C2 and PH domains are ubiquitous eukaryotic targeting motifs that drive membrane docking in response to a second messenger. When activated by second messenger signals, signaling proteins containing these domains dock to specific target membranes and control a wide array of essential cellular pathways. This continuing project investigates the complex signaling circuit at the leading edge of polarized cells, where C2 and PH domains play a dominant role by targeting dozens of signaling proteins to the appropriate membrane surface. The resulting targeting is universally required for cell migration and chemotaxis: the present focus is the leading edge of leukocytes that drive the primary immune response. The membrane targeting reaction of a given C2 or PH domain is optimized to ensure that the domain docks with exquisite specificity, as well as the appropriate affinity and speed, to its target membrane. Subsequently, the lipid-bound domain diffuses randomly in the bilayer plane for the total time and distance needed to undergo productive collisions with effector molecules. Finally, the domain dissociates to terminate activity. One broad goal of this continuing proposal is to elucidate the fundamental molecular mechanisms underlying the membrane targeting reactions of key leading edge C2 and PH domains.
The Specific Aims begin with in vitro studies of isolated C2 and PH domains, then move to in vitro studies of full length proteins, and finally to studies of domains and proteins in live cells.
Aim 1 employs spin label EPR measurements to elucidate the first membrane docking geometries of PH domains.
Aim 2 analyzes the membrane targeting mechanisms of representative C2 and PH domains, and tests a new working model for the protein-lipid interactions that control targeting.
Aim 3 employs innovative single molecule methods to dissect the contributions of multiple membrane docking domains in full length proteins, and to analyze the regulatory mechanisms of full length proteins.
Aim 4 tests the conclusions of in vitro studies in live cells, and develops new biosensors to analyze second messenger signals at the leading edge of polarized cells. Completion of these Aims will reveal key principles of membrane recognition in cell migration, the immune response inflammation, and cancer.

Public Health Relevance

This proposal investigates the ubiquitous C2 and PH domain motifs that regulate the membrane targeting of signaling proteins in a wide array of cellular pathways. Certain C2 and PH domains also play central roles in human diseases such as inflammation and cancer;for example, the highly oncogenic E17K mutation of AKT1 PH domain drives constitutive plasma membrane targeting by an unidentified mechanism, yielding kinase superactivation that accounts for its carcinogenicity. Preliminary results reveal that the E17K mutation enhances membrane targeting by changing the target lipid specificity of the PH domain, thereby defining the molecular mechanism of this important oncogenic mutation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Ainsztein, Alexandra M
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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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