Initiation of cytokinesis is controlled through multiple modes of regulation of the Sid2p-Mob1p kinase complex

The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mob1p is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell.     

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.     

The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1

Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin-dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review, see [1, 2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated partly by their localization; both proteins are thought to be sequestered in the nucleolus in interphase. Cdc14 and Clp1 are released from the nucleolus in mitosis, and in late mitosis conserved signaling pathways termed the mitotic exit network (MEN) and the septation initiation network (SIN) keeps Cdc14 and Clp1, respectively, out of the nucleolus through an unknown mechanism [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, maintains Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the Clp1-Rad24 interaction and causes Clp1 to return prematurely to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring but does not affect other Clp1 functions. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.     

The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network

The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p. The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs. However, a direct interaction between Sid4p and other members of the SIN has not been detected. To understand how Sid4p is linked to other SIN components, we have begun to characterize an S. pombe homolog of the Saccharomyces cerevisiae SPB protein Nud1p. We have determined that this S. pombe Nud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element. We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p. The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction. The N-terminus of Cdc11p is required for Spg1p binding to SPBs. Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway.     

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.     

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.     

Genetic Evidence for Pak1 Autoinhibition and Its Release by Cdc42

Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.     

Schizosaccharomyces pombe Pak-related protein,Pak1p/Orb2p,phosphorylates myosin regulatory light chain to inhibit cytokinesis

p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.     

Dual function of the germinal centre kinase Don3 during mitosis and cytokinesis in Ustilago maydis

Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.     

The meiosis-specific Sid2p-related protein Slk1p regulates forespore membrane assembly in fission yeast

Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In fission yeast, a signaling module termed the septation initiation network (SIN) plays an essential role in the assembly of new membranes and cell wall during cytokinesis. In this study, we have characterized Slk1p, a protein-kinase related to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN-dependent manner. Slk1p also localizes to the forespore membrane during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading frequently to the formation of asci with smaller and/or fewer spores. The ability of slk1Δ cells to sporulate, albeit inefficiently, is fully abolished upon compromise of function of Sid2p, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Interestingly, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1Δ. Thus, it is likely that Slk1p and Sid2p play a role in forespore membrane assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby provide a novel link between the SIN and vesicle trafficking during cytokinesis.     

Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton

The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKalpha-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an alpha-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKalpha-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1delta (PP1delta), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1delta, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the alpha-helical leucine repeats. The former was able to interact with PP1delta only in the phosphorylated state and result in inactivation of PP1delta activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.     

The fission yeast septation initiation network (SIN) kinase, Sid2, is required for SIN asymmetry and regulates the SIN scaffold, Cdc11

The Schizosaccharomyces pombe septation initiation network (SIN) is an Spg1-GTPase-mediated protein kinase cascade that triggers actomyosin ring constriction, septation, and cell division. The SIN is assembled at the spindle pole body (SPB) on the scaffold proteins Cdc11 and Sid4, with Cdc11 binding directly to SIN signaling components. Proficient SIN activity requires the asymmetric distribution of its signaling components to one of the two SPBs during anaphase, and Cdc11 hyperphosphorylation correlates with proficient SIN activity. In this paper, we show that the last protein kinase in the signaling cascade, Sid2, feeds back to phosphorylate Cdc11 during mitosis. The characterization of Cdc11 phosphomutants provides evidence that Sid2-mediated Cdc11 phosphorylation promotes the association of the SIN kinase, Cdc7, with the SPB and maximum SIN signaling during anaphase. We also show that Sid2 is crucial for the establishment of SIN asymmetry, indicating a positive-feedback loop is an important element of the SIN.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

Phosphorylation and spindle pole body localization of the Cdc15p mitotic regulatory protein kinase in budding yeast

Cdc15p is an essential protein kinase and functions with a group of late mitotic proteins that includes Lte1p, Tem1p, Cdc14p and Dbf2p/Dbf20p to inactivate Cdc28p-Clb2p at the end of mitosis in budding yeast [1] [2]. Cdc14p is activated and released from the nucleolus at late anaphase/telophase to dephosphorylate important regulators of Cdc28p-Clb2p such as Hct1p/Cdh1p, Sic1p and Swi5p in a CDC15-dependent manner [3] [4] [5] [6] [7]. How Cdc15p itself is regulated is not known. Here, we report that both the phosphorylation and localization of Cdc15p are cell cycle regulated. The extent of phosphorylation of Cdc15p gradually increases during cell-cycle progression until some point during late anaphase/telophase when it is rapidly dephosphorylated. We provide evidence suggesting that Cdc14p is the phosphatase responsible for the dephosphorylation of Cdc15p. Using a Cdc15p fusion protein coupled at its carboxyl terminus to green fluorescent protein (GFP), we found that Cdc15p, like its homologue Cdc7p [8] in fission yeast, localizes to the spindle pole bodies (SPBs) during mitosis. At the end of telophase, a portion of Cdc15p is located at the mother-bud neck, suggesting a possible role for Cdc15p in cytokinesis.     

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.     

The S. pombe cytokinesis NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment through Pom1/Wee1

Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved. At present, it is assumed that the functions of the core SIN-MEN components are restricted to their characterized roles at the end of mitosis. We now show that the NDR (nuclear Dbf2-related) kinase component of the fission yeast SIN, Sid2-Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2-Mob1 promotes mitotic commitment by directly activating the NIMA (Never In Mitosis)-related kinase Fin1. Fin1's activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards?Wee1.     

Etd1p is a novel protein that links the SIN cascade with cytokinesis

In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast cells, ring constriction is triggered by the septum initiation network (SIN), an SPB-associated GTPase-regulated kinase cascade that coordinates exit from mitosis with cytokinesis. We have identified a novel protein, Etd1p, required to trigger actomyosin ring constriction in fission yeasts. This protein is localised at the cell tips during interphase. In mitosis, it relocates to the medial cortex region and, coincident with cytokinesis, it assembles into the actomyosin ring by association to Cdc15p. Relocation of Etd1p from the plasma membrane to the medial ring is triggered by SIN signalling and, reciprocally, relocation of the Sid2p-Mob1p kinase complex from the SPB to the division site, a late step in the execution of the SIN, requires Etd1p. These results suggest that Etd1p coordinates the mitotic activation of SIN with the initiation of actomyosin ring constriction. Etd1p peaks during cytokinesis and is degraded by the ubiquitin-dependent 26S-proteasome pathway at the end of septation, providing a mechanism to couple inactivation of SIN to completion of cytokinesis.     

Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.     

Fission yeast Clp1p phosphatase affects G2/M transition and mitotic exit through Cdc25p inactivation

The Cdc14 family of phosphatases specifically reverses proline-directed phosphorylation events. In Saccharomyces cerevisiae, Cdc14p promotes Cdk1p inactivation at mitotic exit by reversing Cdk1p-dependent phosphorylations. Cdk1p is a proline-directed kinase whose activity is required in all eukaryotes for the transit into mitosis. At mitotic commitment, Cdk1p participates in its own regulation by activating the mitotic inducing phosphatase, Cdc25p, and inhibiting the opposing kinase, Wee1p. We have investigated the ability of Schizosaccharomyces pombe Clp1p, a Cdc14p homolog, to disrupt this auto-amplification loop. We show here that Clp1p is required to dephosphorylate, destabilize, and inactivate Cdc25p at the end of mitosis. Clp1p promotes recognition of Cdc25p by the anaphase-promoting complex/cyclosome, an E3 ubiquitin ligase. Failure to inactivate and destabilize Cdc25p in late mitosis delays progression through anaphase, interferes with septation initiation network signaling, and additionally advances the commitment to mitotic entry in the next cycle. This may be a widely conserved mechanism whereby Cdc14 proteins contribute to Cdk1p inactivation.