) Many signaling peripheral proteins, including protein kinases C (PKC), are translocated to different cell membranes upon cell activation. Recent studies have shown that the 41embrane targeting and activati6n of diverse peripheral proteins is mediated by a small number of membrane targeting domains, including Cl and C2 domains. During the past granting period, we have studied the in vitro membrane binding and activation mechanisms of PKC isoforms, which have led to a working model that explains much of the temporal and spatial sequences of its membrane targeting and activation. The primary objective in the next granting period is to elucidate the mechanisms of both the in vitro and cellular membrane targeting and activation of different PKC isoforms containing Cl and C2 domains, with an emphasis on understanding how these domains interact with PKC activators and with each other to achieve specific membrane targeting and exquisite PKC regulation. A long-term objective is to apply the principles learned from these studies to the development of therapeutic agents that can specifically modulate the targeting and activation of signaling proteins.
Specific aims for the next granting period are as follows, (1) further elucidation of the in vitro membrane binding and activation mechanisms of conventional PKCs, including PKC-alpha and PKC-gamma, (2) determination of the in vitro membrane binding and activation mechanisms of novel PKCs, including PKC-delta and PKC-epsilon, and (3) determination of the cellular membrane translocation and activation mechanisms of these PKCs. The principal methodologies to be used include: (1) the site-directed mutagenesis and the overexpression 0: PKC isoforms, (2) membrane-binding analysis of PKC isoforms with various model membranes developed in this laboratory and the surface plasmon resonance analysis that allows direct measurement of membrane association and dissociation rates, and (3) cell transfection of PKC isoforms and their mutants that are tagget with green fluorescent protein, followed by two-photon microscopic analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM053987-05S1
Application #
6445138
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
1997-01-01
Project End
2003-02-28
Budget Start
2001-03-01
Budget End
2002-02-28
Support Year
5
Fiscal Year
2001
Total Cost
$39,661
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
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; 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
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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