We have purified p19, a 19-kDa cytosolic protein, from bovine brain and shown that it is a substrate for cAMP-dependent protein kinase. Using an antiserum raised in rabbits, we have demonstrated that the expression of p19 in rats and mice is restricted to brain and testis. The protein is also expressed in cultured neuroblastoma and neuroendocrine tumor cells but not in a variety of other cell lines. The level of expression of p19 in brain is significantly higher in newborn that in adult rats and preliminary immunohistochemical studies suggest that the highest level of expression occurs in immature neurons. The proposed studies are designed to test our hypothesis that p19 play a role in neuronal development. Our first objective is to clone the cDNA encoding p19 and to determine its nucleotide sequence. Using this information, we will search available sequence data bases for potential similarities with previously characterized proteins in order to obtain potential clues regarding the functional identity of p19.
The second aim i s the determine the cellular and subcellular localization of p19 in selected regions of the rat brain during development. This may enable us to better define the potential role of p19 in neuronal differentiation. We also plan to study the phosphorylation and expression of p19 in PC12 cells and neuroblastoma cells when they are induced to differentiate in vitro. In these cells, we will also attempt to suppress the expression of p19 using an antisense RNA strategy in order to determine whether this protein plays a critical role in neuronal differentiation or cell replication. The last objective of this proposal is to isolate the p19 gene and to characterize its structure, with the long-range goal of determining the sequence elements that are responsible for the cell type-specific expression of this gene. The results of these studies promise to provide valuable new insights regarding the role of this phosphoprotein in neuronal development.
