At the leading edge membrane of a migrating cell, three membrane-associated master kinases (PKC?, PI3K?, PDK1) together play a central role in controlling the signaling pathway that regulates chemotaxis up attractant gradients. The present project has provided fundamental insights into key membrane targeting reac- tions, triggered by local Ca2+ and PIP3 signals, that recruit these and other signaling proteins to the leading edge membrane. The resulting membrane-localized signaling network controls cell migration. New studies will focus on the three master kinases and their membrane-based signaling reactions. These studies will elucidate the activation mechanisms of individual kinases and define the pathway connec- tions between them. The lipid bilayer itself is a crucial element of the signaling pathway - the bilayer surface serves as a 2-dimensional scaffold and diffusion platform on which recruited proteins (a) collide with and bind membrane-bound activators and substrates, (b) carry out signaling reactions, and, in some cases (c) combine with other membrane proteins to form higher order signaling complexes. The new Specific Aims investigate the activation mechanisms and regulatory interactions of the three master kinases on planar bilayers. Initial work will analyze each individual membrane-bound kinase to under- stand the sequence of molecular steps leading to kinase activation. Longer term studies will reconstitute all three kinases with their membrane-bound activators and substrates, enabling direct analysis of signal trans- mission and regulatory interactions between membrane-bound components. The project has three Aims:
Aim 1. Delineate the molecular steps by which multiple lipids and activators switch on PKC?.
Aim 2. Elucidate the mechanisms by which lipids, proteins, and oncogenic mutations activate PI3K?.
Aim 3. Define the interactions between kinases that establish pathway connections and activate PDK1. Completion of the Aims will reveal fundamental molecular principles underlying the activation, signal propagation, and feedback connections of the three master kinases, with major impacts for signaling at the leading edge and diverse areas of signaling biology. Furthermore, each targeted master kinase is directly linked to human cancers and inflammatory disease, thus a mechanistic understanding of kinase activation is medically relevant and could facilitate the development of new therapies.

Public Health Relevance

This project focuses on a set of three master kinases that control a wide array of cell pathways, including the migration of leukocytes in the primary immune response. The project will reveal the molecular activation mechanisms of individual kinases, as well as pathway connections between kinases. High activity levels of each kinase are directly linked to human cancers and inflammatory diseases, thus a mechanistic understanding will facilitate the development of new therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
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Flicker, Paula F
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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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