At the leading edge of a migrating leukocyte, receptor and Ca2+ signals regulate a membrane-associated pathway that orchestrates chemotaxis up attractant gradients. At the heart of this chemosensory pathway is a core PIP3 signaling network also found in pathways controlling cell growth and oncogenesis. Elucidation of the signaling mechanisms underlying this vital core network is essential to a molecular understanding of leukocyte chemosensing and other cellular processes, with biomedical significance for inflammation and cancer. Past progress on this continuing project has yielded fundamental insights into the membrane targeting and activation mechanisms of individual proteins isolated from the core PIP3 signaling network. Ongoing and future work will employ in vitro single molecule methods to directly observe groups of membrane-bound core components operating in regulatory modules and complexes on supported lipid bilayers, while simultaneously elucidating their mechanisms of signal transduction. Key in vitro findings will be tested in live cells. The new Specific Aims will investigate the molecular mechanisms of signaling reactions that (i) trigger synthesis of the PIP3 output signal by the master lipid kinase phosphatidylinositol-3-kinase (PI3K), or (ii) inhibit PIP3 production to control the amplitude and lifetime of PIP3 signals. The project has three Aims.
Aim 1. Elucidate the mechanisms by which receptors and G proteins activate PI3K and PIP3 production. Receptor and G protein signals together stimulate PI3K and PIP3 production, but the mechanism of this synergistic activation remains unresolved. In a long-standing debate, two opposing models differ on whether G protein-triggered membrane recruitment of PI3K, or activation of the membrane-bound enzyme, is primarily responsible for the large net increase in lipid kinase activity.
This aim will reveal how G proteins and receptors generate synergy when simultaneously activating PI3K.
Aim 2. Define the mechanisms by which Ca2+ signals regulate PI3K and shape PIP3 signals. Pre- liminary single molecule studies have detected two stable, membrane-bound complexes formed between key pathway regulatory elements: (a) Calmodulin (CaM) and HRas form a previously unknown complex that blocks HRas activation of PI3K. (b) Phosphoinositide-dependent kinase (PDK) and protein kinase C (PKC) form a stable complex hypothesized to inhibit both kinases. Studies of both complexes will elucidate their assembly and regulatory mechanisms, and their roles in shaping PIP3 signals.
Aim 3. Test the key predictions of in vitro mechanistic models in live cells. Single molecule studies of reconstituted systems can provide deep insights, but it is important to test key findings in the native pathway.
This aim employs unique advantages of the leukocyte leading edge to carry out such tests in live cells. Completion of these Aims will advance the mechanistic understanding of a crucial signaling network with direct relevance to the innate immune response, inflammation, oncogenesis, and pharmaceutical development.

Public Health Relevance

This project focuses on the membrane-localized signaling network that produces PIP3 as its output signal and controls multiple cellular processes, including (i) the leukocyte chemosensory pathway that guides cell move- ment during the innate immune response and inflammation, and (ii) cell growth pathways involved in oncogen- esis. The project will reveal the molecular mechanisms of receptor?G protein?PI3K modules that activate PIP3 production, and of novel inhibitory complexes that limit the amplitude and lifetime of PIP3 signals. A deeper mechanistic understanding of the PIP3 signaling network will help guide the development of new anti-cancer and anti-inflammatory pharmaceuticals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chao, Chien-Chung
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Buckles, Thomas C; Ziemba, Brian P; Masson, Glenn R et al. (2017) Single-Molecule Study Reveals How Receptor and Ras Synergistically Activate PI3K? and PIP3 Signaling. Biophys J 113:2396-2405
Ziemba, Brian P; Burke, John E; Masson, Glenn et al. (2016) Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway. Biophys J 110:1811-1825
Ziemba, Brian P; Swisher, G Hayden; Masson, Glenn et al. (2016) Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module. Biochemistry 55:6395-6405
Lin, Yuan; Protter, David S W; Rosen, Michael K et al. (2015) Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. Mol Cell 60:208-19
Li, Jianing; Ziemba, Brian P; Falke, Joseph J et al. (2014) Interactions of protein kinase C-? C1A and C1B domains with membranes: a combined computational and experimental study. J Am Chem Soc 136:11757-66
Falke, Joseph J; Ziemba, Brian P (2014) Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells. Chem Phys Lipids 182:73-9
Ziemba, Brian P; Li, Jianing; Landgraf, Kyle E et al. (2014) Single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase C-?. Biochemistry 53:1697-713
Lai, Chun-Liang; Srivastava, Anand; Pilling, Carissa et al. (2013) Molecular mechanism of membrane binding of the GRP1 PH domain. J Mol Biol 425:3073-90
Ziemba, Brian P; Pilling, Carissa; Calleja, Veronique et al. (2013) The PH Domain of Phosphoinositide-Dependent Kinase-1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium with Important Implications for Kinase Domain Activation: Single-Molecule and Ensemble Studies. Biochemistry 52:4820-9
Ziemba, Brian P; Falke, Joseph J (2013) Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core. Chem Phys Lipids 172-173:67-77

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