DYNAMICS OF C-FOS PROTEIN INTERACTIONS

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The fos protein is a nuclear, DNA binding protein which is a transcription regulator and is induced in a wide range of cell types by many different transmembrane signals, including growth factor stimulation. Recently, it has been shown that fos makes a specific complex with the jun protein through interactions of alpha helical regions containing heptad repeats of leucines (leucine zipper). The zipper interaction juxtaposes basic amino acid motifs of both proteins which form a sequence specific DNA binding domain. We propose to determine the structural features of fos which give specificity for heterodimerization with jun. These could reside within the alpha helix, or elsewhere, and will be analyzed by the method of site directed mutagenesis and in vitro assay of reticulocyte lysate translation products. We have also shown that when nerve growth factor (NGF) stimulates model PC12 pheochromocytoma cells to differentiate down a neuronal pathway, fos is induced. Peak fos transcription is 30 min. post NGF. Following fos induction, other genes which express neuronal specific functions are also expressed. One of these, the tyrosien hydroxylase (TH) gene, is induced transcriptionally 1 hr. following NGF treatment. The TH promoter has a DNA element which binds authentic fos-jun complex and also binds an in vivo PC12 product which is induced by NGF over the period 1-4 hr post treatment. This is the same interval over which TH transcription is positively and negatively regulated. We will analyze the induced PC12 for factors which may regulate TH expression. This will include identification of the factor)s) which interact with the TH gene promoter, analysis of the particular role of fos, and a search for new, neuronal-specific members of the fos and jun families. Thus, the Specific Aims of the proposal are: Aim 1. To determine the structural features of fos which confer specificity of heterodimerization on the fos and jun proteins. Aim 2. To determine the role of the immediate early response genes (including the fos family members) in regulation of transcription of delayed early genes in PC12 cells.