The oncoprotein v‐Myb is a potent inducer of myeloid leukemias, and its cellular homolog c‐Myb plays a crucial role in the regulation of hematopoiesis. Both proteins function as transcriptional regulators. We demonstrate that this function is mediated at least in part by the nuclear co‐activator CREB binding protein (CBP). This protein interacts directly with both c‐Myb and v‐Myb and potentiates Myb‐specific transcription as measured on the mim‐1 promoter. In contrast, dominant negative mutants of CBP lead to repression, as does E1A, an antagonist of CBP function. Phosphorylation of c‐Myb does not appear to be required for interaction with CBP, thus indicating that the binding may be constitutive. Furthermore, the C/EBP family member NF‐M, which cooperates with c‐Myb in transactivating the mim‐1 promoter through an adjacent DNA binding site, is co‐activated by CBP in a Ras‐dependent manner. Not only the individual activities of c‐Myb and NF‐M are stimulated by CBP, but also their synergistic transcriptional function, while it is negatively regulated by dominant negative forms of CBP. These data suggest that CBP is recruited by both Myb proteins and NF‐M and potentiates their transcriptional activity. We suggest that CBP can bridge between c‐Myb and NF‐M, thus providing an explanation for the strong synergism between these two proteins.

C 2 -␣-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N-and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1-13 to be sufficient for C-mannosylation. Sitedirected mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position ϩ3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

The 75-kDa nuclear proto-oncoprotein c-Myb plays a crucial role in the regulation of cell growth and differentiation in the hematopoietic system and has been shown to be involved in the etiology of various hematopoietic tumors (3,10,21,41,44,62). c-Myb is a member of a class of transcription factors characterized by a DNA binding domain consisting of three imperfect repeats (R1, R2, and R3 in c-Myb) of 50 to 52 amino acids (18,23,24,26,30,31,46,47,53,55,68). A number of genes, including the mim-1 (47), c-myc (16, 71), c-myb (49), CD4 (63), cdc2 (33), CD34 (43), PR264/SC35 (65), and T-cell receptor ␦ (22) genes, have been shown to be Myb responsive. In several cases, c-Myb was found to activate transcription in cooperation with other factors such as Ets-2, core-binding factor, and NF-M, the chicken homolog of mammalian C/EBP␤, or other members of the C/EBP family of transcription factors (9,15,22,48).c-Myb is phosphorylated at multiple sites in vivo, and at least some of these sites seem to be of functional importance. Ser-11 and -12 have been mapped as in vivo phosphorylation sites in chicken c-Myb and can be modified in vitro by casein kinase II (CKII), resulting in reduced DNA binding (40). Interestingly, tumors in which the c-myb locus is involved show a strong selection against the N terminus of the protein, and the corresponding deletions always include the CKII phosphorylation sites (27,45,57,60,61,69,70). Taken together, these data suggest a modulatory role of the N-terminal domain for c-Myb function, with CKII as a potential regulator. In addition to the CKII sites, a number of other phosphorylation sites appear to be present in c-Myb and in p48 v-myb , but little information about their functional relevance is available (2,4,42).In this study, we have analyzed the effects of CKII phosphorylation site mutants on DNA binding, transactivation, and cooperativity with NF-M. We describe an efficient method to study DNA binding of native full-length c-Myb. This method should be useful in testing Myb mutants which have been difficult to analyze previously, e.g., by expression in bacteria or in reticulocyte lysate. Mutations of Ser-11 and -12 of c-Myb were found to alter DNA binding affinity and transactivation when tested on the mim-1 promoter or an artificial Myb response element A (MRE-A)-thymidine kinase (tk) promoter. In addition, we found that the promoter of the murine neutrophil elastase (NE) gene responded to Myb and that the effects of the CKII phosphorylation site mutants paralleled the observations regarding the mim-1 promoter. Interestingly, the effect of mutation of Ser-11 and -12 was overcome at least in part by an NF-M-like activity. This was not the result of direct interaction of the two proteins. We propose that a third factor is required for efficient cooperativity. MATERIALS AND METHODSCell culture. CV-1 cells (African green monkey kidney derived) and COS-7 cells (CV-1 cells transformed by simian virus 40 large T antigen) were cultured in Dulbecco's modified Eagle medium supplemented with 10...