Differential Regulation of Phosphatidylinositol 4-Phosphate 5 Kinase Beta during Hypertonic and Oxidative Stress




Chen, Mark

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Of the total amount of phospholipids in the cell, phosphoinositides account for only a small fraction, yet they play an indispensable role in regulating cellular homeostasis. The phosphatidylinositol derivative, PI(4,5)P2 (PIP2)is an essential mediator of cellular processes such as signal transduction, membrane traffic, actin cytoskeleton, ion homeostasis, growth and apoptosis. Despite the importance of this signaling molecule in controlling a diverse array of functions, little is known about the regulation of the kinases that produce PIP2 as well as its precursor, phosphatidylinositol 4 phosphate (PI4P). Our laboratory found that hypertonic and oxidative stress increases and decreases PIP2 levels, respectively. In this thesis, I sought to understand how these two different types of stresses alter PIP2 by examining the effects on the phosphatidylinositol 5-phosphate 4 kinases that generate PIP2. Using RNAi and in vitro kinase activity assays, I found that PIP5Kbeta is regulated by both stimuli in opposite directions and is responsible for the net change in PIP2 levels. Hypertonic stress activates PIP5Kbeta through PP1 phosphatase dependent ser/thr dephosphorylation which leads to increased kinase activity and plasma membrane localization. The PIP2 produced by PIP5Kbeta during this event leads to an increase in actin polymerization and stress fiber formation. Conversely, oxidative stress leads to a decrease in PIP5Kbeta activity, membrane detachment and stress fiber dissolution. Oxidative stress induces simultaneous tyrosine phosphorylation and ser/thr dephosphorylation, establishing that tyrosine phosphorylation decreases PIP5Kbeta activity and is the dominant modification of PIP5Kbeta that overrides the activating effects of ser/thr dephosphorylation. I further identified the tyrosine kinase Syk as the kinase responsible for PIP5Kbeta phosphorylation during oxidative stress. The phosphorylation site was mapped to position Y105 which was shown to control both activity and localization of PIP5Kbeta. Oxidative stress also increased PI4P which is likely to be at least partly due to activation of a PI4K. Using RNAi and wortmannin to selectively inhibit the activity of different PI4K isoforms, I sought to identify the PI4K isoform that is responsible for this increase.

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