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.

Structural complexity in isoprenoid glycerol dialkyl glycerol tetraether lipid cores of Sulfolobus and other archaea revealed by liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometry of membrane lipid cores from Sulfolobus species reveals isomeric forms of ring-containing isoprenoid glycerol dialkyl glycerol tetraether components not previously recognised via the use of NMR and liquid chromatography-mass spectrometry techniques. Equivalent isomerism was confirmed for the components in other hyperthermophilic genera and in sediments which contain the lipids of mesophilic archaea. The recognition of the isomeric structures in distinct archaeal clades suggests that profiles of tetraether lipids reported previously may have oversimplified the true lipid complexity in archaeal cultures and natural environments. Accordingly, the extent of variation in tetraether structures revealed by the work should direct more informative interpretations of lipid profiles in the future. Moreover, the results emphasise that tandem mass spectrometry provides a unique capability for assigning the structures of intact tetraether lipid cores for co-eluting species during chromatographic separation.

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.