Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast

Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.     

Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs)

Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK) [1]. Metazoan CAK is itself a Cdk, Cdk7, whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks [1]. The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme [2] [3]. Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S. pombe [3]. We therefore tested the ability of Csk1, a putative regulator of Mcs6 [4], to activate Cdk-cyclin complexes in vitro. Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6. Surprisingly, Csk1 also activated Cdc2 in complexes with either Cdc13 or Cig2 cyclins. When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation. Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest. Mcs6, which is essential, may therefore have required functions other than Cdk activation.     

Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.     

Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.     

The highly conserved protein methyltransferase, Skb1, is a mediator of hyperosmotic stress response in the fission yeast Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for viability or mating of S. pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions. Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress. We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin. We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity. Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.     

A novel member of the Swi6p family of fission yeast chromo domain-containing proteins associates with the centromere in vivo and affects chromosome segregation

We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates. Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins. Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization. The S. pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome. Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p. Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.     

Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast

A great deal is now known about how cells regulate entry into mitosis, but only recently have the mechanisms controlling exit from mitosis and cytokinesis begun to be revealed. In the budding yeast Saccharomyces cerevisiae, Mob1p interacts with the Dbf2p kinase and cells containing mutations in these genes arrest in late anaphase [1] [2]. Proteins related to Mob1p are present in both plants and animals, but information about Mob1p function has been obtained only from budding yeast. Here, we describe the identification and characterization of Mob1p from Schizosaccharomyces pombe. Mob1p associates with the Sid2p kinase and like Sid2p, Mob1p is required for the initiation of cytokinesis, but not for mitotic exit. Mob1p localizes to the spindle pole body (SPB) and to the cell-division site during cell division, suggesting that it might be involved in transducing the signal to initiate cell division from the SPB to the division site. Mob1p is required for Sid2p localization, and Mob1p localization requires the function of the cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 genes, suggesting that together with Sid2p, Mob1p functions at the end of the signaling cascade required to regulate the onset of cytokinesis at the end of mitosis.     

The fission yeast kinetochore component Spc7 associates with the EB1 family member Mal3 and is required for kinetochore-spindle association

A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins, which have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. Here, we show that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore-microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains.     

The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability

Spindle formation in fission yeast occurs by the interdigitation of two microtubule arrays extending from duplicated spindle pole bodies which span the nuclear membrane. By screening a bank of temperature-sensitive mutants by anti-tubulin immunofluorescence microscopy, we previously identified the sad1.1 mutation (Hagan, I., and M. Yanagida. 1990. Nature (Lond.). 347:563-566). Here we describe the isolation and characterization of the sad1+ gene. We show that the sad1.1 mutation affected both spindle formation and function. The sad1+ gene is a novel essential gene that encodes a protein with a predicted molecular mass of 58 kD. Deletion of the gene was lethal resulting in identical phenotypes to the sad1.1 mutation. Sequence analysis predicted a potential membrane-spanning domain and an acidic amino terminus. Sad1 protein migrated as two bands of 82 and 84 kD on SDS-PAGE, considerably slower than its predicted mobility, and was exclusively associated with the spindle pole body (SPB) throughout the mitotic and meiotic cycles. Microtubule integrity was not required for Sad1 association with the SPB. Upon the differentiation of the SPB in metaphase of meiosis II, Sad1-staining patterns similarly changed from a dot to a crescent supporting an integral role in SPB function. Moderate overexpression of Sad1 led to association with the nuclear periphery. As Sad1 was not detected in the cytoplasmic microtubule-organizing centers activated at the end of anaphase or kinetochores, we suggest that Sad1 is not a general component of microtubule-interacting structures per se, but is an essential mitotic component that associates with the SPB but is not required for microtubule nucleation. Sad1 may play a role in SPB structure, such as maintaining a functional interface with the nuclear membrane or in providing an anchor for the attachment of microtubule motor proteins.     

The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.     

p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast.

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.     

Microtubule affinity-regulating kinase 1 (MARK1) is activated by electroconvulsive shock in the rat hippocampus

Electroconvulsive shock (ECS) induces phosphorylation and dephosphorylation of many signaling molecules in the rat brain. While studying phosphorylated proteins in the rat brain after ECS, we observed a 100-kDa protein that cross-reacted with anti-phospho-p70 S6 kinase antibody, which was subsequently purified and identified as microtubule affinity-regulating kinase 1 (MARK1). Purified MARK1 was phosphorylated at the Ser and Thr residues. MARK1 activation and subsequent Tau phosphorylation in the hippocampus after ECS was confirmed by an in-gel kinase assay using tau protein as a substrate. MARK1 was maximally activated between 2 and 5 min after ECS, and Tau phosphorylation at Ser262 was also increased at 2 min and lasted to 1 h after ECS. Taken together, we concluded that ECS activated MARK1 and subsequently phosphorylated Tau at Ser262. Both MARK1 activity and Tau phosphorylation were increased in the rat hippocampus after chronic ECS where axonal remodeling was apparent. In order to investigate the physiologic stimuli which are involved in the activation of MARK1, SH-SY 5Y cells were treated with brain-derived neurotrophic factor or 60 mm KCl. Both stimuli were capable of inducing MARK activation.     

Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1 (LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis

Triacylglycerols (TAGs) are major storage materials that accumulate in developing seeds and serve as carbon and energy reserves for germination and growth of the seedling. One of the critical reactions in TAG biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACSs). Of the nine LACSs identified in Arabidopsis, only LACS9 is known to reside in the plastid, the site of de novo fatty acid synthesis, and is considered the major LACS isoform involved in plastidial fatty acid export for TAG formation. Because the lacs9 null mutant did not show any detectable phenotype, it was hypothesized that at least one additional LACS enzyme must be active in the plastid. Expression analyses to identify potential plastid-localized LACSs involved in TAG biosynthesis revealed that, in addition to LACS9, isoforms LACS1, LACS2, LACS4 and LACS8 are transcribed in the seed. LACS8 showed the highest expression level in the embryo and a high sequence similarity with LACS9, and was therefore characterized further and shown to be associated with the ER, not the plastid. Furthermore, disruption of LACS8 in the lacs8 mutant and lacs8 lacs9 double mutant, and over-expression of LACS8, did not affect the seed fatty acid content. In contrast, 11 and 12% decreases in fatty acid content were detected in lacs1 lacs9 and lacs1 lacs8 lacs9 seeds, respectively, indicating that LACS1 and LACS9 have overlapping functions in TAG biosynthesis. This result is surprising because, unlike LACS9, LACS1 is localized in the ER and has been shown to be involved in cuticular lipid synthesis.     

The p21-activated kinase, Shk1, is required for proper regulation of microtubule dynamics in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is required for the proper establishment of cell polarity in the fission yeast, Schizosaccharomyces pombe. We showed recently that loss of the essential Shk1 inhibitor, Skb15, causes significant spindle defects in fission yeast, thus implicating Shk1 as a potential regulator of microtubule dynamics. Here, we show that cells deficient in Shk1 function have malformed interphase microtubules and mitotic microtubule spindles, are hypersensitive to the microtubule-destabilizing drug thiabendazole (TBZ) and cold sensitive for growth. TBZ treatment causes a downregulation of Shk1 kinase activity, which increases rapidly after release of cells from the drug, thus providing a correlation between Shk1 kinase function and active microtubule polymerization. Consistent with a role for Shk1 as a regulator of microtubule dynamics, green fluorescent protein (GFP)-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles, as well as to cell ends and septum-forming regions of fission yeast cells. We show that loss of Tea1, a cell end- and microtubule-localized protein previously implicated as a regulator of microtubule dynamics in fission yeast, exacerbates the growth and microtubule defects resulting from partial loss of Shk1 and that Shk1 localizes to illicit growth tips produced by tea1 mutant cells. Our results demonstrate that Shk1 is required for the proper regulation of microtubule dynamics in fission yeast and implicate Tea1 as a potential Shk1 regulator.     

Dim1p is required for efficient splicing and export of mRNA encoding lid1p, a component of the fission yeast anaphase-promoting complex

Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression.