Casein Kinase II Phosphorylation Site Mutations in c-Myb Affect DNA Binding and Transcriptional Cooperativity with NF-M

Abstract

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...

Exon 4-encoded acidic domain in the epithelium-restricted Ets factor, ESX, confers potent transactivating capacity and binds to TATA-binding protein (TBP)

“…Interestingly, an N-terminal 6 amino acid element of exon 4 (129-TSSSED-134) harbors two overlapping casein kinase II consensus sequences (S/TXXD/E). Since transactivation by Myb and PU.1 are known to be modulated by casein kinase II phosphorylation (OelgeschlaÈ ger et al, 1995;Pongubala et al, 1993), the mutations S131R and S132A were introduced into the GAL4(DBD)-ESX (1 ± 156) fusion construct to address the possibility that ESX activation is dependent upon serine phosphorylation within exon 4. As shown in Figure 3, replacement of these exon 4 serines (S131R/ S132A) had no signi®cant impact on transactivation relative to the unmutated GAL4(DBD)-ESX (1 ± 156) fusion control, suggesting, at least in the context of the GAL4(DBD) activation assay, that serine phosphorylation is not involved in transactivation by exon 4.…”

Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IκB homologue cactus

“…A similar mechanism has been proposed to modulate the DNA binding activity of c-Jun and c-Myb [22,23]. PEST sequences have not yet been identified with a particular pathway of proteolysis, so it is possible that CKII phosphorylation can affect either the ubiquitin-or trypsin-mediated forms of degradation [21].…”

Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells

“…pcDNA3.1͞Zeo͞hMBN and pcDNA3.1͞ Zeo͞hMBNS203A vectors were verified by DNA sequencing. Expression vectors for c-Myb (CMV-c-Myb) and for PU.1 (pECE-PU.1) (17) were obtained from B. Lüscher, Medizinische Hochschule Hannover, Germany (24) and R. Maki, The Burnham Institute, La Jolla, CA (15), respectively.…”