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.

Solution structure of calmodulin-W-7 complex: the basis of diversity in molecular recognition.

The solution structure of calcium-bound calmodulin (CaM) complexed with an antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), has been determined by multidimensional NMR spectroscopy. The structure consists of one molecule of W-7 binding to each of the two domains of CaM. In each domain, the W-7 chloronaphthalene ring interacts with four methionine methyl groups and other aliphatic or aromatic side-chains in a deep hydrophobic pocket, the site responsible for CaM binding to CaM-dependent enzymes such as myosin light chain kinases (MLCKs) and CaM kinase II. This competitive binding at the same site between W-7 and CaM-dependent enzymes suggests the mechanism by which W-7 inhibits CaM to activate the enzymes. The orientation of the W-7 naphthalene ring in the N-terminal pocket is rotated approximately 40 degrees with respect to that in the C-terminal pocket. The W-7 ring orientation differs significantly from the Trp800 indole ring of smooth muscle MLCK bound to the C-terminal pocket and the phenothiazine ring of trifluoperazine bound to the N or C-terminal pocket. These comparative structural analyses demonstrate that the two hydrophobic pockets of CaM can accommodate a variety of bulky aromatic rings, which provides a plausible structural basis for the diversity in CaM-mediated molecular recognition.

NMR spectroscopic study of single-stranded DNA fragments of d(CGGCGAAAGCCG) and d(CGGCAAAAGCCG).

We have investigated the structures of two kinds of single-stranded DNA fragments, d(CGGCGAAAGCCG) and d(CGGCAAAAGCCG), by use of NMR spectroscopy. It was found that the former takes a hair-pin like structure stabilized by hydrogen bonding of G/C base pairs in the stem region, while the latter takes a rather extended helical structure. The stable conformation of the former DNA is considered to originate from the stability of the sequence-specific conformation of the loop region rather than the stem region.

Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.

The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by > 500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.

Interaction of a protooncogene product, Myb with DNAs.

The DNA-binding domain of Myb consists of three imperfect tandem repeats and the third one which is essential for sequence-specific binding was established to have a helix-turn-helix-related motif. DNA sequences recognized by Myb have been reported to contain TAACPy sequence. Here we have examined the details of Myb-binding sequence. Using DNAs with a single mutation on the various sites of two specific DNAs and some fragments of the DNA-binding domain of Myb, we have found that (i) in a specific DNA which contains only one AAC sequence, each AAC nucleotide is found to be essential for the specific binding of Myb, while any other mutations cause no serious binding loss, (ii) in a specific DNA which contains two AAC sequences separately, one AAC is not so important in the binding, and (iii) for the specific binding with DNA, at least both repeats 2 and 3 of Myb are required. These findings suggest that repeat 3 containing a helix-turn-helix-related structure recognizes the core AAC sequence and repeat 2 supports this recognition by interactions with phosphate groups of DNA.

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.

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.