Hirotake Kitaura

MM-1, a novel c-Myc-associating protein that represses transcriptional activity of c-Myc.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering themyc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.

TOK-1, a Novel p21Cip1-binding Protein That Cooperatively Enhances p21-dependent Inhibitory Activity toward CDK2 Kinase

A p21Cip1/Waf1/Sdi1 is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1α and TOK-1β, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1α, but not TOK-1β, directly bound to the C-terminal proximal region of p21, and both were expressed at the G1/S boundary of the cell cycle. TOK-1α also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1α enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1α is thus thought to be a new type of CDK2 modulator.

AMY‐1, a novel C‐MYC binding protein that stimulates transcription activity of C‐MYC

The c‐myc proto‐oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C‐MYC, the product of c‐myc, are attributed to protein–protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C‐terminal region of C‐MYC, while p107, Bin1 and TBP bind to the N‐terminal region covering myc boxes. The N‐terminal region is involved in all the biological functions of C‐MYC, and different proteins are therefore thought to interact with the N‐terminal region of C‐MYC to display different functions.

Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation

Background: Prefoldin, a molecular chaperone composed of six subunits, prevents misfolding of newly synthesized nascent polypeptides. Results: Prefoldin inhibited aggregation of pathogenic Huntingtin and subsequent cell death. Conclusion: Prefoldin suppressed Huntingtin aggregation at the small oligomer stage. Significance: Prefoldin plays a role in preventing protein aggregation in Huntington disease. Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.

Negative regulation of the Wnt signal by MM-1 through inhibiting expression of the wnt4 gene.

We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc through TIF1beta/KAP1, a transcriptional corepressor, and that the c-fms gene was a target gene involved in this pathway. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, to further identify target genes of MM-1, DNA microarray analysis was carried out by comparing expression levels of genes in MM-1 knockdown and parental cells, and the wnt4 gene, a member of the Wnt-beta-catenin pathway, was identified as a target gene of MM-1. Increased expression level of the wnt4 gene, accumulation and translocation of beta-catenin to the cytoplasm and nucleus, and upregulation of TCF/Lef-1, a target protein of the Wnt-beta-catenin pathway, were found in MM-1 knockdown cells. Reporter assays using various deletion constructs of the wnt4 gene promoter showed that MM-1 recognized the region spanning -286 to -229 from a transcription start site, and MM-1 complex was found to bind to this region by chromatin immunoprecipitation and gel mobility shift assays. Furthermore, it was found that Egr-1 and MM-1 were bound to this region and that both proteins mutually down-regulate promoter activity of the wnt4 gene. Since the c-myc gene is the target gene of the Wnt-beta-catenin pathway, these findings suggest that MM-1 inhibits c-Myc by a dual mechanism.

DJ-1, an oncogene and causative gene for familial Parkinson's disease, is essential for SV40 transformation in mouse fibroblasts through up-regulation of c-Myc

MINT‐7988969: DJ‐1 (uniprotkb:Q99497) binds (MI:0407) to LT SV40 (uniprotkb:P03070) by pull down (MI:0096) MINT‐7988948: LT SV40 (uniprotkb:P03070) physically interacts (MI:0914) with DJ‐1 (uniprotkb:Q99LX0) and p53 (uniprotkb:P02340) by anti bait coimmunoprecipitation (MI:0006)

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

Prefoldin Plays a Role as a Clearance Factor in Preventing Proteasome Inhibitor-induced Protein Aggregation

Background: Prefoldin consisting of six subunits prevents protein misfolding. Results: Prefoldin prevented proteasome inhibitor- and endoplasmic reticulum stress-induced protein aggregation, and mutation of a prefoldin subunit resulted in loss of its activity. Conclusion: In addition to its protein folding activity, prefoldin protects cells from aggregated protein-induced cell death. Significance: Prefoldin is a quality control protein against protein aggregation. Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases.

Activation of c-myc promoter by c-myc protein in serum starved cells

The function of the c‐myc protein, the product of a proto oncogene, is not clearly understood although many reports, including ours, suggest that the c‐myc protein plays several roles in the regulation of transcription and DNA replication. Here we examined the effects of c‐myc protein on transcription from the c‐myc promoter, and by inference its role in auto‐regulation, after introducing into cultured cells a c‐myc expression vector and a CAT reporter gene linked to the promoter and upstream region of the human c‐myc gene. To minimize the effects of the endogenous c‐myc protein on the exogenously added CAT reporter gene, the transfected cells were treated under serum‐free conditions. The results show that CAT expression from the myc promoter increased in a dose‐dependent manner after addition of the c‐myc expression vector, and that it also required the presence of a c‐myc binding sequence previously identified 2 kb upstream from c‐mycs first exon. Moreover, the domains of the c‐myc protein important for transactivation were determined by use of various deletions mutants of c‐myc cDNA. The results showed that the N‐terminal portion in the c‐myc protein was necessary for transactivation beside the C‐terminal portion containing basic region, helix‐loop‐helix, and leucine zipper.

Transcriptional activation of the cholecystokinin gene by DJ-1 through interaction of DJ-1 with RREB1 and the effect of DJ-1 on the cholecystokinin level in mice.

DJ-1 is an oncogene and also causative gene for familial Parkinson’s disease. DJ-1 has multiple functions, including transcriptional regulation. DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found that the cholecystokinin (CCK) gene is a transcriptional target gene for DJ-1. CCK is a peptide hormone and plays roles in contraction of the gallbladder and in promotion of secretion of pancreatic fluid. CCK is co-localized with dopamine in the substantia nigra to regulate release of dopamine. Reduced expression of CCK mRNA was observed in DJ-1-knockdown cells. The Ras-responsive element (RRE) and Sp1 site were essential for promoter activity, and DJ-1 stimulated promoter activity by binding to RRE-binding protein 1 (RREBP1). The complex of DJ-1 with RREB1 but not with Sp1 bound to the RRE. Furthermore, the reduced CCK level in the serum from DJ-1-knockout mice compared to that from wild-type mice was observed. This is the first report showing that DJ-1 participates in peptide hormone synthesis.