Signal transduction through growth factor receptor complexes regulates cell proliferation and when signaling elements are genetically altered, cell transformation is a frequent consequence. Among the large number of signal transducing proteins downstream from activated growth factor receptors is the tyrosine kinase substrate phospholipase C-gamma 1 (PLC-gamma 1), an enzyme which generates second messenger molecules that regulate intracellular calcium levels and protein kinase C activity. The research proposed in this application addresses the regulation of PLC-gamma 1 and its role in growth factor signalling. The first objective concerns structure/function analysis of the PLC-gamma 1 molecule. Mutagenesis of various structural domains within the molecule will be used to explore the role of individual domains in the control of PLC enzyme activity. Src homology domains, located in the center of the PLC-gamma 1 sequence, will be explored as a possible source of intramolecular regulation of basal and growth factor-stimulated enzyme activity. The experiments will involve analysis of PLC-gamma 1 mutants in cultured cells and of selected PLC-gamma 1 mutant molecules expressed in the baculovirus system. Additionally, we will collaborate with crystallography group to produce x-ray quality crystals of this protein for structural determination. The second and third areas of investigation concern the biological function of PLC-gamma 1 in cells and in the intact animal. The latter is addressed by disrupting the gene for PLC-gamma 1 in mice, which produces an embryonic lethal phenotype. We propose experiments to rescue this phenotype and to induce PLC-gamma 1 gene disruption in the adult animal. PLC-gamma 1 null cell lines have been generated from transgenic animals. These cells lines will be used to address the role of PLC-gamma 1 in growth factor-induced proliferation in cell culture systems and to analyze the behavior of PLC-gamma 1 mutants within the context of the intact cell.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA075195-01
Application #
2382778
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1997-08-19
Project End
2002-02-28
Budget Start
1997-08-19
Budget End
1998-02-28
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Crooke, Cornelia E; Pozzi, Ambra; Carpenter, Graham F (2009) PLC-gamma1 regulates fibronectin assembly and cell aggregation. Exp Cell Res 315:2207-14
Liao, Hong-Jun; de Los Santos, Josue; Carpenter, Graham (2006) Contrasting role of phospholipase C-gamma1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors. Exp Cell Res 312:807-16
Linggi, Bryan; Carpenter, Graham (2006) ErbB receptors: new insights on mechanisms and biology. Trends Cell Biol 16:649-56
Tvorogov, Denis; Wang, Xue-Jie; Zent, Roy et al. (2005) Integrin-dependent PLC-gamma1 phosphorylation mediates fibronectin-dependent adhesion. J Cell Sci 118:601-10
Liao, Hong-Jun; Kume, Tsutomu; McKay, Catriona et al. (2002) Absence of erythrogenesis and vasculogenesis in Plcg1-deficient mice. J Biol Chem 277:9335-41
Tvorogov, Denis; Carpenter, Graham (2002) EGF-dependent association of phospholipase C-gamma1 with c-Cbl. Exp Cell Res 277:86-94
Chattopadhyay, Ansuman; Carpenter, Graham (2002) PLC-gamma1 is required for IGF-I protection from cell death induced by loss of extracellular matrix adhesion. J Cell Sci 115:2233-9
Wang, X T; McCullough, K D; Wang, X J et al. (2001) Oxidative stress-induced phospholipase C-gamma 1 activation enhances cell survival. J Biol Chem 276:28364-71
Liao, H J; Ji, Q S; Carpenter, G (2001) Phospholipase C-gamma1 is required for the induction of immediate early genes by platelet-derived growth factor. J Biol Chem 276:8627-30
Mehlmann, L M; Chattopadhyay, A; Carpenter, G et al. (2001) Evidence that phospholipase C from the sperm is not responsible for initiating Ca(2+) release at fertilization in mouse eggs. Dev Biol 236:492-501

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