Sanae M.M. Iguchi-Ariga

DJ‐1 has a role in antioxidative stress to prevent cell death

Deletion and point (L166P) mutations of DJ‐1 have recently been shown to be responsible for the onset of familial Parkinsons disease (PD, PARK7). The aim of this study was to determine the role of DJ‐1 in PD. We first found that DJ‐1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ‐1 knockdown by short interfering RNA rendered SH‐SY5Y neuroblastoma cells susceptible to hydrogen peroxide‐, MPP+‐ or 6‐hydroxydopamine‐induced cell death and that cells harbouring mutant forms of DJ‐1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ‐1. These results clearly showed that DJ‐1 has a role in the antioxidative stress reaction and that mutations of DJ‐1 lead to cell death, which is observed in PD.

Identification of heterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase and the effect of phosphorylation on the transcriptional repression function of HP11

Pim‐1, a protooncogene product, is a serine/threonine kinase and is thought to play a role in signal transduction in blood cells. Few phosphorylated target proteins for Pim‐1, however, have been identified. In the present study, two‐hybrid screening to clone cDNAs encoding proteins binding to Pim‐1 was carried out, and a cDNA for heterochromatin protein 1γ (HP1γ) was obtained. Binding assays both in yeast and in vitro pull‐down using the purified HP1γ and Pim‐1 expressed in Escherichia coli showed that Pim‐1 directly bound to the chromo shadow domain of HP1γ. HP1γ was also associated with Pim‐1 in human HeLa cells and the serine clusters located at the center of HP1γ were phosphorylated by Pim‐1 in vitro. Furthermore, a transcription repression activity of HP1γ was further stimulated by the deletion of the serine clusters targeted by Pim‐1. These results suggest that Pim‐1 affects the structure or silencing of chromatin by phosphorylating HP1.

DJ-1 binds to mitochondrial complex I and maintains its activity

Parkinsons disease (PD) is caused by neuronal cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for onset of PD. DJ-1, a causative gene product of a familial form of Parkinsons disease, PARK7, plays roles in transcriptional regulation and anti-oxidative stress. The possible mitochondrial function of DJ-1 has been proposed, but its exact function remains unclear. In this study, we found that DJ-1 directly bound to NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and was colocalized with complex I and that complex I activity was reduced in DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and that DJ-1 plays a role in maintenance of mitochondrial complex I activity.

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.

Cell Cycle-dependent Switch of Up- and Down-regulation of Human hsp70 Gene Expression by Interaction between c-Myc and CBF/NF-Y

A CCAAT box-binding protein subunit, CBF-C/NF-YC, was cloned as a protein involved in the c-Myc complex formed on the G1-specific enhancer in the human hsp70gene. CBF-C/NF-YC directly bound to c-Myc in vitro andin vivo in cultured cells. The CBF/NF-Y·c-Myc complex required the HSP-MYC-B element as well as CCAAT in the hsp70G1-enhancer, while the purified CBF subunits recognized only CCAAT even in the presence of c-Myc. Both the HSP-MYC-B and CCAAT elements were also required for the enhancer activity. In transient transfection experiments, the CBF/NF-Y·c-Myc complex, as well as transcription due to the G1-enhancer, was increased by the introduction of c-Myc at low doses but decreased at high doses. The repression of both complex formation and transcription by c-Myc at high doses was abrogated by the introduction of CBF/NF-Y in a dose-dependent manner. Furthermore, the CBF/NF-Y·c-Myc complex bound to the G1-enhancer appeared in the early G1 phase of the cell cycle when c-Myc was not higly expressed and gradually disappeared after the c-Myc expression reached its maximum. The results indicate that the cell cycle-dependent expression of the hsp70 gene is regulated by the intracellular amount of c-Myc through the complex formation states between CBF/NF-Y and c-Myc.

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.

Oxidative status of DJ-1-dependent activation of dopamine synthesis through interaction of tyrosine hydroxylase and 4-dihydroxy-L-phenylalanine (L-DOPA) decarboxylase with DJ-1.

Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-l-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H2O2, 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO2H and SO3H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD.

Molecular cloning of human and mouse DJ-1 genes and identification of Sp1-dependent activation of the human DJ-1 promoter☆

DJ-1 has been identified as a novel oncogene that transforms mouse NIH3T3 cells in cooperation with activated ras. Subsequently, two other groups have identified SP22 or CAP-1, rat homologs of human DJ-1, as a sperm protein targeted by some toxicants leading to male infertility, and another group has also reported that RS, the same as human DJ-1, is a component of an RNA-binding protein complex. To characterize the expression or functional importance of DJ-1, the genomic DNAs of both human and mouse DJ-1 were cloned and characterized. Both genomic DNAs comprise 7 exons spanning about 16-24 kb, in which 2-6 exons encode the DJ-1 protein. The human DJ-1 gene was mapped at chromosome 1p36.2-p36.3, a region that has been shown to be a hot spot of chromosome abnormalities in several tumor cells. To analyze the promoter of the human DJ-1 gene, a series of deletion constructs of the region upstream of exon 2 were linked to the luciferase gene, and their luciferase activities were measured in human HeLa cells. Of the many putative transcription regulatory sequences, the Sp1 site present at -100 from the transcription initiation site contributed to the major promoter activity, and Sp1 was identified as a protein binding to this site by a mobility shift assay using HeLa nuclear extract.

Pim‐1 translocates sorting nexin 6/TRAF4‐associated factor 2 from cytoplasm to nucleus

Pim‐1, an oncogene product of serine/threonine kinase, has been found to play roles in apoptosis induction/suppression, cell‐cycle progression and transcriptional regulation by phosphorylating the target proteins involved in these processes. The target proteins phosphorylated by Pim‐1, including p100, Cdc25A, PAP‐1 and heterochromatin protein 1, have been identified. The precise functions of Pim‐1, however, are still poorly understood. In this study, we identified tumor necrosis factor receptor‐associated factor 4‐associated factor 2/sorting nexin 6 (TFAF2/SNX6) as a Pim‐1‐binding protein, and we found that TFAF2/SNX6 was phosphorylated and translocated from the cytoplasm to nucleus by Pim‐1. This translocation of the protein was not affected by Pim‐1‐dependent phosphorylation. Since sorting nexins, including TFAF2/SNX6, have been reported to be located in the cytoplasm or membrane by association with several receptors of tyrosine‐ or serine/threonine‐kinase, this is the first report of TFAF2/SNX6 being located in the nucleus after binding to Pim‐1.