The long-term goal of the proposed research is to understand the molecular mechanisms that regulate the phosphoinositide-dependent kinase, PDK-1. The discovery of PDK-1 in 1997 unveiled a linchpin of cellular signaling. This kinase, alone, provides the 'on' switch for the catalytic function of diverse members of the AGC superfamily of kinases, including Akt (protein kinase B), protein kinase C family members, p70S6 kinase, among many others. Although the function of PDK-1 has been known since its discovery, the molecular mechanisms of this pivotal kinase are largely unexplored. The goal of the proposed research is to understand the molecular mechanisms governing the regulation, function, and subcellular location of PDK-1. The central hypothesis guiding this proposal is that the subcellular location and macromolecular interactions of PDK-1 control its physiological function.
Three Specific Aims are: 1. Regulation of PDK-1 by Membrane Interactions The goal of this section is to understand the spatial and temporal regulation of PDK-1 in live cells. Specifically, we delve into the central questions of 1] where in the cell is PDK-1 active, 2] what are the cellular inputs that control this activity, and 3] how sustained is substrate phosphorylation? We will take advantage of novel fluorescence methodologies to monitor both the activity and subcellular location of PDK- 1 in real time in response to cellular stimuli. 2. Biochemical and Structural Analysis of PDK-1 The goal of this section is to understand the biochemical mechanisms controlling PDK-1 activity. Specifically, we ask the questions 1] what is the mechanism of membrane binding of PDK-1? 2] what mechanisms govern the catalytic activity of PDK-1, and 3] what is the structure of PDK-1? 3. Regulation of PDK-1 by Protein: Protein Interactions The central hypothesis of this aim is that protein: protein interactions are key regulators of PDK-1 function in vivo. We 1] address the mechanisms by which PDK-1 recognizes Akt, testing the hypothesis that the interaction is regulated by the PH domain of each kinase, 2] propose to screen for binding partners of the PH domain and linker region of PDK-1, 3] test the hypothesis that PDK-1 is in a complex with a phosphatase, and 4] test the hypothesis that PDK-1 is tethered at the centrosome in a signaling complex with AKAP 350.
|Grzechnik, Agnieszka T; Newton, Alexandra C (2016) PHLPPing through history: a decade in the life of PHLPP phosphatases. Biochem Soc Trans 44:1675-1682|
|Bradley, Elizabeth W; Carpio, Lomeli R; Newton, Alexandra C et al. (2015) Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation. J Biol Chem 290:16272-80|
|Sierecki, Emma; Newton, Alexandra C (2014) Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase. Biochemistry 53:3971-81|
|Wang, Pingping; Zhou, Zhihong; Hu, Anchang et al. (2014) Both decreased and increased SRPK1 levels promote cancer by interfering with PHLPP-mediated dephosphorylation of Akt. Mol Cell 54:378-91|
|Newton, Alexandra C; Trotman, Lloyd C (2014) Turning off AKT: PHLPP as a drug target. Annu Rev Pharmacol Toxicol 54:537-58|
|Reyes, Gloria; Niederst, Matt; Cohen-Katsenelson, Ksenya et al. (2014) Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output. Proc Natl Acad Sci U S A 111:E3957-65|
|O'Neill, Audrey K; Niederst, Matthew J; Newton, Alexandra C (2013) Suppression of survival signalling pathways by the phosphatase PHLPP. FEBS J 280:572-83|
|O'Hayre, M; Niederst, M; Fecteau, J F et al. (2012) Mechanisms and consequences of the loss of PHLPP1 phosphatase in chronic lymphocytic leukemia (CLL). Leukemia 26:1689-92|
|Warfel, Noel A; Newton, Alexandra C (2012) Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP): a new player in cell signaling. J Biol Chem 287:3610-6|
|Warfel, Noel A; Niederst, Matt; Stevens, Michael W et al. (2011) Mislocalization of the E3 ligase, ?-transducin repeat-containing protein 1 (?-TrCP1), in glioblastoma uncouples negative feedback between the pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) and Akt. J Biol Chem 286:19777-88|
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