Browsing by Subject "Cell Physiological Processes"
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Item Biochemical and Cellular Imaging Studies of a Novel CDC42-Dependent Formin Pathway(2006-05-16) Seth, Abhinav; Rosen, Michael K.The Rho GTPases are important regulators of actin cytoskeletal dynamics during processes such as cell migration, cell polarization and cell division. Different Rho family members exert their effects on actin through specific downstream effectors including members of the WASP and Diaphanous-Related Formin (DRF) protein families. It is presently unclear if, and by what mechanisms, the level, timing and localization of Rho GTPase activity control and coordinate effector activity to produce different types of cytoskeletal structures and rearrangements. On a molecular level, autoinhibition is a common regulatory mechanism for many Rho GTPase effectors. Relief of autoinhibition of WASP by the Rho family member Cdc42 involves a significant GTPase-induced conformational change. Based on this conformational change, I have created a series of singlemolecule, FRET-based sensors for active Cdc42 that can faithfully report on Cdc42 activity in vitro and in cells. These sensors may be valuable tools for studying the spatio-temporal dynamics of Cdc42 signaling in vivo. The mechanisms of autoinhibition and activation are less well understood for the DRF family of GTPase effectors. DRFs are characterized by a C-terminal Diaphanous Autoregulatory Domain (DAD) that is postulated to regulate the actin assembly activity of the adjacent formin homology 2 (FH2) domain through autoinhibitory interactions with an N-terminal regulatory region, although this has only been shown directly for the DRF mDia1. Here, I show that the actin assembly activity of FRLa, a macrophage-specific DRF, is also autoinhibited by its N-terminal domain. In cells, autoinhibitory interactions also block a novel GTPase-independent membrane localization activity of the N-terminal domain in both FRLa and mDia1. Autoinhibitory control of FRLa activity and localization are specifically relieved by Cdc42. Timelapse microscopy was used to address the potential physiological significance of the Cdc42-FRLa interaction during Fc-gamma receptor mediated phagocytosis in macrophages, a Cdc42-dependent process. The data show that FRLa is required for efficient Fc-gamma receptor mediated phagocytosis and that it is recruited to the phagocytic cup by Cdc42. These results suggest mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate an important role for a novel Cdc42-formin pathway in immune function.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 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.Item Scaffold-Mediated Organization of Signal Transduction Networks(2004-08-19) Bumeister, Ronald; White, Michael A.The Raf-MEK-ERK kinase cascade is a highly conserved signal transduction module involved in cellular processes ranging from proliferation and differentiation to transformation and apoptosis, and is engaged in response to growth factors, cytokines, morphogens, and other extracellular stimuli. Given the ubiquitous nature of this signaling pathway, it is not clear how specialization with respect to various Raf-dependent phenomena is acquired. In theory, functional specificity could be achieved by selectively coupling the core enzymatic components of a cascade to other regulatory pathways depending on the cellular context. Here, by using loss-of-function and partial loss-of-function mutant analysis, we demonstrate that Raf can participate in cell regulatory processes independently of its ability to activate MEK. We also show that scaffolding proteins can serve as specificity determinants in Ras/MAPK signaling, and may function to coordinate the activity of this cascade with additional regulatory pathways required for an appropriate biological response. Thus, we provide evidence for functional diversification both at the level of Raf substrates and at the level of molecular organization by accessory scaffolding molecules.Item Substrate Interaction and Sub-Cellular Localization in Map Kinase Pathways(2005-08-11) Ranganathan, Aarati; Cobb, MelaineProtein kinase cascades control responses to extracellular cues by transmitting signals throughout the cell. Prominent among multifunctional enzymes in kinase cascades are the mitogen-activated protein kinases (MAPKs). Among the various MAPKs identified, the extracellular signal-regulated kinases, ERK2 and ERK5 are two closely related enzymes that have overlapping functions in a number of cellular pathways. Sub-cellular localization and specificity towards substrates are two mechanisms of controlling the function of an enzyme in the cell. My dissertation discusses the insights we have gained into both these regulated processes through our studies on ERK2 and ERK5. Sub-cellular localization of ERK2 is a tightly regulated process. The current model for sub-cellular localization of ERK2 suggests that there is continuous nuclear-cytoplasmic shuttling of the free pool of ERK2. Anchoring of ERK2 in the different compartments of the cell plays a critical role in determining its location. Entry of inactive ERK2 into the nucleus has been reported to occur by an energy- and carrier-independent mechanism. However, export of inactive ERK2 and import of active ERK2 in intact cells seem to occur by an active process. The mechanisms governing these processes have not been investigated. We have used an in vitro permeabilized-cell reconstitution assay in HeLa and BJ fibroblast cells to explore the mechanism of GFP-ERK2 export and His6-tagged thiophosphorylated ERK2 import. Our results identify more levels of regulation within this model. The ERK5 pathway is triggered in response to various stimuli including growth factors and cellular stresses. Compared to other MAPKs, little is known about ERK5 substrate specificity. Our lab had shown previously that ERK5 is capable of stimulating nuclear factor-κΒ (NF-κΒ ). Our data suggested that this function might be attributed in part to ribosomal protein S6 kinase (known as RSK or p90RSK), which was activated by coexpression with ERK5 and a constitutively active form of its MAP2K, MEK5DD. Here we demonstrate that RSK, among the first known substrates of the ERK1/2 MAPKs, is also directly phosphorylated and activated by ERK5. We have used RSK to explore the basis of substrate recognition by ERK5.Item Uncovering the Complexity of RAS Signaling Networks(2004-05-04) Matheny, Sharon A.; Anderson, Richard G.W.The Raf/MEK/MAP Kinase signal transduction cascade is a key Ras effector pathway that mediates diverse cellular responses to environmental cues, and makes a major contribution to Ras-dependent oncogenic transformation. Here we describe two important aspects to understanding the signaling complexity of any multiphenotypic arrangement, namely, the identification and examination of new effectors and the direct authentication of protein-protein interactions. We have identified a new Ras effector, IMP (Impedes Mitogenic signal Propagation) that negatively regulates MAP kinase activation by limiting formation of Raf/MEK complexes. The mechanism of inhibition appears to be through inactivation of the KSR1 adaptor/scaffold protein, demonstrating that in addition to promoting signal transmission, scaffold proteins can function to restrict signal propagation. Ras can inactivate IMP through induction of IMP auto-ubiquitination, facilitating KSR-dependent engagement of MEK by activated Raf. These observations reveal dual Ras effector inputs to the MAP kinase cascade: induction of Raf kinase activity concomitant with derepression of KSR-dependent Raf/MEK complex formation. This relationship provides a mechanism to limit engagement of the MAP kinase cascade in the absence of Ras activation. MAP kinase activation contributes to multiple diverse cellular responses to environment. The capacity to control the amplitude of this response via molecules like IMP likely contributes to flexible and adaptive cellular behavior in the context of complex regulatory signals. Analysis of the primary amino acid sequence of IMP indicated that it may regulated on multiple levels in different ways. We show that IMP can be phosphorylated by ERK and that this correlates with IMP translocation to the nucleus. We also demonstrate that SUMO-1 exerts negative effects on IMP inhibitory activity. Additionally, a predicted UBP-ZnF was examined for regulation of autoubiquitination. Together, these data point to the existence of multiple regulatory inputs that control IMP activity. Finally, we discuss the importance of establishing true binding relationships between associated proteins for the characterization of signaling pathways. Using Ras and its putative effector RalGDS as an example, we describe a simple method to this end. By identifying loss-of-function mutations in both proteins that reciprocally compensate to restore a biological outcome, we confirm that RalGDS is a bona fide effector of Ras, thereby proving the validity of this technique for authenticating other protein-protein interactions.