Yoko Kimura

Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes

Viral infections commonly induce expression of type I interferon (IFN) genes. The induction is transient and involves transcriptional activation wherein a positive factor, IRF-1, binds to upstream regulatory cis elements. In the present study we report the isolation of a cDNA encoding a novel factor, termed IRF-2, that interacts with the same nucleotide sequence elements as IRF-1. Both genes are inducible not only by virus but also by IFN. Unlike IRF-1, IRF-2 does not function as an activator; rather, it suppresses the function of IRF-1 under certain circumstances. Our results suggest that transcription of the IFN and IFN-inducible genes is regulated by two similar trans-acting factors that apparently compete for the same cis-acting recognition sequences, but which have opposite effects.

Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-β gene regulatory elements

Expression of the interferon-beta (IFN-beta) gene is induced by a variety of agents, including viruses. Evidence has been provided that a mouse nuclear factor, termed interferon regulatory factor-1 (IRF-1), specifically binds to the upstream regulatory region of the human IFN-beta gene and mediates virus-induced transcription of the gene. In this study, we describe the molecular cloning and characterization of the mouse and human cDNAs encoding IRF-1. Our results suggest that IRF-1 is also involved in the regulation of other genes such as IFN-alpha and MHC class I genes. Surprisingly, IRF-1 gene expression is dramatically induced by Newcastle disease virus in mouse L929 cells and by Concanavalin A in spleen cells. We show here that the IRF-1 gene possesses virus-inducible promoter.

Ubiquitin is phosphorylated by PINK1 to activate parkin

PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.

Evidence for a nuclear factor(s), IRF-1, mediating induction and silencing properties to human IFN-beta gene regulatory elements.

Transcription of the human interferon‐beta (IFN‐beta) gene is induced by a variety of agents such as viruses, dsRNA and some cytokines. In this study, we describe a nuclear factor, termed interferon regulatory factor‐1 (IRF‐1), that is involved in the transcription of IFN‐beta and possibly other genes. We demonstrate that IRF‐1 functions in virus‐induced transcription by interacting with previously identified, IFN‐beta regulatory DNA elements. Our data suggest that IRF‐1 participates in the transient formation of an induction‐specific complex(es) with the regulatory elements. IRF‐1 may also be involved in silencing the function of the SV40 enhancer juxtaposed to the regulatory elements in uninduced cells.

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders. Cell Death and Differentiation (2001) 8, 977–984

Regulatory mechanisms involved in the control of ubiquitin homeostasis

Ubiquitin (Ub) modification plays an essential role in the regulation of various cellular processes. Ub performs a remarkable array of cellular tasks through the production of a large number of ubiquitinated proteins; such tasks require many Ubs. Ubs are expressed abundantly from several Ub encoding genes, though not in excess. Rather, Ub expression is tightly regulated through various control mechanisms. Recent studies have shown that the cellular Ub level is regulated by balanced activities of deubiquitinating enzymes and their regulators. Here, we review the current understandings of the regulatory mechanisms that control Ub expression and its metabolism and maintain Ub homeostasis.

An Inhibitor of a Deubiquitinating Enzyme Regulates Ubiquitin Homeostasis

The dynamic and reversible process of ubiquitin modification controls various cellular activities. Ubiquitin exists as monomers, unanchored chains, or protein-conjugated forms, but the regulation of these interconversions remains largely unknown. Here, we identified a protein designated Rfu1 (regulator of free ubiquitin chains 1), which regulates intracellular concentrations of monomeric ubiquitins and free ubiquitin chains in Saccharomyces cerevisiae. Rfu1 functions as an inhibitor of Doa4, a deubiquitinating enzyme. Rapid loss of free ubiquitin chains upon heat shock, a condition in which more proteins require ubiquitin conjugation, was mediated in part by Doa4 and Rfu1. Thus, regulation of ubiquitin homeostasis is controlled by a balance between a deubiquitinating enzyme and its inhibitor. We propose that free ubiquitin chains function as a ubiquitin reservoir that allows maintenance of monomeric ubiquitins at adequate levels under normal conditions and rapid supply for substrate conjugation under stress conditions.

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell‐adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin‐binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmons involvement in podosome/invadopodium‐mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.

ATPase Activity of p97/Valosin-containing Protein Is Regulated by Oxidative Modification of the Evolutionally Conserved Cysteine 522 Residue in Walker A Motif *

Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (Δcdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.