BP1 and rapamycin partially blocked the phosphorylation of 4E-BP1. Surprisingly, wortmannin did not block the phosphorylation of 4EBP1. Due to the preliminary nature of the results, more experiments are needed to fully understand the role that PI3K may play in the regulation of protein translation.
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