Regulation of c-Myb through protein phosphorylation and leucine zipper interactions.
c-Myb function is modulated in part by a negative regulation domain which encompasses a leucine zipper (LZ). When E. coli-expressed c-Myb with wild type or mutated LZ proteins are assessed for DNA binding activity, the mutant form is substantially better at DNA binding than the wild type (WT) form. In contrast, the DNA binding activity of the WT protein is increased to an equivalent level of activity of the LZ-mutant when both are expressed in rabbit reticulocyte lysates (RRL) or insect cells. The possibility that phosphorylation overcomes the negative influence of the LZ was investigated. E. coli-expressed mutant, but not wild type c-Myb proteins, were shown to be substrates for Casein Kinase II (CKII) and cAMP-dependent Protein Kinase (PKA). The phosphorylation sites for CKII and PKA were serines 11 and 12, and 8 and 116, respectively. Serines 11 and 12 were found to be phosphorylated in recombinant wild type and mutant c-Myb expressed in insect cells and DNA binding was markedly reduced following phosphatase treatment. Substitution of serines 11 and 12 with glutamic acid and alanine in E. coli-expressed 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.
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D047390:Casein Kinase II; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020598:Proto-Oncogene Proteins c-myb; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D018411:Spodoptera / Q000821:virology; D014158:Transcription, Genetic
D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases