The overall goal of this research is to understand the role of protein fatty acylation in directing subcellular localization and function of normal and oncogenic signaling proteins. Members of the Src family of tyrosine protein kinases will be used as model systems. Membrane attachment of these proteins is critical for their function, and the mechanism responsible for membrane targeting involves covalent modification with palmitate and/or myristate. The experiments proposed in this application are designed to address 3 key questions: 1) How are fatty acylated proteins targeted to specific membrane subdomains? The ability of the dually acylated Src family kinase Fyn to localize to non- ionic detergent-resistant membrane domains will be reconstituted in vitro. Binding of non-acylated, myristoylated, and myristoylated + palmitoylated Fyn to liposomes with specific phospholipid compositions will be quantitated; the effect of incorporating caveolin-1 into the liposomes will be assessed. 2) Can signaling protein function be altered by attachment of different types of membrane targeting motifs? Sequences encoding different fatty acylation/prenylation signals will be fused to c-Raf-1 and the constructs placed under an inducible promoter. The ability of the modified Raf to initiate downstream signaling through the MAP kinase pathway will be determined. 3) What is the role of Fyn and its fatty acylation in normal cell function? We recently determined that oligodendrocytes, the myelin producing cells of the CNS, are dependent on upregulation of Fyn for differentiation. We will test the abilities of wt, dominant negative and fatty acylation mutants of Fyn to promote the oligodendrocyte differentiation process.
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