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DJ-1 is all oncogene and also a causative gene for a familial form of Parkinson's disease. DJ-1 has multiple functions, including anti-oxidative stress reaction and cysteine 106 (C106) of DJ-1 is an essential amino acid for DJ-1 to exert its function. While increased expression and secretion of DJ-1 into serum in patients with various cancers and regulation of p53 and PTEN by DJ-1 have been reported, the molecular mechanism underlying oncogenicity of DJ-I is poorly understood. Here, we analyzed the function of DJ-1 in the PI3'K signaling pathway under an oxidative stress condition, focusing on the interaction of DJ-1 with PTEN. We found that both wild-type (wt) and C106S-DJ-1, a substitution mutant of DJ-1, directly bound to PTEN and inhibited PTEN phosphatase activity but that C106S-DJ-1 more strongly inhibited the activity than did wt-DJ-1. When NIH3T3 cells were treated with H2O2, oxidation of C106 of wt-DJ-1 occurred, accompanied by increased binding, of wt-DJ-1 to PTEN, decreased PTEN activity and increased phosphorylation of AKT. C106S-DJ-1 transformed cells more strongly than did wt-DJ-1 and the transforming activity of DJ-1 was enhanced by H2O2 treatment of cells in which increased binding of DJ-1 to PTEN and decreased PTEN activity were observed. Furthermore, TOF-MS analysis of the oxidative status of C106 suggested that DJ-1 activity requires the presence of the reduced form of C106, which accounts for >50% of the total form. These results suggest that the oxidative status of DJ-1 regulates PTEN activity, leading to cell proliferation and transformation.

We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1s/ KAP1, a transcriptional corepressor. 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, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome. Plasmids. pcDNA3-FLAG-Elongin B, pcDNA3-FLAG- Rpt3, pcDNA3-FLAG-Cullin2, pcDNA3-T7-Rpn12 and pcDNA3-T7-Skp2, corresponding cDNAs starting from the first ATG, were inserted in-frame into the EcoRI-XhoI sites of pcDNA3-FLAG and pcDNA3-T7, respectively. Plasmids of various kinds of c-Myc and MM-1 were described previously (11-13).

DJ-1 was identified by us as a novel oncogene in cooperation with activated ras. Although over-expression of DJ-1 has been reported in several cancer cells, including cells in breast cancer, lung cancer and prostate cancer, the precise mechanism underlying transformation has not been clarified. In this study, we screened proteins by a yeast two-hybrid method and identified Abstrakt as a DJ-1-binding protein. Abstrakt is an RNA helicase, but it has not yet been characterized. Northern blot analysis showed that human Abstrakt was expressed ubiquitously in all tissues. Abstrakt was then found to bind to and to be colocalized in the nucleus with DJ-1 in human cells. Furthermore, Abstrakt was found to stimulate transforming activity of DJ-1 in rat 3Y1 cells transfected with DJ-1 with activated ras. These findings suggest that Abstrakt is a positive regulator for DJ-1.

DJ-1 is a multi-functional protein that plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in onset of Parkinson's disease. Here, we report that DJ- 1 bound to Topors/p53BP3, a ring finger protein binding to both topoisomerase I and p53, in vitro and in vivo and that both proteins were colocalized in cells. DJ-1 and p53 were then found to be sumoylated by Topors in cells. It was also found that DJ-1 bound to p53 in vitro and in vivo and that colocalization with and its binding to p53 were stimulated by UV irradiation of cells. Transcription activity of p53 was found to be abrogated by Topors concomitant with sumoylation of p53 in a dose-dependent manner, and DJ-1 restored its repressed activity by releasing the sumoylated form of p53. These findings suggest that DJ-1 positively regulates p53 through Topors-mediated sumoylation.

We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from cytoplasm to nuclei in a c-Myc-dependent manner. Here, the role of AMY-1 in cell differentiation was examined. AMY-1 expression was up-regulated after differentiation induction of human K562 cells to erythrocyte cells by AraC, while c-Myc expression was rapidly down-regulated. K562 cell lines expressing exogenous AMY-1 were established, and these cells expressed a high level of epsilon-globin mRNA, a marker gene necessary for erythrocyte cell differentiation, without differentiation induction. The addition of AraC rapidly initiated differentiation in these cell lines, which continued to differentiate to erythrocyte cells possessing a high level of hemoglobin even after the decrease in AMY-1 expression. These results suggest that AMY-1 is a trigger for K562 cells to differentiate to erythrocyte cells and that AMY-1 may have a function independent of or different from c-Myc.

The mouse p53 gene, spreading over 16 kb, was divided into 10 segments of approximately 1,000 bp and subcloned into pUC19. The clones were transfected into mouse L cells together with an expression vector of the hygromycin B- or blasticidin S-resistance gene. The transfected cells were cultured in the drug-containing medium to establish cell lines resistant to the drug. Among the established lines, a high percentage of the cells originally transfected with pp53(H-R), possessing the HindIII-EcoRI fragment located downstream from the last coding exon of the p53 gene, harbored plasmid DNAs in an episomal state. The plasmid DNAs recovered from the 53HR cell-lines, transfected with pp53(H-R), were indistinguishable in structure from the original plasmids used for transfection, as assayed by Southern blotting and back-transformation to bacteria. The plasmids in episome of the cells replicated once per cell cycle in S phase in concert with chromosomal DNA. In the HindIII-EcoRI fragment (i.e., the p53(H-R) fragment) which showed a highly efficient replication activity, there exists a putative sequence for p53 recognition near the HindIII site. A DNA bending region with the clusters of AT tracts also exists near the EcoRI site of the same p53(H-R) fragment. In a transient replication system, pp53(H-R) autonomously replicated in episome of transfected cells, while mutant plasmids lacking either the p53 recognition sequence or the bending region did not. The results suggest that the HindIII-EcoRI region downstream from the p53 gene contains an activity of cellular DNA replication and both the p53 recognition sequence and the bending region are necessary for the replication activity.