The overall goal of this proposal is to investigate translational and posttranslational control of the c-myc and """"""""activated"""""""" myc proteins during growth. Based on preliminary studies, the hypothesis is that a factor(s) in the conditioned media induces the non-AUG initiation of c-myc 1 protein through the modification of a protein(s) in the translational initiation complex which then efficiently recognizes the non-AUG codon. These ideas will be tested by 1) characterizing the biochemical nature and specificity of the factor(s) in conditioned media which induces c-myc 1, 2) examining the changes in proteins associated with the translational initiation complex during c-myc 1 induction by two-dimensional gels and RNA-protein UV crosslinking and 3) determining the influence of sequence and secondary structure on the basal and induced levels of non-AUG initiation by translating c-myc in vitro and in vivo after site-directed mutagenesis. In addition, the posttranslational control of c-myc will be investigated by the further characterization of three recently identified phosphorylation sites in a highly conserved region of the N-terminal domain of c-myc. Based on preliminary studies, these phosphorylation events may be controlled by cell cycle and/or growth-regulated processes and may modulate the biological function of c-myc in growth control. These ideas will be tested by 1) determining if myc proteins are substrates for cdc 2 kinase Ix vivo by assaying the kinase activity of the cdc 2 complex immunoprecipitated from normal and transformed cells in different phases of the cell cycle using myc Hl histone and casein proteins as substrates, 2) characterizing the kinase which phosphorylates c-myc protein at the threonine which. is absent in v-myc proteins by determining if growth changes modulate the phosphorylation of this site and examining the properties of this kinase using a myc peptide as a substrate in vitro, 3) determining the influence of phosphorylation of the c-myc N-terminal domain on biological function by overexpressing mutants containing nonphosphorylatable residues in 1OT1/2 cells and measuring their ability to grow in .semisolid media. These studies will not only provide valuable information on the control of normal c-myc proteins in growth control and the activation of c-myc proteins associated with transformation and tumorigenesis, but will also provide information on the basic processes of translational and posttranslational regulation.
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