A point mutation in the MyoD basic domain imparts c-Myc-like properties.

MyoD and c-Myc, members of the large "basic-helix-loop-helix" family of proteins, regulate diverse aspects of both normal and neoplastic growth and specific gene regulation. These two proteins differ at 9 of the 14 amino acids that comprise the basic domains necessary for DNA binding and transcriptional control. Individual amino acids in the MyoD basic domain were mutated to those found at the analogous positions in c-Myc. Four classes of mutants were obtained: (i) those with no effects on MyoD-site binding or activation of MyoD-responsive genes, (ii) those with no effect on MyoD-site binding but with a loss of activation potential, (iii) those with a loss of both DNA binding and activation potential, and (iv) one mutant (mut 9, Leu122----Arg) that left MyoD-site binding unaffected but imparted a new c-Myc-site binding capability. mut 9 competed with wild-type protein for the activation of MyoD-responsive reporter genes but could, like c-Myc, also suppress the adenovirus major-late promoter, which contains a c-Myc binding site. Our studies thus identify specific amino acid residues in the MyoD basic domain that are important for its activity as a DNA-binding transcriptional activator. Most significantly, our results with mut 9 indicate that Leu122 of MyoD is a critical determinant of specific DNA binding and that mutation at this residue can alter this specificity.

Regulation of the junB gene by v-src.

The proteins encoded by cellular and viral src genes are believed to be involved in the transmission of mitogenic signals, the nuclear recipients of which are largely unknown. In this work, we report that four different v-src-transformed cell lines from three different species possess elevated levels of junB transcripts. Transient expression of junB promoter-chloramphenicol acetyltransferase constructs in NIH 3T3 cells was used to demonstrate that the increase in junB transcripts was specifically associated with v-src expression and could not be recapitulated with a c-src, v-H-ras, or v-raf expression vector. Deletion mutants were used to localize the v-src-responsive region in the junB promoter to a 121-nucleotide region encompassing the CCAAT and TATAA elements. This region is distinct from one in the 5' untranslated region of the junB gene which is required to maintain its high-level basal expression. Point mutagenesis of the junB TATAA box completely abolished v-src responsiveness, suggesting that proteins which bind to this element are modified by src transformation. Several v-src and c-src mutants were used to demonstrate that elevated tyrosine kinase activity of src proteins is required for the observed effects on junB expression. Finally, homology between the TATAA box regions of junB and the unrelated but src-responsive gene 9E3/CEF-4 suggests that modulation of gene activity through proteins which bind to this region may be a recurrent, although not exclusive, theme in src transforming action. Our results suggest that src proteins may modulate some nuclear effectors through pathways not involving cellular ras or raf gene products.