Proteins which function in signal transduction often contain recurring motifs which play critical roles in protein-DNA and protein-protein interactions. The focus of this study is the Raf cysteine-rich domain, a motif found in a number of cell signaling enzymes that require lipids for activation. The Raf cysteine-rich domain coordinates two moles of zinc via six cysteine and two histidine residues. The zinc ions most likely provide a structural scaffold to stabilize residues involved in both Ras and phosphatidylserine interactions, that are likely to play a critical role in the activation of the Raf kinase and its subsequent activation of the MAP kinase cascade. The structural and functional properties of the Raf cysteine-rich domain and related motifs are poorly understood compared to other well documented proteins domains such as src homology 2 (SH2) and 3 (SH3) domains. The focus of this proposal is to examine the structural features of the zinc finger 'like' motif contained in Raf-1 kinase using NMR spectroscopic approaches, investigate Raf-Cys binding interactions with its putative physiological activators, Ras and phosphatidylserine, and elucidate the functional role of Raf and phosphatidylserine in the regulation of Raf-1 kinase activity. A multi-disciplinary approach, combining tools of biophysics, biochemistry, molecular biology and cell biology is represented.
The specific aims of this proposal are: 1) To determine the solution structure of the Raf cysteine-rich domain (Raf-Cy) using high resolution NMR spectroscopy). 2) To map Raf-Cys binding interactions with Ras through direct and competition binding studies, NMR spectroscopic analyses, and site-directed mutagenesis approaches. 3) To define recognition elements involved in phosphatidylserine/Raf-Cys interactions using fluorescence spectroscopy, direct binding assays, suite- directed mutagenesis and NMR spectroscopy. 4) To investigate the functional role of phosphatidylserine and Ras in the regulation of Raf kinase activity.
