Regulation of c-Myb activity by tumor suppressor p53.

Heat shock proteins (HSPs) act as chaperones and play important roles during cellular proliferation and apoptosis. Heat shock factors (HSFs) mediate transcriptional induction of HSP genes. Among multiple heat shock transcription factors (HSFs) in vertebrates, HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). Since c-Myb has an important role in cellular proliferation, this regulatory pathway suggests a link between the events of cellular proliferation and the stress response. The c-Myb-induced activation of HSF3 is negatively regulated by the p53 tumor suppressor protein. p53 directly binds to HSF3 and blocks the interaction between c-Myb and HSF3. In addition, p53 stimulates the degradation of c-Myb, which is, at least partly, mediated by induction of Siah in certain types of cells. Thus, c-Myb and p53 regulate the expression of HSPs via HSF3 in opposite ways.

Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle.

The c-myb gene encodes a transcription factor that is central to hematopoietic cell growth. Phosphorylation of c-Myb by casein kinase 2 (CK2) at serines 11 and 12 has been variously implicated in the regulation of DNA binding. However, it is unclear when c-Myb phosphorylation at serines 11 and 12 occurs during the cell cycle and how this is regulated. We generated specific antisera that recognize phosphoserines 11 and 12 of c-Myb. C-Myb protein levels, extent of CK2 phosphorylation and DNA binding were then monitored following mitogenic stimulus and passage through the cell cycle in normal peripheral T-cells and the T leukemia cell line CCRF-CEM. We found that endogenous c-Myb is constitutively phosphorylated at serines 11 and 12. The amount of phosphorylated c-Myb correlates with DNA binding activity in cycling CEM cells but not upon entry of T-cells into the cell cycle. Exogenous expression of c-Myb with substitutions of serines 11 and 12 with glutamic acid or alanine had no effect on the transactivation of a c-Myb responsive reporter. These data strongly suggest that c-Myb is constitutively phosphorylated on serines 11 and 12 by CK2 or like activity and is not regulated during the cell cycle.

p53 suppresses the c-Myb-induced activation of heat shock transcription factor 3.

Expression of heat shock proteins (HSPs) is controlled by heat shock transcription factors (HSFs). Vertebrates express multiple HSFs whose activities may be regulated by distinct signals. HSF3 is specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb), which plays an important role in cellular proliferation. This suggests that the c-Myb-induced HSF3 activation may contribute to the growth-regulated expression of HSPs. Here we report that the p53 tumor suppressor protein directly binds to HSF3 and blocks the interaction between c-Myb and HSF3. In addition, p53 stimulates the degradation of c-Myb through a proteasome-dependent mechanism, which is, at least partly, mediated by induction of Siah in certain types of cells. Induction of p53 by a genotoxic reagent in DT40 cells disrupts the HSF3-c-Myb interaction and down-regulates the expression of certain HSPs. Mutated forms of p53 found in certain tumors did not inhibit c-Myb-induced HSF3 activation. The regulation of HSF3 activity by c-Myb and p53 sheds light on the molecular events that govern HSP expression during cellular proliferation and apoptosis.

Activation of heat shock transcription factor 3 by c-Myb in the absence of cellular stress.

In vertebrates, the presence of multiple heat shock transcription factors (HSFs) indicates that these factors may be regulated by distinct stress signals. HSF3 was specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). These factors formed a complex through their DNA binding domains that stimulated the nuclear entry and formation of the transcriptionally active trimer of HSF3. Because c-Myb participates in cellular proliferation, this regulatory pathway may provide a link between cellular proliferation and the stress response.

CBP as a transcriptional coactivator of c-Myb.

CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A). CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form. We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator. CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro. The domain of CBP that touches c-Myb is also required for binding to CREB. A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay. CBP stimulates the c-Myb-dependent transcriptional activation. Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation. In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation. Our data thus identify CBP as a coactivator of c-Myb. These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously.

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin.

Eukaryotic proteins are frequently produced in Escherichia coli as insoluble aggregates. This is one of the barriers to studies of macromolecular structure. We have examined the effect of coproduction of the E. coli thioredoxin (Trx) or E. coli chaperones GroESL on the solubility of various foreign proteins. The solubilities of all eight vertebrate proteins examined including transcription factors and kinases were increased dramatically by coproduction of Trx. Overproduction of E. coli chaperones GroESL increased the solubilities of four out of eight proteins examined. Although the tyrosine kinase Lck that was produced as an insoluble form and solubilized by urea treatment had a very low autophosphorylating activity, Lck produced in soluble form by coproduction of Trx had an efficient activity. These results suggest that the proteins produced in soluble form by coproduction of Trx have the native protein conformation. The mechanism by which coproduction of Trx increases the solubility of the foreign proteins is discussed.

c-Myb repression of c-erbB-2 transcription by direct binding to the c-erbB-2 promoter.

The c-myb proto-oncogene product (c-Myb) is a transcriptional activator that can bind to the specific DNA sequences. Although c-Myb also represses an artificial promoter containing the Myb binding sites, natural target genes transcriptionally repressed by c-Myb have not been identified. We have found that the human c-erbB-2 promoter activity is repressed by c-Myb or B-Myb in a chloramphenicol acetyltransferase co-transfection assay. Domain analyses of c-Myb suggested that Myb represses the c-erbB-2 promoter activity by competing with positive regulators of the c-erbB-2 promoter. In in vitro transcription assays, Myb proteins containing only the DNA binding domain could repress c-erbB-2 promoter activity. Two Myb binding sites in the c-erbB-2 promoter were critical for transcriptional repression by c-Myb. One of the two Myb binding sites overlaps the TATA box, and DNase I footprint analyses indicated that c-Myb can compete with TFIID. These results suggest that Myb-induced trans-repression of the c-erbB-2 promoter partly involves competition between Myb and TFIID.

Independent control of transcription initiations from two sites by an initiator-like element and TATA box in the human c-erbB-2 promoter.

Transcription of the human c-erbB-2-proto-oncogene starts mainly at two sites, nucleotide positions +1 and -69. The present studies have identified an initiator-like element that specifies the position of transcription initiation at position -69. This initiator-like element contains six GGA repeats and is located just downstream from the transcription start site between positions -68 and -45. In addition, both in vitro and in vivo studies indicated that transcription initiation at position +1 is specified by a TATA box 25 bp upstream from the transcription startpoint. Thus, initiation at two sites in the c-erbB-2 promoter is controlled independently by the initiator-like element and the TATA box.

c-Myb-induced trans-activation mediated by heat shock elements without sequence-specific DNA binding of c-Myb.

The c-myb proto-oncogene product (c-Myb) can transactivate the human hsp70 promoter in a transient cotransfection assay. The present studies have demonstrated that the heat shock element (HSE) in the hsp70 promoter mediates trans-activation by c-Myb. Mutagenesis of the DNA sequence in HSE indicated that the NGAAN motif is necessary for not only the heat shock response but also the c-Myb-induced trans-activation. The HSE in the hsp70 promoter does not contain a c-Myb-binding site, implying that the sequence-specific DNA binding of c-Myb is not required for the HSE-dependent trans-activation by c-Myb. We had demonstrated that a disruption of the leucine zipper motif in the central portion of the c-Myb molecule increased the degree of c-Myb-induced trans-activation of the promoter containing c-Myb-binding sites, suggesting that a putative inhibitor binds to c-Myb through this leucine zipper (Kanie-Ishii, C., MacMillan, E. M., Nomura, T., Sarai, A., Ramsay, R. G., Aimoto, S., Ishii, S., and Gonda, T. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3088-3092). However, disruption of the leucine zipper in c-Myb abolished the HSE-dependent trans-activation by c-Myb, whereas deletion of the transcriptional activation domain containing acidic amino acids in c-Myb did not abolish the HSE-dependent trans-activation by c-Myb. These results suggest that c-HSEs by interacting with unidentified trans-acting factor(s) but not by a direct binding to the promoter through its DNA-binding domain.

Negative autoregulation of c-Myb activity by homodimer formation through the 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.

Transcriptional control by myb oncogene product.

Structure and function of two domains of c-Myb were analyzed. We show 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. Our results suggest that an inhibitor which suppresses transactivation binds to c-Myb through the leucine zipper, and that c-Myb can be oncogenically activated by mis-sense mutation. We also proposed a model, the "tryptophan cluster", for the structure of the Myb DNA-binding domain, in which the three tryptophans form a cluster in the hydrophobic core in each repeat. The results of NMR analysis of repeat 3 revealed that the conserved tryptophans play a key role to make the hydrophobic core.

Transcriptional activation of the c-myc gene by the c-myb and B-myb gene products.

To identify the target genes modulated by the myb gene product (Myb), a co-transfection assay with a Myb expression plasmid was performed. Both c-Myb and B-Myb, another member of the myb gene family, trans-activated the human c-myc promoter. DNAase I footprint analysis using the bacterially expressed c-Myb, identified multiple c-Myb binding sites in the c-myc promoter region. Deletion analysis of the c-myc promoter suggested that some number of Myb binding sites, not a specific Myb binding site, is important for the c-Myb-induced trans-activation of the c-myc promoter. Using the c-myc-chloramphenicol acetyltransferase (CAT) construct as a reporter in a co-transfection assay, the domains of c-Myb required for trans-activation were examined. The functional domains of c-Myb identified using the c-myc promoter were almost the same as those identified previously with the artificial target gene containing Myb binding sites, but unlike the case with the artificial target gene the N-terminal half of the previously identified negative regulatory domains and the C-terminal 136 amino acids were required for the maximal trans-activation of the c-myc promoter. These results indicate that there are some differences in the regulation of Myb-dependent trans-activation in different target genes.

Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure.

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.

Transformation by carboxyl-deleted Myb reflects increased transactivating capacity and disruption of a negative regulatory domain.

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 tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product.

In the DNA-binding domain of the c-myb protooncogene product (c-Myb) which consists of three repeats of 51-52 amino acids, there are 3 perfectly conserved tryptophans in each repeat. Site-directed mutagenesis of these tryptophans showed that any single or multiple mutations of tryptophan to hydrophilic residues or alanine abolished or greatly reduced the sequence-specific DNA-binding activity, but mutations to hydrophobic amino acids retained considerable activity. Raman spectroscopic study showed that these tryptophans were buried in the protein core. These 3 tryptophans are proposed to form a cluster in the hydrophobic core in each repeat. This hypothetical structure is referred to as the "tryptophan cluster," and it may represent a characteristic property of a group of DNA-binding proteins including the myb- and ets-related proteins.

Binding of the c-myb proto-oncogene product to the simian virus 40 enhancer stimulates transcription.

The proto-oncogene c-myb encodes a nuclear protein which binds to DNA. Here we find that bacterially synthesized c-myb protein binds to one site of the simian virus 40 enhancer. The c-myb protein purified from the human T-cell line, Molt4, was also shown to recognize the same sequence. In co-transfection experiments with a c-myb expression plasmid, tandem repeats of a c-myb-binding sequence were shown to function as a c-myb-dependent enhancer. These results indicate the c-myb protein is a simian virus 40 enhancer-binding protein that can positively regulate transcription.

Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene.

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.

Leucine zipper structure of the protein CRE-BP1 binding to the cyclic AMP response element in brain.

By screening a lambda gt11 library with the multimerized sequence of the cAMP response element (CRE), we isolated human clones encoding the CRE binding protein, CRE-BP1, from a human brain cDNA library. CRE-BP1 expressed in Escherichia coli bound not only to the CRE element of the somatostatin and fibronectin genes, but also to the CRE element of the adenovirus E4 gene, suggesting that the protein was not distinguishable from the adenovirus transcription factor, ATF. The human CRE-BP1 clone encoded a 54.5 kd protein similar at its carboxy terminus to the leucine zipper motifs found in other enhancer binding proteins such as C/EBP and c-jun/AP-1. CRE-BP1 mRNA was expressed in all of the cells examined and was abundant in brain. The structure of CRE-BP1 and its recognition elements suggest that cellular response to extracellular stimuli is controlled by a family of transcription factors that bind to related cis-active elements and that contain several highly conserved domains.