Proliferative diseases, such as cancer, and inflammatory diseases, such as rheumatoid arthritis, result from aberrations in cellular signaling pathways. Many cellular proteins that participate in cellular signaling pathways, most notably several forms of phospholipase A2 (PLA2) and protein kinase C (PKC), interact with the cell membrane (interfacial binding) in response to cellular and lipid second messengers. Although studies to date have shown that the interfacial binding plays a key role in the regulation of these proteins, little is known about the mechanism of their interfacial binding. The primary objective of this research program is to understand the molecular basis of the interfacial binding of PLA2 and PKC isoforms with a special emphasis on determining the differences in their interfacial binding mechanisms. A long-term objective is to apply the principles learned from these studies to the development of therapeutic agents that can specifically block the uncontrolled activation of particular PLA2 and PKC isoforms.
Specific aims during this proposed period are: (1) to identify the protein residues of mammalian secretory PLA2s that are involved in the interfacial binding mechanisms, (2) to determine the interfacial binding mechanism of cytosolic PLA2 and identify its interfacial binding residues, (3) to elucidate the mechanisms whereby three different types of PKC interact with membranes, determine how the physical state of membrane affects the PKC activation, and identify PKC residues that are involved in the interfacial binding and activation, and finally (4) to determine the mechanism and functional roles of protein and lipid domain formation in the interfacial binding of PLA2 and PKC. The principal methodologies to be used include: (1) many recombinant DNA techniques for the site-directed mutagenesis and over-expression of PLA2s and PKCs, (2) kinetic and membrane-binding analyses of PLA2s and PKCs using the polymerized mixed liposome system developed in this laboratory which allows the systematic structure-function studies of interfacial binding, and (3) phospholipid monolayer technique elaborated for the analysis of interfacial binding mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053987-04
Application #
6138511
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
1997-01-01
Project End
2001-02-28
Budget Start
2000-01-01
Budget End
2001-02-28
Support Year
4
Fiscal Year
2000
Total Cost
$166,767
Indirect Cost
Name
University of Illinois at Chicago
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Stahelin, Robert V; Subramanian, Preeti; Vora, Mohsin et al. (2007) Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain. J Biol Chem 282:20467-74
Cho, Wonhwa; Stahelin, Robert V (2006) Membrane binding and subcellular targeting of C2 domains. Biochim Biophys Acta 1761:838-49
Stahelin, Robert V; Hwang, Jeong H; Kim, Jin-Hahn et al. (2005) The mechanism of membrane targeting of human sphingosine kinase 1. J Biol Chem 280:43030-8
Subramanian, Preeti; Stahelin, Robert V; Szulc, Zdzislaw et al. (2005) Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2 alpha and enhances the interaction of the enzyme with phosphatidylcholine. J Biol Chem 280:17601-7
Stahelin, Robert V; Wang, Jiyao; Blatner, Nichole R et al. (2005) The origin of C1A-C2 interdomain interactions in protein kinase Calpha. J Biol Chem 280:36452-63
Malkova, Sarka; Long, Fei; Stahelin, Robert V et al. (2005) X-ray reflectivity studies of cPLA2{alpha}-C2 domains adsorbed onto Langmuir monolayers of SOPC. Biophys J 89:1861-73
Cho, Wonhwa; Stahelin, Robert V (2005) Membrane-protein interactions in cell signaling and membrane trafficking. Annu Rev Biophys Biomol Struct 34:119-51
Stahelin, Robert V; Digman, Michelle A; Medkova, Martina et al. (2005) Diacylglycerol-induced membrane targeting and activation of protein kinase Cepsilon: mechanistic differences between protein kinases Cdelta and Cepsilon. J Biol Chem 280:19784-93
Stahelin, Robert V; Ananthanarayanan, Bharath; Blatner, Nichole R et al. (2004) Mechanism of membrane binding of the phospholipase D1 PX domain. J Biol Chem 279:54918-26
Blatner, Nichole R; Stahelin, Robert V; Diraviyam, Karthikeyan et al. (2004) The molecular basis of the differential subcellular localization of FYVE domains. J Biol Chem 279:53818-27

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