Analysis of the novel Lyn-associated cytoskeletal modular protein, LACM

A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to "round up", though this was not observed with either protein alone or AFAP11 O. LACM localization was observed in the immortalized erythroid progenitor cell lines J2E and R11 and also in phenylhydrazinetreated mouse spleen cell progenitors from 'normal' and Lyn-I - mice. LACM was shown to interact with the SH2 and SH3 domains of Vav2 and the SH2 domain of Grb4 at sites of tyrosine phosphorylation on LACM. These sites on LACM were determined to be at Y136 for Vav2 association and Y566 for Grb4 association. The carboxy-terminal SH3 domain of Vav2 was found to interact with the same polyproline motif on LACM as Lyn indicating a competitive association with the molecules. In summary, the data presented in this thesis reports on the identification and characterization of a novel adaptor protein that is involved in signaling from Lyn tyrosine kinase through to the adaptor protein, Grb4 and the guanine nucleotide exchange factor, Vav2. Both Grb4 and Vav2 are proteins known to be involved in cell signaling to the actin-based cytoplasm and, therefore, LACM may be involved in modulating cytoskeletal changes within cells.