Browsing by Subject "MAP Kinase Kinase Kinases"
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Item BTB-Kelch Proteins and TAk1 Kinase N Immune Function(2006-12-19) Liu, Hong-Hsing; Chen, Zhijian J.BTB-kelch proteins are putative components of E3 ligases with a BTB domain at the N terminus and several kelch repeats at the C terminus. KLHL6 and mKELCH are two members of this family. Conditional ablation of Klhl6 in B cells resulted in mild developmental phenotypes in bone marrow precursors. The number of peripheral B cells was decreased by half, and responded defectively in germinal center formation after antigen stimulations. mKELCH is a novel protein cloned from hearts. Knocked-in LacZ expressed predominantly at muscles and several photosensitive organs. TAK1 is a member of MAPKKK. Deletion of TAK1 prevented the maturation of CD4+ or CD8+ single positive thymocytes, leading to reduction of T cells in peripheral tissues. Thymocytes lacking TAK1 failed to activate NF-κB and JNK, and were prone to apoptosis upon stimulation. All three mouse models have provided important evidences in elucidating biological functions for each protein in vivo.Item Identification of Intracellular Signaling Pathways Regulated by the TAO Family of Mammalian STE20p Kinases(2006-05-16) Raman, Malavika; Cobb, Melanie H.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.Item Kinetics and Regulation of Protein Kinases(2015-06-23) Piala, Alexander Townshend; Rosenbaum, Daniel M.; Goldsmith, Elizabeth J.; Phillips, Margaret A.; De Brabander, Jef K.One of the major functions of kinases in biological systems is the relay of signal from an effector to a downstream target. Kinases are regulated by a diversity of mechanisms, both internal and external to the kinase. When the regulation of these kinases is somehow abrogated, disease can result. As such, understanding of these regulatory mechanisms could serve as useful in not only understanding cellular signal propagation, but also the disease states that can arise from their malfunction. Taking advantage well-developed projects in the Goldsmith lab, three systems were studied: two kinases and one kinase cascade. The MAP3K Thousand and One Kinase 2 was the subject of an inhibitor discovery program using high-throughput screening of a large small-molecule library. Secondly, the mechanism by which the kinase With No Lysine(K) 1 responds to chloride concentration in order to modulate downstream signaling was studied. Finally, a cascade comprising the MAP3K Apoptosis Signaling Kinase 1, MAP/ERK Kinase 6, and p38a was studied through modeling approaches using data derived from mass spectrometry. In each case, mechanisms for regulation were discovered. Tight-binding inhibitors of TAO2 were found that may prove useful in both in in vitro signaling studies as well as future in vivo work. WNK1 was shown to be regulated by direct chloride binding, which inhibits its auto-phosphorylation and thusly its auto-activation. Finally, important kinetic parameters for MAPK cascade signaling were determined, and it was demonstrated that additional species outside the cascade do not appear necessary for switch-like behavior under physiological-like in vitro conditions. These diverse regulatory mechanisms showcase how protein kinases manage to synthesize diverse inputs into physiologically meaningful downstream signals.Item Mechanism and Regulation of ERK2 Subcellular Localization(2004-05-04) Whitehurst, Angelique Wright; Cobb, Melanie H.Dynamic changes in the localization of activated proteins can be obligatory events in signaling networks that control cell behavior. ERK1/2 activation contributes to regulated processes such as proliferation, differentiation and survival through the phosphorylation of multiple nuclear and cytoplasmic substrates. The pleiotropic effects of ERK1/2 activation suggest that regulated compartmentalization of the kinases and substrates may contribute to the fidelity of phenotypic changes in response to specific cell stimuli. Therefore, elucidating the mechanism of translocation as well as how this process is controlled is important for understanding how MAP kinases transmit signals. In vitro studies using a permeabilized cell system indicate that nuclear import of ERK2 is not regulated by soluble transport factors, but requires access to nucleoporins. While this process is not influenced by classical import machinery, it can be modulated by anchoring proteins that bind to ERK2 and sequester the kinase in the cytoplasm. One of these proteins, PEA-15, prevents ERK2 import in an in vitro system by inhibiting the kinases' ability to interact with nucleoporins. In vivo assays of phosphorylated ERK1/2 show discrete subcellular localization patterns in response to different stimuli that are independent of the level of ERK1/2 activation. Under conditions in which ERK1/2 is concentrated in the cytoplasm, the nuclear substrate of the kinase, c-Fos, is not expressed, while the cytoplasmic substrate of ERK1/2, p90RSK, is phosphorylated.