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• W2019982171 abstract "Article15 March 1997free access Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe Lin Wu Lin Wu Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Paul Russell Corresponding Author Paul Russell Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Lin Wu Lin Wu Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Paul Russell Corresponding Author Paul Russell Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Author Information Lin Wu1 and Paul Russell 1 1Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA, 92037 USA The EMBO Journal (1997)16:1342-1350https://doi.org/10.1093/emboj/16.6.1342 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the ∼70 kDa Nim1 (Cdr1) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1+ (nim1 interacting factor-1). Nif1 is a novel ∼75 kDa protein containing a leucine zipper motif. The Nif1–Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. Δnif1 mutants are ∼10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to Δnim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in Δnim1 or Δwee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase. Introduction Studies of the fission yeast Schizosaccharomyces pombe have played a major role in uncovering how eukaryotic cells regulate the onset of mitosis. The focus of the mitotic control is Cdc2–Cdc13, the M-phase-inducing cyclin-dependent kinase that consists of the 34 kDa Cdc2 catalytic subunit and the ∼60 kDa Cdc13 cyclin-B subunit (Booher et al., 1989; Moreno et al., 1989). The activity of Cdc2–Cdc13 is maintained in an inhibited state during interphase mainly through the activities of Wee1 and Mik1 tyrosine kinases, which phosphorylate tyrosine-15 of the Cdc2 subunit (Russell and Nurse, 1987b; Gould and Nurse, 1989; Featherstone and Russell, 1991; Lundgren et al., 1991; Parker et al., 1992). Wee1 is the dominant component of the Wee1–Mik1 pair, as shown by the observation that wee1− mutants have a wee phenotype (Nurse, 1975), undergoing mitosis and cell division at half the length of wild type, whereas the mik1− mutation has no effect on cell size (Lundgren et al., 1991). However, simultaneous inactivation of wee1+ and mik1+ causes a lethal premature mitosis (Lundgren et al., 1991), often referred to as mitotic catastrophe (Russell and Nurse, 1986), showing that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for viability in S.pombe. The inhibitory activities of Wee1–Mik1 tyrosine kinases are counteracted by Cdc25–Pyp3 tyrosine phosphatases, which directly carry out the dephosphorylation of tyrosine-15 of Cdc2 (Russell and Nurse, 1986; Gould and Nurse, 1989; Millar et al., 1991, 1992). Cdc25 is the dominant element of the pair: loss of Cdc25 activity causes G2 arrest, whereas pyp3− phenotypes are apparent only in strains that are partially defective for Cdc25 activity (Millar et al., 1992). Genetic studies have established that the timing of mitosis is largely determined by the counteractive activities of Wee1 and Cdc25 (Russell and Nurse, 1986, 1987b), a balance which shifts in the favor of Cdc25 as cells reach the appropriate size to undergo mitosis. The protein kinases and phosphatases that regulate Cdc2–Cdc13 are themselves controlled by phosphorylation (Dunphy, 1994). Studies in fission yeast, Xenopus, humans and other species have established that Cdc25 undergoes activating phosphorylation around the time of the G2–M transition (Izumi et al., 1992; Kumagai and Dunphy, 1992; Hoffmann et al., 1993; Kovelman and Russell, 1996). Although it appears that Cdc2–cyclin B kinases are capable of carrying out at least some of the activating phosphorylations in vitro (Hoffmann et al., 1993; Izumi and Maller, 1993), it remains to be resolved whether Cdc2–cyclin B kinases perform these phosphorylations in vivo. Indeed, recent studies of Xenopus oocyte extracts have led to the identification of a second protein kinase, Plx1, that activates Cdc25 in vitro (Kumagai and Dunphy, 1996). In fission yeast, it is clear that Cdc25 remains in a weakly active state in cdc2 temperature-sensitive mutants that are arrested in late G2, establishing that the activations of Cdc2–Cdc13 and Cdc25 are mutually dependent in vivo (Kovelman and Russell, 1996). Studies of Xenopus and human cells have established that Wee1 is also regulated around the time at which cells undergo mitosis, in this case phosphorylation inhibits Wee1 (Tang et al., 1993; McGowan and Russell, 1995; Mueller et al., 1995; Watanabe et al., 1995). As is the case for Cdc25 regulation, Cdc2–cyclin B has been proposed to have a role in the inhibitory phosphorylation of Wee1, although this remains to be proven. These discoveries regarding Cdc25 and Wee1 regulation have led to a model in which activation of Cdc2–cyclin B involves autocatalytic feedback loops, whereby Cdc2–cyclin B promotes its own activation by directly or indirectly catalyzing the activating phosphorylation of Cdc25 and inhibitory phosphorylation of Wee1. In fission yeast, there is a second mechanism of regulating Wee1 that is distinct from the M-phase inactivation process mentioned above. The discovery of this regulatory process arose from the cloning of the gene nim1+ as a high-copy suppressor of cdc25-22, a temperature-sensitive mutation of cdc25+(Russell and Nurse, 1987a). cdc25-22 is suppressed by loss of Wee1 activity (Fantes, 1979), and a series of genetic tests strongly suggested that Nim1 overproduction rescued cdc25-22 by inhibiting Wee1 (Russell and Nurse, 1987a). The identification of Nim1 as a mitotic inducer was confirmed by finding that Δnim1 mutants undergo division at an elongated cell length and that loss of Nim1 activity severely enhances the cell cycle delay phenotype of cdc25-22 cells grown at the permissive temperature (Russell and Nurse, 1987a). Biochemical studies eventually established that Nim1 kinase, also known as Cdr1 (Young and Fantes, 1987; Feilotter et al., 1991), inhibits Wee1 via direct phosphorylation occurring in the C-terminal catalytic domain of Wee1 (Coleman et al., 1993; Parker et al., 1993; Wu and Russell, 1993). Having established that Nim1 functions as a mitotic inducer by negatively regulating Wee1, we have since extended our studies of Nim1 regulation. In this report, we describe how we have identified proteins that specifically interact with Nim1 by use of the yeast two-hybrid screening system (Durfee et al., 1993). The most common clone isolated in the screen, nif1+, encodes a novel protein. Nif1 overproduction delays mitosis, whereas mutational inactivation of Nif1 advances mitosis. These findings strongly suggest that Nif1 functions as a mitotic inhibitor via a direct interaction with Nim1 protein kinase. Results Isolation of S.pombe cDNA clones encoding proteins that specifically interact with Nim1 A yeast two-hybrid screen was carried out to identify proteins that interact with Nim1 (Figure 1). The bait, encoded on plasmid pAS2-Nim1, consisted of full-length Nim1 protein kinase fused to the C-terminal end of the Gal4 DNA binding domain. Gal4–Nim1 was tested for interaction with a S.pombe cDNA library fused to the Gal4 activation domain in plasmid pACT (Durfee et al., 1993). Approximately 2000 Y190/pAS2-Nim1 cells transformed with the pACT S.pombe cDNA library grew on SSC-Trp-Leu-His + 25 mM 3-amino-triazole (AT) plates. A transformation efficiency test indicated that 2×107 Trp+ Leu+ transformants were obtained in this experiment, thus the 25 mM 3-AT selection provided an ∼104 enrichment. Transcriptional activation of the lacZ gene was detected in 134 of the His+ clones. pACT-cDNA plasmids that retested as positive were recovered from 17 of these transformants. Southern hybridization showed that 16/17 (∼95%) of these cDNAs were derived from the same gene, which we named nif1+ (Nim1 interacting factor 1). The specific interaction of Nif1 with Nim1 as compared with several control bait proteins is shown in Figure 1. Analysis of the second gene, nif2+, will be presented elsewhere. Figure 1.Nif1 specifically interacts with Nim1 in the yeast two-hybrid assay. Strain Y190 was transformed with pACT-Nif1 together with pAS2-Nim1, pAS2-CDK2, pAS2-p53, pAS2-lamin or pAS2-SNF1 as indicated in the left panel. Transformants were incubated either on a SSC-Trp-Leu plate without 3-AT (middle panel), which only selects for the presence of the plasmids, or on a SSC-Trp-Leu-His + 25 mM 3-AT plate, which also selects for transcriptional activation of HIS3 reporter gene (right panel). Only the combination of pAS2-Nim1 and pACT-Nif1 promoted the formation of colonies on plates containing 25 mM 3-AT. Download figure Download PowerPoint The nif1+ cDNA was used to probe membranes containing overlapping cosmid and P1 clones spanning the complete S.pombe genome (Hoheisel et al., 1993). A 6 kb HindIII fragment from P1 clone 28C4p, containing the complete genomic sequence of nif1+, was cloned into pBluescript. nif1+ encodes a novel protein of 681 amino acids with a predicted mol. wt of ∼75 kDa (Figure 2). Nif1 has no extensive regions of homology to any proteins with known function. Nif1 contains a leucine zipper motif at amino acids 505–526. The leucine zipper motif has been implicated in protein dimerization. Three PEST regions were found in Nif1 (amino acids 78–107, 234–252 and 280–298). PEST regions are commonly found in proteins that are rapidly degraded (Rogers et al., 1986). Figure 2.The nucleotide sequence of nif1+ and predicted amino acid sequence of Nif1 protein. nif1+ encodes a protein of 681 amino acids with a predicted mol. wt of 75 kDa. The in-frame stop codon TAA in the 5′ untranslated region is underlined. The boxed TCA codon indicates the 5′ end of the 2.2 kb nif1+ cDNA clones obtained from the screen; the boxed TTT sequence indicates the 5′ end of 1.8 kb nif1+ cDNA clones that were also obtained in the screen. The putative leucine zipper motif in the Nif1 protein sequence is also boxed (amino acids 505–526). The three putative PEST regions are underlined: amino acids 78–107, 234–252 and 280–298. The nif1+ accession number is U64574. Download figure Download PowerPoint Nif1 associates with Nim1 in S.pombe Experiments were carried out to determine whether Nim1 and Nif1 interact in fission yeast cells. Nim1 was co-expressed with Nif1 fused to glutathione-S-transferase (GST) or unfused GST. Schizosaccharomyces pombe cells were lysed under stringent conditions and the supernatants were incubated with glutathione–Sepharose to purify GST fusion proteins. The bound proteins were analyzed by immunoblotting. Nim1 was specifically detected in the GST–Nif1 sample, whereas no Nim1 signal was detected in the GST sample (Figure 3). These findings strongly suggest that Nif1 and Nim1 proteins interact in S.pombe cells. Figure 3.Nif1 associates with Nim1 in S.pombe cells. A S.pombe strain (PR109) was co-transformed with pREP1-Nim1 and pREP2-GST-Nif1 or pREP1-Nim1 and pREP2-GST. Transformants were grown in EMM2 − B1 for 24 h to induce protein expression. Cells were lysed as described in Materials and methods. (A) Nim1 associates with GST–Nif1 in S.pombe. Cell extracts were incubated with glutathione beads. After washing with stringent buffer, bound proteins were analyzed by immunoblotting using affinity-purified α-Nim1 antibody. Nim1 was detected in association with GST–Nif1 (lane 1), but not with GST (lane 2). Lysates from Δnim1 cells (PR387) were used as an additional control (lane 3). (B) Immunoblot using total cell extracts showed that Nim1 was expressed in cells expressing Nim1 and GST–Nif1 (lane 1) and Nim1 and GST (lane 2), but not in Δnim1 cells (lane 3). (C) The same membrane in (A) was reprobed with α-GST antibody. GST–Nif1 (lane 1) and GST (lane 2) were detected. A large amount of the ∼23 kDa GST was also detected in cells expressing GST–Nif1 (lane 1), perhaps indicating that GST–Nif1 is cleaved in vitro or is otherwise unstable. No proteins related to GST were detected in the Δnim1 control sample (lane 3). Download figure Download PowerPoint An important region of Nim1 is required for its association with Nif1 The ∼30 kDa C-terminal non-catalytic domain of Nim1 (amino acids 354–593) is not required for Nim1 function in vivo (Russell and Nurse, 1987a; Feilotter et al., 1991). In particular, expression of a truncated form of Nim1 containing amino acids 1–354 from a multicopy plasmid causes a wee phenotype and rescues cdc25-22 (Russell and Nurse, 1987a). Experiments were carried out to determine whether Nif1 interacts with the functional N-terminal domain of Nim1. In the two-hybrid assay, Nif1 interacted with the Nim1(1–354) construct, but failed to interact with the Nim1(1–291) construct (Figure 4A). These findings suggested that the 291–354 region of Nim1 is required for the interaction with Nif1. Further support for this conclusion was provided by the observation that Nim1(258–593) interacts with Nif1 in the yeast two-hybrid assay. Figure 4.The Nif1–Nim1 two-hybrid interaction requires an essential region in the non-catalytic domain of Nim1 protein kinase. (A) Amino acids 291–354 of Nim1 are required for the Nif1–Nim1 interaction and for Nim1 mitotic inducer activity. The organization of Nim1 protein kinase is illustrated in the left panel. The protein kinase homology domain is located in amino acids 1–257. Full-length Nim1(1–593), Nim1(1–354) and Nim1(258–593) interacted with Nif1 in the two-hybrid assay, whereas Nim1(1–291) failed to interact with Nif1. Likewise, full-length Nim1 and Nim1(1–354) rescued cdc25-22, whereas Nim1(1–291) failed to rescue cdc25-22. The results of the kinase assay experiments are also summarized here. (B) Expression of GST–Nim1(1–354) and GST–Nim1(1–291) in bacteria. The soluble lysates of bacteria expressing GST–Nim1(1–354) (lane 1) or GST–Nim1(1–291) (lane 2) were incubated with glutathione beads. Bound proteins were separated by SDS–PAGE and visualized by Coomassie blue staining. Note that a small amount of truncated or degraded GST–Nim1(1–354) that runs near the position of GST–Nim1(1–291) was detected in lane 1. (C) In vitro autophosphorylation of GST–Nim1. Purified GST–Nim1(1–354) and GST–Nim1(1–291) were incubated in kinase assay buffer and [γ-32P]ATP as described (Wu and Russell, 1993). Autoradiography revealed that only GST–Nim1(1–354) became phosphorylated in this assay. (D) Phosphorylation of Wee1 by GST–Nim1 in vitro. Wee1 was produced in Sf9 insect cells and purified on Ni2+-NTA beads as described (Wu and Russell, 1993). Wee1 was incubated alone or with the purified GST–Nim1 fusion proteins in kinase assay conditions. Phosphorylation of Wee1 by Nim1 causes Wee1 to migrate with reduced electrophoretic mobility (Wu and Russell, 1993). The electrophoretic mobility of Wee1 was analyzed by immunoblotting using affinity-purified α-Wee1 antibody 7373. Only GST–Nim1(1–354) caused Wee1 to migrate with reduced electrophoretic mobility (lane 3), indicating that GST–Nim1(1–354) phosphorylates Wee1, whereas GST–Nim1(1–291) is unable to phosphorylate Wee1. Download figure Download PowerPoint Experiments were carried out to determine whether the 291–354 region is important for Nim1 mitotic inducer activity. This analysis revealed that the 291–354 region is essential for the mitotic induction activity of Nim1 (Figure 4A). Specifically, the Nim1(1–354) construct rescued the cdc25-22 mutation and caused a wee phenotype when expressed in wild-type cells, consistent with previous studies (Russell and Nurse, 1987a), whereas the Nim1(1–291) construct failed to rescue cdc25-22 and had no effect on cell size in wild-type cells. These findings were followed by an analysis of whether the 291–354 region of Nim1 is important for Nim1 in vitro protein kinase activity. GST–Nim1(1–354) and GST–Nim1(1–291) fusion proteins were expressed in bacteria and purified (Figure 4B). GST–Nim1(1–354) retained a vigorous autophosphorylation activity, whereas GST–Nim1(1–291) was inactive in this assay (Figure 4C). The truncated forms of Nim1 were also tested for their ability to phosphorylate Wee1. Histidine-tagged Wee1 was expressed in insect cells and isolated by Ni2+-NTA affinity purification. The purified Wee1 was incubated with the GST–Nim1 fusion proteins in kinase assay conditions. The electrophoretic mobility of Wee1 was then analyzed by immunoblotting. Wee1 incubated with GST–Nim1(1–354) exhibited reduced electrophoretic mobility (Figure 4D), this was due to phosphorylation of Wee1 carried out by Nim1 (Wu and Russell, 1993). In contrast, the mobility of Wee1 was unaffected by incubation with GST–Nim1(1–291), indicating that GST–Nim1(1–291) was unable to phosphorylate Wee1 (Figure 4D). These results show that the Nif1–Nim1 interaction is dependent on a short region of Nim1 protein, amino acids 291–354, that is crucial for Nim1 function. Chromosomal disruption of nif1+ causes a reduction of cell size at division A nif1+ gene disruption was performed in a diploid strain (Figure 5A). Two constructs were made, differing in the orientation of the ura4+ marker used for the disruption. Sporulation of the disrupted diploid cells gave rise to Ura+ haploid cells, indicating that nif1+ is not essential. The disruption of nif1+ was confirmed by Southern hybridization (Figure 5B). The growth rate of Δnif1 cells was indistinguishable from that of wild type (data not shown). However, as shown in Figure 5C, the Δnif1 cells underwent division at a reduced cell length (13.7 ± 0.7 and 13.6 ± 0.9 μm) as compared with an isogenic wild-type strain (15.3 ± 0.8 μm). Figure 5.Δnif1 mutation causes a reduction of cell size at division. (A) Map of the nif1::ura4+ disruption constructs. (B) Southern hybridization analysis of genomic DNA from a nif1+/nif1+ diploid (lane 1), a nif1+/nif1::ura4+(1) diploid (lane 2), a nif1+ haploid (lane 3) and a nif1::ura4+(1) haploid (lane 4), confirming the nif1+ disruption. DNA was digested with EcoRV and probed with the 6 kb nif1+ HindIII fragment. (C) Cell division size data for nif1::ura4+ disruptant haploids and an isogenic nif1+ control. Download figure Download PowerPoint Nif1 overexpression phenotypes strongly suggest that Nif1 inhibits Nim1 The discovery that mutational inactivation of Nif1 causes cells to undergo mitosis at a reduced cell size suggested that Nif1 may function as a mitotic inhibitor, perhaps by inhibiting Nim1. This possibility was explored further by examining whether Nif1 overexpression affected mitotic timing. Expression of the nif1+ open reading frame was placed under the control of the thiamine-repressible nmt1 promoter in plasmid pREP1 (Maundrell, 1993). High expression of Nif1 in a wild-type background had no effect on growth rate or colony-forming ability (data not shown). However, high Nif1 expression caused significant cell elongation, with cells dividing at 19.4 ± 0.9 μm (Table I), a size that is very similar to Δnim1 cells (Russell and Nurse, 1987a). Cells transformed with control pREP1 vector divided at 14.5 ± 0.8 μm (Table I). These results strongly suggest that Nif1 functions as a mitotic inhibitor. Table 1. Cell size at division Strain Plasmid Length at division (μm) h− leu1-32 ura4-D18 pREP1 14.5 ± 0.8 h− leu1-32 ura4-D18 pREP1-Nif1 19.4 ± 0.9 h− leu1-32 ura4-D18 Δnim1 pREP2 19.3 ± 1.4 h− leu1-32 ura4-D18 Δnim1 pREP2-Nif1 20.4 ± 1.5 This finding was followed by an investigation of the effect of Nif1 overproduction in a cdc25-22 strain. Cells carrying the cdc25-22 mutation, which are moderately elongated at the permissive temperature of 25°C, are very sensitive to mutational inactivation of Nim1 (Feilotter et al., 1991). Thus, if Nif1 inhibits Nim1, then overproduction of Nif1 should exacerbate the cdc25-22 cell cycle delay phenotype. Clonal isolates of cdc25-22 cells transformed with pREP1-Nif1, which carries the nmt1:nif1+ construct, were grown in nmt1-repressing medium (EMM2 + B1) or in nmt1-inducing medium (EMM2 − B1) for 24 h at 20°C and then incubated for 4 h at 32°C. As predicted by the model in which Nif1 acts as a Nim1 inhibitor, overproduction of Nif1 caused the cdc25-22 cells to be highly elongated as compared with cdc25-22 cells transformed with pREP1 (Figure 6A). In fact, cdc25-22 cells transformed with pREP1-Nif1 were unable to form colonies when incubated on EMM2 − B1 plates at 25°C, whereas pREP1 transformants of cdc25-22 cells exhibited excellent growth in the same conditions (Figure 6B). Both pREP1 and pREP1-Nif1 transformants of cdc25-22 cells readily formed colonies when incubated in media that repressed the activity of the nmt1 promoter. These data confirmed that overexpression of Nif1 causes a delay of mitosis. Figure 6.Nif1 overproduction delays mitosis. (A) A cdc25-22 strain (PR196) transformed with pREP1-Nif1, which contains a nmt1:nif1+ construct, was grown on nmt1-repressing medium (EMM2 + B1) or nmt1-inducing medium (EMM2 − B1) for 24 h at 20°C and then incubated for 4 h at 32°C. Cells expressing Nif1 (right panel) became highly elongated as compared with those which repressed Nif1 expression (left panel). (B) Nif1 overexpression is lethal in cdc25-22 cells incubated at 25°C. Transformant cdc25-22 cells described in (A) were incubated on EMM2 + B1 and EMM2 − B1 media at 25°C. The cdc25-22 cells expressing Nif1 were unable to form colonies. (C) Overexpression of Nif1 does not rescue the lethal mitotic catastrophe caused by inactivation of Wee1 and Mik1. A wee1-50 Δmik1 strain (PR754) was transformed with pREP1 or pREP1-Nif1. At the permissive temperature of 25°C, cells are viable regardless of the expression of Nif1 (upper panel). Nif1 failed to rescue lethality when cells were incubated at the restrictive temperature of 35°C (lower panel). Download figure Download PowerPoint If Nif1 delays mitosis by a mechanism that exclusively involves Nim1, then Nif1 overexpression should cause no phenotype in a Δnim1 background. Consistent with this prediction, we found that Nif1 overexpression had no effect on the ability of Δnim1 cells to form colonies. Cell size measurements showed that there was no significant difference in cell division length between Δnim1 cells harboring the pREP2 control vector (19.3 ± 1.4 μm) and Δnim1 cells carrying pREP2-Nif1 (20.4 ± 1.5 μm) when grown in EMM2 − B1 (Table I). These results suggest that Nif1 and Nim1 operate through the same pathway. Overproduction and inactivation of Nim1 have no effect in a Δwee1 background, a finding consistent with the conclusion that Wee1 is the exclusive target of Nim1 regulation (Russell and Nurse, 1987a). If Nif1 specifically inhibits Nim1, then overproduction of Nif1 should have no effect in Δwee1 cells. This prediction was confirmed: Δwee1 cells transformed with pREP1-Nif1 or pREP1 plasmids exhibited identical wee phenotypes when grown in EMM2 − B1 (data not shown). Mik1 and Wee1 are redundant protein kinases that phosphorylate Cdc2 on tyrosine-15. Simultaneous inactivation of Wee1 and Mik1 causes a lethal mitotic catastrophe phenotype (Lundgren et al., 1991). If Nif1 specifically inhibits Nim1, then overexpression of Nif1 should not rescue the lethality of wee1-50 Δmik1 cells at 35°C. Indeed, Nif1 overexpression failed to rescue wee1-50 Δmik1 mitotic catastrophe (Figure 6C), providing further support for the conclusion that Nif1 overexpression delays mitosis by inhibiting Nim1. Inhibition of Nim1 by Nif1 in an insect cell expression system These studies suggested that Nif1 may have a direct role in regulating the ability of Nim1 to phosphorylate and inhibit Wee1. As a first step in exploring this mechanism of regulation, we asked whether Nif1 was capable of inhibiting Nim1 in an insect cell expression system. Previous studies showed that Nim1 is able to phosphorylate and inactivate Wee1 in insect cells (Coleman et al., 1993; Parker et al., 1993; Wu and Russell, 1993). The phosphorylation of Wee1 is accompanied by a reduction of the mobility of Wee1 in SDS–PAGE. A recombinant baculovirus was constructed to direct the expression of HA epitope-tagged Nif1 protein. Infection of Sf9 cells with this virus led to the production of Nif1 as a protein that migrated with the apparent mol. wt of ∼97 kDa (Figure 7A). Co-infection of Sf9 cells with viruses encoding Wee1 and Nim1 caused the Wee1 to migrate with reduced mobility (Figure 7B, lanes 1 and 2), confirming earlier findings. This effect was largely abolished by the additional infection of cells with the virus encoding Nif1 (Figure 7B, lanes 3–5). The ability of Nif1 to block the Nim1-mediated phosphorylation of Wee1 was dependent on the dose of Nif1 virus. These findings strongly suggest that Nif1 is able to inhibit Nim1 by a direct mechanism, a suggestion consistent with the detection of an in vivo physical interaction involving Nif1 and Nim1. Figure 7.Nif1 inhibits the ability of Nim1 to phosphorylate Wee1 in Sf9 insect cells. (A) Expression of Nif1 in Sf9 cells. Sf9 cells were infected with a recombinant baculovirus expressing Nif1-6His2Ha as described in Materials and methods. Cells were harvested 55 h post-infection and lysed in SDS sample loading buffer. Total protein extract from control cells (lane 1) or cells expressing Nif1 (lane 2) was separated by SDS–PAGE and analyzed by immunoblotting using anti-HA monoclonal antibody 12CA5. Nif1-6His2Ha was detected as a series of bands migrating at the ∼97 kDa region of the gel. (B) Nif1 inhibits Nim1′s ability to phosphorylate Wee1 in Sf9 cells. Sf9 cells were infected with recombinant baculoviruses expressing Wee1 alone (lane 1), Wee1 and Nim1 (lane 2), or Wee1, Nim1 and increasing amounts of Nif1 as indicated (lanes 3–5). The multiplicity of infection (MOI) was 10 for Wee1 and 20 for Nim1 in all infections. For Nif1, the MOI was 20 (1×, lane 3), 40 (2×, lane 4) and 80 (4×, lane 5). Cells were harvested at 55 h post-infection. Samples were treated as described above. Wee1 was detected by immunoblotting using affinity-purified anti-Wee1 antibody 7451. Infection of cells with viruses expressing Wee1 and Nim1 caused Wee1 phosphorylation, as indicated by the slower mobility of Wee1 in SDS–PAGE (lane 2). Infection of cells with increasing amounts of recombinant viruses expressing Nif1 inhibited the ability of Nim1 to phosphorylate Wee1, as indicated by the fast mobility of Wee1 in SDS–PAGE (lanes 3–5). Download figure Download PowerPoint Discussion We have used the yeast two-hybrid system to identify a protein, Nif1, that specifically interacts with Nim1 protein kinase. A series of findings strongly suggest that the Nif1–Nim1 interaction is specific and has functional significance in regulating Nim1 activity in vivo. First, in the two-hybrid assay, Nif1 cDNA clones were repeatedly isolated, accounting for ∼95% of all positive clones. Second, GST–Nif1 fusion protein expressed in S.pombe was found to co-precipitate with Nim1. Third, the Nif1–Nim1 interaction is dependent on a short region of Nim1 protein adjacent to the kinase homology domain that is essential for in vitro and in vivo activities of Nim1. Fourth, gene disruption of nif1+ causes advancement of mitosis at a small cell size, while Nif1 overproduction causes a delay of mitosis. Fifth, by a variety of genetic tests, Nif1 overproduction causes a phenotype that is phenotypically equivalent to the Δnim1 mutation. Specifically, Nif1 overproduction is synthetically lethal with cdc25-22, has no genetic interaction with Δnim1 or Δwee1 mutations, and fails to rescue wee1-50 Δmik1 mitotic catastrophe. Sixth, in an insect cell expression system, Nif1 reduces the phosphorylation of Wee1 by Nim1. These findings strongly support a model in which Nif1 functions as an inhibitor of mitosis via a direct interaction with Nim1. It remains to be determined exactly how Nif1 regulates Nim1 activity in vivo. Perhaps the simplest possibility is that Nif1 directly inhibits Nim1 kinase by interacting with a domain requ" @default.
• W2019982171 created "2016-06-24" @default.
• W2019982171 creator A5012431295 @default.
• W2019982171 creator A5079948759 @default.
• W2019982171 date "1997-03-15" @default.
• W2019982171 modified "2023-09-23" @default.
• W2019982171 title "Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe" @default.
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