Search

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V(267)NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check.

Several constitutively active mutant forms of the common beta subunit of the human IL-3, IL-5 and GM-CSF receptors (hbetac), which enable it to signal in the absence of ligand, have recently been described. Two of these, V449E and I374N, are amino acid substitutions in the transmembrane and extracellular regions of hbetac, respectively. A third, FIDelta, contains a 37 amino acid duplication in the extracellular domain. We have shown previously that when expressed in primary murine haemopoietic cells, the extracellular mutants confer factor-independence on cells of the neutrophil and monocyte lineages only, whereas V449E does so on all cell types of the myeloid and erythroid compartments. To study the in vivo effects and leukaemic potential of these mutants, we have expressed all three in mice by bone marrow reconstitution using retrovirally infected donor cells. Expression of the extracellular mutants leads to an early onset, chronic myeloproliferative disorder marked by elevations in the neutrophil, monocyte, erythrocyte and platelet lineages. In contrast, expression of V449E leads to an acute leukaemia-like syndrome of anaemia, thrombocytopaenia and blast cell expansion. These data support the possibility that activating mutations in hbetac are involved in haemopoietic disorders in man.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

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.

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.

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.