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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063235-13A1
Application #
8631326
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Flicker, Paula F
Project Start
2001-04-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
13
Fiscal Year
2014
Total Cost
$316,036
Indirect Cost
$94,943
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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; 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
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
Chen, Huai-Chun; Ziemba, Brian P; Landgraf, Kyle E et al. (2012) Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study. PLoS One 7:e33640
Ziemba, Brian P; Knight, Jefferson D; Falke, Joseph J (2012) Assembly of membrane-bound protein complexes: detection and analysis by single molecule diffusion. Biochemistry 51:1638-47
Falke, Joseph J (2012) Lipid targeting domain with dual-membrane specificity that expands the diversity of intracellular targeting reactions. Proc Natl Acad Sci U S A 109:1816-7
Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J (2011) The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3). Biochemistry 50:9845-56

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