Takeharu Nishimoto

Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis.

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re‐replication. We show here that the N‐terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S–G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1‐Cul4‐mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF‐Skp2, which recognizes the Cy‐motif‐mediated Cyclin E/A‐cyclin‐dependent kinase‐phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S–G2 phases. The Cul4‐containing E3 is active during ongoing replication, while SCF‐Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N‐terminal amino acids. PCNA is essential for Cul4‐ but not Skp2‐directed degradation during DNA replication and following ultraviolet‐irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re‐replication.

Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the production of MPM‐2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10‐fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo‐metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.

Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome

Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1s interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p ofSaccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [3H]GTP and [3H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature‐sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non‐permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non‐permissive temperature, p34cdc2 was dephosphorylated and M‐phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non‐permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase‐microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.

The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N‐Ran), which was predicted to be primarily in the GDP‐bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N‐Ran disrupts normal nuclear activity and found that T24N‐Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N‐Ran‐blocked interphase extracts to assemble nuclei from de‐membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild‐type GTP‐bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two‐hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3–54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide‐releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670–200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1‐Ran pathway.

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.