Identification of Intracellular Signaling Pathways Regulated by the TAO Family of Mammalian STE20p Kinases

TAO1, 2 and 3 are a sub-family of mammalian Ste20p protein kinases. They have been shown to regulate activation of p38 MAPK by phosphorylating and activating MEK3 and 6. Little is known about the precise cellular roles for these TAO protein kinases, or whether they function together or individually within the cell. Recently, genome-wide screens have identified these protein kinases as important mediators of vital cellular processes such as proliferation and apoptosis. Determining the mechanisms that govern the activity of these protein kinases and the pathways that utilize them is of utmost importance for understanding important aspects of cell signaling. My research focused on determining physiological stimuli that activated TAO protein kinases and the consequence of this activation on downstream signaling. This approach, in conjunction with two-hybrid screening led to the elucidation of two pathways that utilized TAO kinases. TAO2 interacted with Gas and Gbeta gamma subunits in yeast two-hybrid screens. TAO2 phosphorylated Gas on threonine 9 in the N-terminus, and this phosphorylation was inhibited when the a subunit was activated by GTPgamma S. TAO2 also interacted with Gbeta gamma in detergent-soluble membrane extracts from cells. At present, the biological significance of this interaction is unclear. I also showed that TAO1, 2 and 3 are activated significantly by agents that damage DNA. The kinetics of activation mirrors that of p38 MAPK. I subsequently demonstrated that over-expression of kinase-deficient TAOs inhibited the activation of p38 by UV and hydroxyurea. The relative contribution of MEK3 and 6 in the activation of p38 by these agents was also determined. Knockdown of TAO 1-3 protein levels by siRNA oligonucleotides against these protein kinases also mimicked the dominant-negative results. TAO kinases interact with one another and p38 and this may be one manner in which signaling is made selective and efficient. The UV-induced G2/M checkpoint is diminished when TAO kinase expression levels are reduced by siRNA. Finally we show that TAOs may be substrates of the ataxia telangiectasia mutated (ATM) and ATM and Rad50-related (ATR) DNA damage kinases, as activation of TAO2 is diminished in cells from a patient with AT, which do not express ATM. These findings show that TAO kinases regulate critical events in cell-cycle arrest by DNA damage by acting as intermediates in p38 activation by ATM/ATR.