The goal of this project is the determination of the mechanism and result of connections between the microfilament system and the plasma membrane. Previously we had analyzed the system of non-erythroid spectrins as a model for this subcortical network. We now propose to extend our research to the analysis of 36,000 Mr tyrosine kinase substrates termed calpactins. These proteins which exist as monomers or as complexes with a 10,000 Mr light chain interact with the cytoskeletal proteins actin and spectrin and are modulated by Ca++ and phospholipid. We will identify other members of this family using a technique which relies on its ability to interact with actin and phospholipid, and a detailed comparison of the structure and function of these proteins will be made. A detailed analysis of Ca++-binding, including affinity modulators will be undertaken. We will analyze the functional relatedness of the light chain with the S-100 proteins. Monoclonal and polyclonal antibodies will be raised to defined regions (peptides) of calpactin for use in analysis of its function. The tissue and subcellular distribution using Western Blotting and immunofluorescence microscopy. The in vivo target of calpactins will be determined using cross-linking reagents followed by immunoprecipitation. The calpactin heavy and light chain genes will be expressed in bacteria and protein products will be purified and tested for biological activity. We will attempt to identify the region of calpactin needed for Ca++-binding. The effect of tyrosine phosphorylation of calpactin will be tested by determining whether there is any difference in the ability of phosphorylated calpactin to interact with phospholipid, Ca++ or spectrin. These studies will further our understanding of the subcortical filament network and its relationship to cell transformation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032866-07
Application #
3282054
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-05-01
Project End
1991-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
7
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Campos-Gonzalez, R; Glenney Jr, J R (1991) Immunodetection of the ligand-activated receptor for epidermal growth factor. Growth Factors 4:305-16
Turner, C E; Glenney Jr, J R; Burridge, K (1990) Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol 111:1059-68
Glenney Jr, J R; Zokas, L (1989) Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol 108:2401-8
Glenney Jr, J R (1989) Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus. J Biol Chem 264:20163-6
Glenney Jr, J R; Kindy, M S; Zokas, L (1989) Isolation of a new member of the S100 protein family: amino acid sequence, tissue, and subcellular distribution. J Cell Biol 108:569-78
Glenney Jr, J R; Zokas, L; Kamps, M P (1988) Monoclonal antibodies to phosphotyrosine. J Immunol Methods 109:277-85
Glenney, J; Zokas, L (1988) Antibodies to the N-terminus of calpactin II (p35) affect Ca2+ binding and phosphorylation by the epidermal growth factor receptor in vitro. Biochemistry 27:2069-76
Davidson, F F; Dennis, E A; Powell, M et al. (1987) Inhibition of phospholipase A2 by ""lipocortins"" and calpactins. An effect of binding to substrate phospholipids. J Biol Chem 262:1698-705
Glenney Jr, J R; Tack, B; Powell, M A (1987) Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta. J Cell Biol 104:503-11
Powell, M A; Glenney, J R (1987) Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src. Biochem J 247:321-8

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