Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.
Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.
Oelgeschläger
M
M
Institut für Molekularbiologie, Medizinische Hochschule Hannover, Germany.
Krieg
J
J
Lüscher-Firzlaff
J M
JM
Lüscher
B
B
D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019272:Leukocyte Elastase; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010196:Pancreatic Elastase / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; C075191:nuclear factor NF-M; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase