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The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac., (Copyright 1998 by The American Society of Hematology.)

The c-myb gene is the cellular homologue of the v-myb oncogenes carried by the avian leukaemia viruses AMV and E26. It encodes a transcription factor (c-Myb), as does each of the viral oncogenes, which recognises the core DNA sequence C/T-A-A-C-G/T-G via a repeated helix-turn-helix-like motif. c-myb is expressed in immature haemopoietic cells, as well as immature cells of the gastro-intestinal epithelium and is down-regulated with differentiation. Enforced expression of activated or even normal forms of Myb can transform haemopoietic cells, most often of the myeloid lineage, in vitro and in vivo. Although many genes have been identified which are likely to be regulated by c-Myb, the critical target genes involved in Myb's transforming activity are not known. Together with data showing increased c-myb expression in colonic tumours, these observations raise the possibility that c-myb may play a role in human malignant disease.

We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB). The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology. The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis. In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

The oncogenic activation of c-myb has been associated with structural alterations to the Myb protein. Although such alterations can increase the ability of Myb to transform haemopoietic cells, it has been unresolved whether over-expression of wild type (WT) c-Myb can lead to transformation. We show here that infection with a retrovirus that expresses WT i.e. full length c-Myb leads to transformation of primary haemopoietic cells (as indicated by clonogenic assays). The transformed cells are similar to those obtained with carboxyl-truncated (CT) c-Myb in that they show phenotypic and morphological characteristics of early myeloid cells and remain dependent on exogenous growth factors. Cells expressing WTMyb form lower numbers of colonies on average and have a greater tendency to spontaneously differentiate than those expressing truncated c-Myb. Additionally, our results show that transformation by both forms of Myb is dependent on the density at which the infected cells are cultured, and that low levels of transformation can be increased by addition of conditioned medium from myb transformed cells grown at high density. This implies that transformation can be enhanced by the effects of an autocrine growth factor. Moreover, the production of, or sensitivity to, such a factor may be influenced by Myb itself, since CT Myb-infected cells cultured at low densities show higher levels of transformation than WT Myb-infected cells.

The product of the c-myb proto-oncogene, c-Myb, binds DNA and can enhance transcription of genes bearing copies of the DNA sequence it recognises. Deletion or disruption of a negative regulatory domain (NRD) in the carboxyl portion of c-Myb results in enhanced transactivating capacity and in parallel, leads to activation of its ability to transform haemopoietic cells. Since mutational analysis has shown that one critical element within the NRD is a leucine zipper motif, we have sought to identify cellular proteins that can interact with the c-Myb leucine zipper. Using fusion proteins containing this region as an affinity reagent, we have identified two nuclear proteins, p67 and p160, that bind to the wild-type, but not to a mutated c-Myb leucine zipper. These two proteins were shown to be related by comparison of peptides generated by partial digestion. While p160 was found to be ubiquitous amongst different murine haemopoietic cell lines, and was also present in NIH3T3 fibroblasts, p67 was detected in a restricted set of immature myeloid cells. Intriguingly p160, but not p67, could also bind to the c-Jun leucine zipper.

The trans-activating and transforming capacities of the c-myb proto-oncogene product (c-Myb) are negatively regulated through a leucine zipper structure in its negative regulatory domain. We show here tht in cotransfection assays, maximal Myb-induced trans-activation occurs with relatively low amounts of wild-type c-Myb, while higher levels of c-Myb result in reduced Myb-induced trans-activation. By contrast, this apparent negative autoregulation is not observed with a c-Myb mutant containing an impaired leucine zipper. Data presented here suggest that this negative autoregulation of trans-activation by wild-type c-Myb is a consequence of homodimer formation by c-Myb through its leucine zipper and of the inability of c-Myb dimers to bind DNA. These findings point to a novel mechanism of regulation of a transcription factor.

Expression of the protooncogene c-Myb protein was assessed in normal mucosa and in tumor samples resected from six patients. We found that the tumor samples always expressed higher levels of full length Myb protein than the normal tissue. This contrasts with the situation in c-myb-associated hemopoietic malignancies of the mouse and chicken, in which Myb proteins are generally amino or carboxyl truncated. Tissues from five patients with colonic adenomatous polyps were also examined and found to express levels of Myb that were, in general, intermediate between those found in normal tissues and tumors. Of particular interest is that the more dysplastic polyps displayed higher Myb levels. In one patient with carcinoma and multiple colonic polyps, some polyps had intermediate levels of Myb, whereas one polyp with carcinoma in situ expressed tumor-like levels of Myb. To directly test the hypothesis that Myb expression may be important in determining the rate of colonic cell proliferation, we examined three colonic carcinoma cell lines and one polyp cell line. We found that the cell lines with the most rapid doubling times exhibited the highest Myb levels. In addition, we show that antisense myb oligonucleotides retard the proliferation of one of these colonic cell lines which expresses the highest level of Myb.

The negative regulatory domain of the c-myb protooncogene product (c-Myb) normally represses transcriptional activation by c-Myb. We show here that a leucine-zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. We also demonstrate that this leucine-zipper structure can interact with cellular proteins. Our results suggest that an inhibitor that suppresses transactivation binds to c-Myb through the leucine zipper and that c-Myb can be oncogenically activated by missense mutation.

c-myb is expressed predominantly in proliferating immature hemopoietic cells and, like v-myb, functions as a transcriptional activator that displays sequence-specific DNA binding. Oncogenic activation of the c-myb protein (Myb) is associated with carboxyl-terminal and/or amino-terminal truncations, the former of which also potentiates Myb's transcriptional activation capacity. We show here that a carboxyl-truncated Myb protein binds with a 7-fold higher affinity to an oligonucleotide bearing a Myb recognition sequence than does the full-length form. In addition, data are presented which show that Myb binds with different apparent affinities to variants of the recognition site and that Myb binds independently to adjacent sites regardless of the orientation of these sites.

Oncogenic forms of the c-myb protein (Myb) often exhibit amino-terminal and/or carboxyl-terminal truncations. When the transcriptional activity of these proteins was examined it was found that carboxyl-truncated Myb is more effective as a transcriptional activator than full-length or amino-truncated Myb. In order to determine the effect of such truncations on sequence-specific DNA binding, we synthesized murine Myb in vitro and assessed DNA binding by using a mobility-shift assay. Compared with the full-length protein no difference in binding was observed following deletion of the amino terminus, despite the removal of much of the first repeat of the DNA-binding domain. However, the specific DNA-binding capacity of carboxyl-truncated Myb was 4-6 times greater than that of the full-length protein; moreover, DNA binding was independent of a 'leucine zipper' motif present in Myb. These observations suggest that the increased transforming and transactivating potential of carboxyl-truncated Myb is due, at least in part, to increased sequence-specific DNA binding.

Carboxyl-truncated forms of the product of the c-myb proto-oncogene (Myb) are encoded by the v-myb oncogene, the rearranged c-myb genes of certain murine cell lines and a transforming recombinant c-myb retrovirus. We report here an examination of the abilities of a series of carboxyl deletions of Myb to transform hemopoietic cells. Increasing degrees of truncation resulted in increasing transforming capacity until the deletions removed the region responsible for transactivation by Myb. Because the effects of these deletions on transformation paralleled their previously described effects on the transactivating capacity of Myb but did not correlate with their ability to repress transcription, our results imply that removal of a domain which negatively regulates transactivation is responsible for oncogenic activation of carboxyl-truncated forms of Myb. Moreover, these data support the view that activated forms of myb transform by increasing and/or deregulating the expression of other genes.

The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNA-binding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly, revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products.

The expression of c-myb proteins in transformed and normal murine cells was investigated. Two c-myb proteins, p75c-myb and p90c-myb, were detected in normal thymocytes and cell lines with intact c-myb genes. These most likely differ by the inclusion of additional amino acids encoded by an alternatively spliced c-myb mRNA. The use of this alternative exon is therefore not a feature exclusively of those cells with viral integrations in their c-myb gene. Smaller c-myb proteins in myeloid leukemic cell lines with rearranged c-myb genes were also characterized. Viral integration into the 5' region of the c-myb gene in the W265 and W274 cell lines leads to the synthesis in each case of two amino-terminally truncated c-myb proteins. By contrast, in NFS60 cells, viral integration into a more 3' region of the c-myb locus (but upstream of an alternate exon) leads to the production of a single p50c-myb protein.

To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5' region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in all cell lines but others are unique; all are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription.

To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well-characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy-terminally truncated c-myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c-myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony-stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF-independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte-macrophage-CSF-encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice.

Murine retroviruses which encode c-myb proteins that have either complete or truncated carboxy (C) termini were used to infect haemopoietic cells from murine fetal liver. Using an agar colony assay, we could show that infection with the virus encoding the truncated protein resulted in the persistence of colony-forming cells well beyond the short period for which such cells are present in uninfected cultures. The resultant colonies failed to give rise to cell lines; however, clonal cell lines occasionally emerged from the original infected liquid cultures. The virus which encoded a myb protein with a complete C-terminus was virtually inactive in the colony assay; surprisingly, however, this virus could enhance proliferation in liquid cultures and has led to the generation of at least one cell line. In addition to demonstrating 'activation' of c-myb by C-terminal truncation, our results imply that an unaltered c-myb protein can also contribute to cellular transformation and that a second event is required to establish myb-transformed cells as a permanent cell line.