This project involves the role of two related substrates for protein kinase C (PKC) in mediating the many cellular effects resulting from activation of this family of kinases by hormones, neurotransmitters and other agonists. The PKC substrates under study are MARCKS and its smaller related protein, the MARCKS-like protein or MLP. Ongoing projects include structure-function studies of the protein and its mutant derivatives in two major systems, development of the mouse central nervous system, and early embryogenesis in Xenopus laevis. These studies have involved creating gene knockouts for MARCKS and MLP in the mouse, and using """"""""knockout"""""""" oligonucleotides and protein overexpression to perturb early Xenopus development. In addition, transgenic complementation of the knockout mice with mutant proteins is continuing in order to analyze structure-function relationships in development. Similar studies are being performed in a cell transfection system, which seeks to examine the effect of the wild-type and mutant proteins on cellular adhesion and migration on various matrices. A novel aspect of this project involves the development of ES cell lines in which MARCKS and MLP have been knocked out, and which also express an immunohistochemical marker such as beta-galactosidase. These are being used to determine the cell-specific or substrate-specific nature of the knockout phenotype, by using the ES cells to create chimeric mice. Finally, we are currently investigating the possibility that mutations in the MARCKS and MLP genes are involved in human neural tube defects, particularly at the level of increasing a genetic predisposition to environmental causes of these defects. The human genes encoding MARCKS and MLP have been resequenced in 72 normal individuals as part of the environmental genome project, and the same genes from subjects with anencephaly have been resequenced to attempt to identify mutations that might correlate with this devastating birth defect. To date, some of the identified sequence variants are statistically correlated with anencephaly in one ethnic group. Further subjects are being studied to determine the significance of this and other potential associations.
