The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.     

Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint

We report on the function of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 did not accumulate at functional telomeres, but localized to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 formed foci that colocalized with other DNA-damage-response factors. This response was strictly dependent on ATM (ataxia telangiectasia mutated) and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition resulted in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype was independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals.     

S. pombe sck2+, a second homologue of S. cerevisiae SCH9 in fission yeast, encodes a putative protein kinase closely related to PKA in function

The Schizosaccharomyces pombe sck2 gene, originally identified as SPAC22E12.14c in the genome-sequencing project, encodes a putative protein kinase highly similar to Saccharomyces cerevisiae Sch9p and S. pombe Sck1p, both of which can suppress loss of cAMP-dependent protein kinase (PKA) if over-produced. Over-expression of sck2 suppressed typical phenotypes of PKA-defective cells, including ectopic mating, slow growth and short cell morphology. Wild-type cells over-expressing sck2 behaved like the PKA-hyperactive mutant. Disruption of sck2 caused no obvious phenotype, but it intensified de-repression for sexual development when combined with the disruption of sck1. The pka1 sck1 sck2 triple disruptant could grow but only very slowly. Whereas disruption of sck1 enhanced the inefficiency of Δpka1 spores in germination, disruption of sck2 did not. These results suggest that the molecular function of Sck2p largely overlaps with that of Sck1p, but also that they differ somewhat either quantitatively or qualitatively. Received: 30 December 1997 / 20 January 1998     

The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC

Cohesin is a protein that plays a key role in the cohesion and separation of sister chromatids. During the duplication of chromatids, cohesin holds sister chromatids together until the onset of anaphase, and thereby prevents the premature separation of sister chromatids which would otherwise jeopardize the faithful segregation of chromosomes. To investigate the molecular mechanisms of sister chromatid cohesion, we have isolated multicopy suppressors of a temperature-sensitive (ts) mutation in the SCC1/MCD1/RHC21 gene which encodes a component of the cohesin complex in budding yeast. Isolation of multicopy suppressors of rhc21-sk16 and further genetic analyses revealed that several distinct biological pathways are involved in the regulation of SCC1/MCD1/RHC21 function. Firstly, PDE2 and BCY1, each of which inhibits the activity of protein kinase A (PKA), suppressed the temperature sensitivity of the rhc21-sk16 mutant . Secondly, PDE2 suppressed the temperature sensitivity of the cdc16-1 mutant. These results suggest that SCC1/MCD1/RHC21 is negatively regulated by the PKA pathway via the anaphase promoting complex (APC). Thirdly, ZDS1, a multicopy suppressor of cdc28-1N, and its homologue ZDS2 were isolated as multicopy suppressors of rhc21-sk16. Furthermore, the rhc21-sk16 mutant did not grow in the presence of the cdc28-1N mutation. Hence, SCC1/MCD1/RHC21 is positively regulated by the mitotic CDK, CDC28. Finally, SCC1/MCD1/RHC21 was found to interact genetically with CDC20, an activator of APC. Overexpression of CDC20 suppressed the temperature sensitivity of rhc21-sk16, and rhc21-sk16 was shown to be synthetically lethal with cdc20-1. In addition, the growth of the rhc21-sk16 mutant was inhibited by overproduction of the anaphase inhibitor Pds1p, whose degradation is mediated by Cdc20p in APC-dependent proteolysis. The functional relationships between SCC1/MCD1/RHC21 and PKA, CDK or APC are discussed. Received: 11 March / 22 June 1999     

IL2-dependent phosphorylation of 40S ribosomal protein S6 is controlled by PI-3K/mTOR signalling in CTLL2 cells

Growth factors and hormones activate global and selective protein translation by phosphorylation and therefore activation of p70 S6 kinase through a wortmannin-sensitive phosphoinositide-3 kinase (PI-3K) antiapoptotic pathway and a rapamycin-sensitive signalling pathway of mTOR. Here we demonstrate that the phosphorylation of 40S ribosomal protein S6, a physiological substrate p70 S6 kinase, was highly increased by growth-stimulation of the cytolytic T cells (CTLL2) with interleukin 2 (IL2), which was accompanied with the increased phosphorylation of p70 S6K. The activity of p70 S6K and phosphorylation of the S6 protein was completely blocked by rapamycin and significantly decreased upon treatment of the cells with wortmannin, indicating an involvement of the PI-3K pathway in concert with the signalling pathway of mTOR in IL2-dependent phos-phorylation of ribosomal protein S6. The phosphorylation and activity of PKB/Akt in IL2-stimulated CTLL2 cells were rapamycin-insensitive and reduced upon wortmannin treatment of the cells, confirming a requirement for PI-3K for Akt activity. The data support the hypothesis that Akt may act downstream to PI-3K and upstream to mTOR in an IL2-mediated signal transduction pathway that controls phosphorylation of the regulatory protein S6 in CTLL2 cells.     

Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1

Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.     

The yeast phosphotyrosyl phosphatase activator protein,yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA

We previously reported the isolation of mutants hypersensitive to the genotoxic agent 4-nitroquinoline-1-oxide, a potent inducer of oxidative stress. One of the mutants was defective in a gene designated yPTPA1, encoding a protein related to the human phosphotyrosyl phosphatase activator hPTPA, which is believed to play a role in activating the serine/threonine phosphatase PP2A. Yeast yptpa1 mutants are also sensitive to the UVA component of sunlight known to produce reactive oxygen species, suggesting a role for yPtpa1 in oxidative stress response. We now report the characterization of another 4-nitroquinoline-1-oxide-sensitive mutant, EBY20. We show that this mutant is defective in the SIT4 gene encoding a catalytic subunit of the PP2A phosphatases and that sit4 mutants exhibit hypersensitivity to 4-nitroquinoline-1-oxide and UVA, but not to UVC at 254 nm. Like the yptpa1 mutants, sit4 mutants are also defective in the repair of 4-nitroquinoline-1-oxide-induced DNA lesions. Genetic analysis revealed that both yPtpa1 and Sit4 function in the same pathway to protect cells against the lethal effects of 4-nitroquinoline-1-oxide and UVA. Moreover, we demonstrate that yPtpa1-affinity columns specifically retain Sit4, confirming a previous report that these two proteins indeed belong to a complex. Cellular localization studies using GFP-tagged proteins reveals that yPtpa1 is localized to the cytoplasm and the nucleus, while the Sit4 protein shows an intense staining spot in the cytoplasm and diffused staining in this organelle. We suggest that the yPtpa1–Sit4 complex may participate in a novel mechanism that mediates repair of oxidative DNA damage caused by 4-nitroquinoline-1-oxide and UVA.     

Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint

HIV-1 Vpr induces cell cycle G2 arrest, morphological changes and cell death in human and fission yeast cells. The cellular targets for G2 arrest were expected to be the inhibitory phosphorylation sites of Cdc2, as G2 arrest correlates with hyperphosphorylation and decreased activity of Cdc2 in both human and fission yeast cells. In this study, we present direct evidence of genetic suppression of Vpr-induced G2 arrest by cdc2 mutations. Mutations in cdc2 (cdc2-1w and cdc2-3w) reduce the ability of Vpr to induce G2 arrest. A strain with a mutation changing the Tyr15 of Cdc2 to the non-phosphorylated Phe (Y15F) eliminated Vpr-induced G2 arrest indicating that Tyr15 of Cdc2 is the sole target for induction of G2 arrest by Vpr. Although the G2 arrest induced by DNA damage also proceeds through phosphorylation of Tyr15, the rad1, rad3, rad9 and rad17 mutations, which eliminate the G2 checkpoint for DNA damage, did not block the G2 arrest induced by Vpr. Furthermore, Vpr expression did not alter sensitivity of these rad mutants to UV radiation. Thus, the pathways for the induction of G2 arrest by DNA damage and Vpr are not identical. Interestingly, Vpr still induces cell death and morphological changes in the Y15F Cdc2 strain indicating that G2 arrest is not required for morphological changes and cell death. This conclusion was further supported by the observation that mutations in Vpr, which have lost their ability to induce G2 arrest, retained the ability to kill cells.     

ELKS, a protein structurally related to the active zone protein CAST, is involved in Ca2+-dependent exocytosis from PC12 cells

The active zone protein CAST binds directly to the other active zone proteins RIM, Bassoon and Piccolo, and it has been suggested that these protein-protein interactions play an important role in neurotransmitter release. To further elucidate the molecular mechanism, we attempted to examine the function of CAST using PC12 cells as a model system. Although PC12 cells do not express CAST, they do express ELKS, a protein structurally related to CAST. Endogenous and exogenously expressed ELKS, RIM2 and Bassoon were colocalized in punctate signals in PC12 cells. Over-expression of full-length ELKS resulted in a significant increase in stimulated exocytosis of human growth hormone (hGH) from PC12 cells, similar to the effect of full-length RIM2. This increase was not observed following over-expression of deletion constructs of ELKS that lacked either the last three amino acids (IWA) required for binding to RIM2 or a central region necessary for binding to Bassoon. Moreover, over-expression of the NH(2)-terminal RIM2-binding domain of Munc13-1, which is known to inhibit the binding between RIM and Munc13-1, inhibited the stimulated increase in hGH secretion by full-length RIM2. Furthermore, this construct also inhibited the stimulated increase in hGH secretion induced by full-length ELKS. These results suggest that ELKS is involved in Ca(2+)-dependent exocytosis from PC12 cells at least partly via the RIM2-Munc13-1 pathway.     

Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- promoting complex at mitosis

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction in Xenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.     

The collagen-like component of the complement system, C1q, is recognized by 7 S autoantibodies and is functionally impaired in synovial fluids of patients with rheumatoid arthritis.

Cross-reactivity between type II collagen (CII) and C1q, the collagen-like subunit of the first component of complement, has been demonstrated in synovial fluid (SF) from rheumatoid arthritis (RA) patients. Many authors have studied autoimmunity to CII in RA, but little work has been done on autoimmunity to C1q in RA. In the data presented here, we have been able to show that in addition to native C1q, an altered form of C1q is present in SF from RA patients. Furthermore, a low molecular weight form of C1q is present in RA SF, although its role, if any, in the pathogenesis of RA is unclear. The presence in these RA SF of C1q-specific antibodies (IgG and IgM) has been studied and we have partially characterized the antibody moieties involved. As well as binding to C1q and fragments representing the collagen-tails from C1q, 7 S IgG autoantibodies against C1q also bind to a C1q molecule altered in vitro by incubation with reactive oxygen species and to the non-apeptide KGEQGEPGA (representing residues 26-34 from the C1q A-chain), which has previously been shown to suppress the onset of CII-induced arthritis in an animal model.