The long-term objective of this project is to establish the role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cell migration, a prerequisite for cancer invasion and metastasis. GAPDH is a multifunctional actin binding protein overexpressed in many human tumors. The GPDH actin-binding region has been identified in preliminary studies by the principal investigator.
Specific Aim 1 is to identify individual amino acids within this region that are important for actin binding. This will be accomplished in two ways. First, site-directed mutagenesis will be used to determine which residues are critical for F actin crosslinking by GAPDH in vitro. Second, fusion proteins consisting of wild type and mutant forms of the GAPDH actin binding domain linked to green fluorescent protein will be expressed in prostate cancer cells to determine which residues are critical fo actin binding.
Specific Aim 2 is to determine whether inhibition of GAPDH-acti interaction inhibits cell migration. In one series of experiments, prostate cancer cells will be microinjected with a peptide known to inhibit F actin gelation by GAPDH and then analyzed with time-lapse videomicroscopy and computer image analysis. In a second set of experiments, Dictyostelium discoideum will be transformed with the GAPDH sequence mutated at specific residues in the actin binding domain and will then be analyzed with videomicroscopy and other established migration assays. Dictyostelium is a slime mold that has been used extensively as a model system for studying cell migration. Since Dictyostelium is haploid, its endogenous GAPDH gene can be knocked out by homologous recombination.
Specific Aim 3 is to determine whethe regions of GAPDH other than the actin-binding domain are important for GAPDH-actin interaction. This will be accomplished by first determining whethe a fusion protein consisting of the entire GAPDH molecule linked to green fluorescent protein colocalizes with actin filaments in prostate cancer cells. Large portions of GAPDH will then be deleted from the fusion protein to determine which regions other than the actin-binding domain, if any, are critical for actin localization.
