REGULATION OF C-MYB THROUGH PROTEIN-PHOSPHORYLATION AND LEUCINE-ZIPPER INTERACTIONS

c-Myb function is modulated in part by a negative regulation domain wh ich encompasses a leucine zipper (LZ). When E. coli-expressed c-Myb wi th wild type or mutated LZ proteins are assessed for DNA binding activ ity, the mutant form is substantially better at DNA binding than the w ild type (WT) form. In contrast, the DNA binding activity of the WT pr otein is increased to an equivalent level of activity of the LZ-mutant when both are expressed in rabbit reticulocyte lysates (RRL) or insec t cells. The possibility that phosphorylation overcomes the negative i nfluence of the LZ was investigated. E. coli-expressed mutant, but not wild type c-Myb proteins, were shown to be substrates for Casein Kina se II (CKII) and cAMP-dependent Protein Kinase (PKA). The phosphorylat ion sites for CKII and PKA were serines 11 and 12, and 8 and 116, resp ectively. Serines 11 and 12 were found to be phosphorylated in recombi nant wild type and mutant c-Myb expressed in insect cells and DNA bind ing was markedly reduced following phosphatase treatment. Substitution of serines 11 and 12 with glutamic acid and alanine in E. coli-expres sed Myb demonstrated that these amino terminal residues influence the negative effect on DNA binding exerted by the LZ. Collectively, these observations support the notion that phosphorylation of serines 11 and 12 positively modulate DNA binding.