|Budget Amount *help
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1996: ¥2,800,000 (Direct Cost: ¥2,800,000)
The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are largely unknown. This research demonstrates, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds specifically with Raf-1 and Bcr, the proto-oncogene products involved in the Ras-MAP kinase signaling pathway. This restricted region, termed 14-3-3 box-1, is one of the structural regions whose sequence is highly conserved among species, and is also identified as the site of interaction with phosphorylated tryptophan and tyrosine hydroxylases, the rate-limiting enzymes in the pathway of monoamine biosynthesis. To examine whether box-1 might bind with other target proteins, the eta isoform fused to glutathion-S-transferase or its mutant which lacks box-1 was mixed with rat brainstem extracts, incubated under phosphorylation conditions in the presence of [gamma-^<32>P] ATP and calmodulin, and the protein complex recovered by precipitation with glutathione-Sepharose was analyzed by SDS-gel electrophoresis and autoradiography. This experiment detected many phosphoproteins co-precipitated with the eta protein, but with the box-1-deletion mutant. These results suggest that box-1 may serve as a common binding site for many target proteins and enzymes including Raf-1, Bcr, and tryptophan and tyrosine hydroxylases.