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J. Biol. Chem., Vol. 281, Issue 15, 9935-9941, April 14, 2006

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Identification of Functional Domains within the Septation Initiation Network Kinase, Cdc7^*

Sapna Mehta and Kathleen L. Gould¹

From the Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received for publication, April 11, 2005 , and in revised form, January 6, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The septation initiation network (SIN) serves to coordinate cytokinesis with mitotic exit in the fission yeast Schizosaccharomyces pombe. SIN components Spg1 and Cdc7 together play a central role in regulating the onset of septation and cytokinesis. Spg1, a Ras-like GTPase, localizes to the spindle pole bodies (SPBs) throughout the cell cycle. It is converted to its GTP-bound (active) state during mitosis, only to become inactivated at one SPB during anaphase and at both SPBs as cells exit mitosis. Cdc7 functions as an effector kinase for Spg1, binding to Spg1 in its GTP-bound state, and therefore is present at both SPBs during mitosis and asymmetrically at only one during anaphase. Interestingly, the kinase activity of Cdc7 does not vary across the cell cycle, suggesting the possibility that Cdc7 kinase activity is independent of Spg1 binding. Consistent with this, we found that Cdc7 associates with Spg1 only during mitosis. To learn more about the essential role of Cdc7 kinase in the SIN and its regulation, we undertook a structure/function analysis and identified independent functional domains within Cdc7. We found that a region adjacent to the kinase domain is responsible for Spg1 association and identified an overlapping but distinct SPB localization domain. In addition Cdc7 associates with itself and exists as a dimer in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell division is a fundamental biological event that underlies the growth and development of all organisms. Critical events in the process of cell division include DNA replication, mitosis (where genetic material is faithfully segregated), and cytokinesis (which results in the actual separation of daughter cells). In a normal cell cycle, cytokinesis takes place only after chromosome segregation has been completed, thus safeguarding genomic integrity. A failure to temporally and spatially regulate this event is catastrophic for the cell, resulting in aneuploidy, cell death, or cancer (1).

Conserved GTPase-driven signaling pathways have emerged that serve to coordinate mitotic exit with cytokinesis (2, 3). In the fission yeast Schizosaccharomyces pombe, this network, referred to as the septation initiation network (SIN),² controls actomyosin ring constriction and septum deposition after mitosis. Loss of function mutations in sin genes result in elongated multinucleate cells due to continuation of nuclear division and cell growth in the absence of deposition of septal material (2, 3). In contrast, hyperactivation of the SIN (caused by mutations in inhibitors of SIN activity) leads to formation of multiple septa without cell separation. SIN components include a GTPase Spg1 (4, 5), a two-component GTPase-activating protein for Spg1 composed of Byr4 and Cdc16 (6), and four protein kinases: Plo1 (7), Cdc7 (8), Sid1 and its regulatory subunit Cdc14 (9), and Sid2 and its regulatory subunit Mob1 (10–12). All SIN components reside on the SPB either constitutively or transiently at some stage during the cell cycle. Two proteins, Sid4 and Cdc11, function together as a platform to assemble the SIN and its regulators on the SPB (13–16). FRAP (fluorescence recovery after photobleaching) analysis of SIN components demonstrate that Sid4 and Cdc11 are stable proteins, as opposed to the regulatory molecules Spg1 and Sid2, which are extremely dynamic (16). These findings further support the role for Sid4-Cdc11 in serving as a scaffold to which other SIN components associate dynamically.

Cdc7 is a member of the STE11/MEKK family of kinases (17–20). Most kinases belonging to the MEKK family share a similar structure with the kinase domain at the C terminus and a regulatory domain at the N terminus (21). They also have been shown to bind to small G-proteins (21). Cdc7 differs from the typical member of the family in that the kinase domain of Cdc7 resides at its N terminus. However, like other family members, it binds to and is an effector for a GTPase, in this case Spg1 (5, 22). Activation of the SIN pathway is triggered by activation of Spg1, and increased production of Spg1 leads to the formation of septa at any point in the cell cycle (5). Interestingly, Spg1 activation at the SPB is asymmetric. It exists in the GDP-bound state during interphase at the single SPB, and during metaphase it is GTP-bound at both SPBs only to be "inactivated" or GDP-bound at one SPB during anaphase B (22). Cdc7 is only recruited to the pole that is occupied by GTP-bound Spg1; hence it also is asymmetrically localized during anaphase. The asymmetric localization of SIN components appears to be important for proper SIN regulation, because Cdc7 is associated with both SPBs during anaphase in cells with deregulated septum formation (7, 23).

Cdc7 kinase plays a crucial role in propagation of the SIN, as it serves to relay the activation signal from Spg1-GTP to its downstream effectors. To understand how Cdc7 is regulated we have carried out a detailed structure/function analysis of Cdc7. We have mapped the regions within Cdc7 required for SPB localization as well as binding to Spg1. Additionally we found that Cdc7 self-associates via an intermolecular interaction.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains, Media, and Genetic Methods—S. pombe strains used in this study (Table 1) were grown in yeast extract or minimal medium with appropriate supplements (24). DNA transformations were done by electroporation (25) or lithium acetate transformation. Regulated expression of genes from various strengths of the nmt promoter in the pREP series of vectors was achieved by growth in the presence of thiamine (promoter repressed) and then removing the thiamine by washing into medium lacking thiamine (promoter nonrepressed) (26). Saccharomyces cerevisiae strain PJ69-4A was used for two-hybrid analysis (27) and transformed by a lithium acetate method (28). leu⁺trp⁺ transformants were scored for positive interactions by plating on synthetic dextrose medium lacking adenine and histidine. -Galactosidase reporter enzyme activity in the two-hybrid strains was measured using the Galacto-Star chemiluminescent reporter assay system according to the manufacturer's instructions (Tropix Inc., Bedford, MA) with the exception that cells were lysed by glass bead disruption. Each sample was measured in triplicate.

In Vitro and Lysate Binding Assays—His₆-Cdc7-(250–535) was produced from bacterial cell lysates and purified over nickel-nitrilotriacetic acid-agarose columns (Qiagen) according to manufacturers instructions. GST, GST-Spg1Q69L, and GST-Spg1T42A were purified as described previously (16). Purified proteins were dialyzed against native binding buffer (NBB: 20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 0.1% Nonidet P-40). 150 µM GST-Spg1Q69L or GST-Spg1T42A was mixed with 200 µM His₆-Cdc7-(250–535), and the volume was brought up to 1.0 ml using NBB. The proteins were allowed to bind in solution at 4 °C for 1 h. GST-Spg1 proteins were pulled down by incubation with glutathione-agarose beads for 40 min, washed several times with NBB, and washed once with NBB containing 250 mM salt. GST-Spg1 and associated proteins were run on a 10% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. For lysate binding experiments GST and GST-Spg1 proteins were left bound to glutathione-agarose beads. S. pombe lysates were prepared as described below, except the Nonidet P-40 buffer was supplemented with 1 mM MgCl₂ and 50 µM GTP. Bound proteins were eluted from beads by incubating in GST elution buffer (50 mM Tris, pH 8.0, 200 mM NaCl, 0.1% Nonidet P-40, 1 mM MgCl₂, 50 µM GTP and 100 mM Glutathione) for 1 h at 4 °C. Eluate was then either immunoblotted or subjected to immunoprecipitation as described below.

Immunoprecipitations, Sucrose Gradients, and Immunoblots—Whole cell S. pombe lysates were prepared in Nonidet P-40 buffer (29), and immunoprecipitations were carried out using either anti-HA (12CA5), anti-Myc (9E10), or polyclonal anti-GFP antibodies as described (30). Immunoprecipitates were run on a 4–12% NuPAGE in MOPS buffer. For sucrose gradient analysis, a 200-µl volume of protein lysate was layered onto a 5-ml 5–20% sucrose gradient prepared in Nonidet P-40 buffer. Gradients were ultracentrifuged at 40,000 rpm for 19 h in a Beckman SW50.1 rotor (Berkeley, CA). Sedimentation markers were fractionated on gradients prepared and spun in parallel. Fractions were collected from the bottom of the gradient, resolved on a 10% SDS-PAGE, and transferred by electroblotting to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). Immunoblotting was done with anti-HA (12CA5; 2 mg/ml), anti-Myc (9E10; 2 mg/ml), or anti-GFP (1:1000 dilution of serum) antibodies. Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (0.4 mg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:50,000, visualized by ECL using SuperSignal (Pierce). For Figs. 1 and 4C immunoblotting was performed using the Odyssey infrared imaging system and protocol (LI-COR). Goat anti-mouse Alexa® 680 (LI-COR) was used at a 1:5000 dilution. Images were exported from Odyssey software in TIFF format.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Cdc7 associates with Spg1 only during mitosis. A, the cdc7-myc13 spg1-ha3 strain (KGY5401) was grown to mid-log phase and synchronized in G2 by centrifugal elutriation. Samples were taken at regular time intervals and processed for staining with 4,6-diamidino-2-phenylindole (Binucleates) and septation indexing as a measure of cell cycle progression. B, protein lysates were prepared from each sample, and anti-HA antibodies were used to immunoprecipitate Spg1 from lysates that were normalized to contain equal amount of protein as determined by immunoblotting for Cdc2 with anti-PSTAIRE antibodies (top panel). The amount of Spg1-HA3 immunoprecipitated was detected by immunoblotting with 12CA5 antibodies (second panel: *, indicates a background band that is present in 246 control; arrow, points to Spg1-HA3 band). Cdc7-Myc13 (indicated by the arrow) in Spg1-HA3 immunoprecipitates was detected by immunoblotting with 9E10 antibodies (third panel: *, indicates background band that is present in Spg1-HA3 KGY2167 and wild-type KGY246 controls). The amount of Cdc7-Myc13 in the lysates was detected by immunoblotting with 9E10 antibodies (bottom panel).

View larger version (59K): [in this window] [in a new window]   FIGURE 2. Amino acids 250–535 of Cdc7 are sufficient to bind Spg1. A, spg1 bait plasmid was co-transformed with various cdc7 or control prey plasmids. Transformants were grown on medium lacking histidine and adenine to score for positive interactions. cdc7 constructs; their ability to grow with the spg1 construct is indicated. B, approximately equal amounts of fusion proteins GST-Spg1Q69L and GST-Spg1T42A were incubated with recombinant His6-Cdc7-(250–535) or control protein His6-Prp19-(1–73). His6-Cdc7-(250–535) associated with GST-Spg1Q69L, but not GST or GST-Spg1T42A, when pulled down with glutathione-agarose beads, as visualized by Coomassie staining; His6-Prp19-(1–73) did not associate with either. C, the spg1-myc13 strain (KGY4071) was transformed with pREP1 vectors producing GFP-Cdc7-(250–535). Transformants were grown in the presence (lane 1) and absence of thiamine (lane 2), and protein lysates were subjected to immunoprecipitation (IP) with polyclonal anti-GFP serum. Immunoprecipitates were run on a 4–12% bis-Tris gel and immunoblotted with polyclonal anti-GFP antibody (left panel) or 9E10 monoclonal anti-Myc antibody (right panel). wt, control GFP immunoprecipitates from untagged wild type (KGY246) cells. D, wild-type cells (KGY246) expressing GFP-Cdc7-(250–535) under the control of the nmt1 promoter (left panel) or the nmt81 promoter (right panel) were grown to mid-log phase in the absence of thiamine at 32 °C for 18 h. Cells were fixed in methanol and stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei (left panel). As Cdc7GFP loses its fluorescence upon fixation, GFP-Cdc7-(250–535) images of live cells were captured (right panel). E, Spg1-GFP (KGY2678), Sid4-GFP (KGY2628), and Sid2-GFP (KGY2945) cells expressing either empty vector (pREP1NTAP) or NTAP-Cdc7-(250–535) under the control of the nmt1 promoter in pREP1 were grown to mid-log phase in the absence of thiamine at 32 °C for 18 h. Images of live cells were captured to visualize GFP-tagged proteins.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cdc7 Associates with Spg1 Only during Mitosis—Cdc7 has been shown to associate with GTP-bound but not GDP-bound Spg1 and is presumed to function as its effector. Most G-protein effector kinases are activated upon binding to their cognate G-proteins (31, 32). However, the kinase activity of Cdc7 has been shown to remain constant throughout the cell cycle despite the observation that Spg1 is in its GTP-bound form at the SPB only during mitosis (22). Given this anomaly, we decided to address whether Cdc7 is always associated with Spg1 even when not at the SPB. We generated a synchronous population of cells by centrifugal elutriation that produced endogenously tagged Cdc7-Myc₁₃ and Spg1-HA₃. Samples were collected at regular intervals and examined for septation and nuclear division as indicators of the cell-cycle stage (Fig. 1A). Protein lysates were prepared, and the presence of Cdc7-Myc₁₃ in Spg1 immunoprecipitates was assessed by immunoblotting (Fig. 1B). A control spg1-HA₃ strain lacking endogenously tagged Cdc7 and a wild type untagged strain were used as controls. A small percentage of Cdc7-Myc₁₃ was present in Spg1-HA₃ immunoprecipitates prepared from mitotic (at minutes 30, 45, and 60) but not from interphase cells. This is also the stage at which Spg1 is in its GTP-bound form (22). In uninucleate cells that exited from mitosis (at minutes 90, 105 and 120), no Cdc7-Myc₁₃ was detectable. Lack of Spg1-HA₃-Cdc7-Myc₁₃ association was not due to lower amounts of Spg1-HA₃ in immunoprecipitates, higher concentrations of Cdc7-Myc₁₃ in mitotic samples (Fig. 1B, bottom panel), or higher overall protein levels (Fig. 1B, top panel). Taken at face value, these results indicate that unlike in other G-protein-associated kinases, the binding to Spg1 does not influence Cdc7 kinase activity.

The N Terminus of Cdc7 Contains an Spg1 Binding Site—Given that the association of Cdc7 with Spg1 at the SPB is a likely SIN-activating step, we addressed what regions of Cdc7 are responsible for binding to Spg1. We first analyzed a series of cdc7 fragments in a directed two-hybrid assay. Residues 250–535 were found to contain a strong interaction domain (Fig. 2A). Because Cdc7 only associates with Spg1 in its GTP-bound form (22), we tested whether His₆-Cdc7-(250–535) bound Spg1 in a nucleotide-dependent manner. For this we examined the ability of bacterially produced His₆-Cdc7-(250–535) to interact with recombinant Spg1 mutants in vitro. His₆-Cdc7-(250–535) was able to bind GST-Spg1Q69L, a constitutively active mutant (5), but not GST-Spg1T42A, an effector domain mutant (5) (Fig. 2B). We also tested whether Cdc7-(250–535) and Spg1-Myc₁₃ could co-immunoprecipitate from S. pombe protein lysates. To this end, GFP-tagged Cdc7-(250–535) was overproduced in a spg1-myc₁₃ strain. Anti-GFP immunoprecipitates contained Spg1-Myc₁₃ (Fig. 2C), confirming that residues 250–535 of Cdc7 were sufficient to interact with Spg1.

We noticed that when GFP-Cdc7-(250–535) was overproduced, it generated a SIN phenotype (Fig. 2D). This could be because it prevented endogenous Cdc7 from localizing to the poles and thus from binding Spg1 or because it titrated Spg1 from the SPBs. To distinguish between these two possibilities, we first examined the localization of GFP-Cdc7-(250–535) when it was overproduced. Inconsistent with the first possibility, GFP-Cdc7-(250–535) localized to the cytosol (Fig. 2D). Furthermore, overproduction of Cdc7-(250–535) displaced endogenous Spg1-GFP from the SPB (Fig. 2E). The SPB itself was intact under these conditions, because the localization of the SIN anchor Sid4-GFP and the downstream SIN component Sid2-GFP, which dynamically associates with Cdc11, were unaffected (Fig. 2E). These data taken together indicate that amino acids 250–535 of Cdc7 are sufficient for binding to Spg1 in vitro and in vivo and, when present in sufficient quantity, to sequester Spg1 away from SPBs. Further, these results indicate that Cdc7 residues outside of its Spg1 binding domain are required for Cdc7 localization to the SPB.

Amino Acid Residues 360–870 Mediate SPB Localization of Cdc7—To determine which region(s) of Cdc7 target it to the SPB, we expressed N- and C-terminal truncations of Cdc7 fused to GFP. Because Cdc7 is an essential gene we tested these truncations in cells that also contained endogenous Cdc7. Full-length GFP-Cdc7 localized to the SPB in a cell cycle-dependent manner. However, unlike endogenous Cdc7, it localized to the SPB symmetrically during anaphase, as has been noted previously when Cdc7 is overexpressed (22). A kinase-dead mutant of Cdc7, GFP-Cdc7K38R, and also GFP-Cdc7-(250–1062) and GFP-Cdc7-(360–1062) constructs lacking the kinase domain were capable of localizing to the SPB (Fig. 3). Hence, neither the kinase domain nor kinase activity is required for Cdc7 SPB localization. We next tested C-terminal truncations to narrow the region responsible for Cdc7 SPB localization. Although GFP-Cdc7-(360–870) could localize to the SPB, further loss of N- or C-terminal residues resulted in abrogation of SPB localization (Fig. 3B). Hence the region that is responsible for SPB association resides within residues Cdc7-(360–870).

To correlate the ability of Cdc7 constructs to localize and bind Spg1 with overall Cdc7 function, GFP-Cdc7 truncation mutants were tested for the ability to rescue growth of cdc7–24 cells at 36 °C (Fig. 3A). Full-length and C-terminally truncated GFP-Cdc7-(1–900) rescued this growth (Fig. 3A), and as expected, all Cdc7 fragments lacking the kinase domain did not. The minimal rescuing fragment, GFP-Cdc7-(1–900), contains both the SPB localization and Spg1-association domains. These results indicate that C-terminal residues Cdc7-(900–1062) are dispensable for Cdc7 function.

Cdc7 Self-associates in Vivo—It has been shown that Cdc15, the S. cerevisiae ortholog of Cdc7, interacts with itself. To determine whether Cdc7 associates with itself, co-immunoprecipitation analyses were performed on a diploid strain in which one allele of cdc7 was tagged with the Myc₁₃ epitope and the other was tagged with the HA₃ epitope. Anti-HA immunoprecipitates from the dually tagged diploid strain, but not the singly tagged strains, contained Cdc7-Myc₁₃ and vice versa (Fig. 4A). These results established that Cdc7 is capable of associating with itself in vivo, suggesting that Cdc7 exists in cells as an oligomer. Because Spg1 associates with Cdc7, we asked whether it, also, was present in an oligomeric complex in vivo. We constructed a diploid strain containing both Spg1-Myc₁₃ and Spg1-HA₃ and performed immunoprecipitations using anti-HA and anti-Myc antibodies. In these experiments anti-HA immunoprecipitates from diploid strains did not contain Spg1-Myc₁₃ and vice versa (Fig. 4B). Therefore Spg1, unlike Cdc7, does not appear to oligomerize in vivo.

View larger version (60K): [in this window] [in a new window]   FIGURE 3. SPB localization domain of Cdc7. A, characterization of Cdc7 truncation mutants. To test for rescue of the growth defect of cdc7–24 (KGY2061) mutants, cells carrying vector alone or deletion mutants of Cdc7 tagged with GFP and expressed from the low-strength nmt81 promoter of pRE81GFP vector were grown on selective plates without thiamine at a permissive temperature (25 °C) and then shifted to the restrictive temperature of 36 °C. Colony formation at the restrictive temperature was indicative of rescue as indicated by the + symbol. –, indicates no growth; ND, not determined. To observe SPB localization of these deletions, cells containing GFP-Cdc7 truncations were grown overnight in the presence of thiamine, washed in media without thiamine, and allowed to grow to mid-log phase in the absence of thiamine at 32 °C for 18 h. B, GFP-Cdc7 images were captured in live cells.

To estimate the oligomeric state of endogenous Cdc7, lysates from asynchronously growing Cdc7-Myc₁₃ and control untagged strains were prepared under native conditions and sedimented on sucrose gradients. The presence of Cdc7-Myc₁₃ in each fraction was determined by immunoprecipitating and immunoblotting for the Myc epitope. Molecular size standards were run in parallel on an identical sucrose gradient. The majority of Cdc7-Myc₁₃, co-sedimented in fractions 9 and 10 with the molecular mass marker phosphorylase B, which forms a trimer of 292.8 kDa. Because the predicted molecular mass of a Cdc7-Myc₁₃ dimer is 285 kDa, this suggests that Cdc7 exists primarily as a dimer. Some Cdc7-Myc₁₃ sediments lower in the gradient, and this could represent Cdc7 in complex with other proteins (such as Spg1) and/or in higher order complexes with itself (Fig. 4D). Importantly, Cdc7-Myc₁₃ was not observed in any fraction that would be consistent with a monomeric form (140 kDa) of Cdc7-Myc₁₃.

View larger version (55K): [in this window] [in a new window]   FIGURE 4. Cdc7 self-associates in vivo. A, anti-Myc (right) and anti-HA (left) immunoprecipitates (IP) from the indicated strains (cdc7-myc13/cdc7-HA3 KGY4426, cdc7-myc13 KGY4071, and cdc7-HA3 KGY3498) were blotted with anti-Myc (upper panels) and anti-HA (lower panels) antibodies. *, indicates Cdc7-Myc13 bands. B, anti-Myc (right) and anti-HA (left) immunoprecipitates from the indicated strains (diploid-spg1-myc13/spg1-HA3 KGY5781, spg1-HA3 KGY2167, and spg1-myc13 KGY4071) were blotted with anti-Myc (lower panels) and anti-HA (upper panels) antibodies. –, indicates an empty lane. C, protein lysates prepared from cdc7-myc13/cdc7-HA3 diploid strain (KGY4426) were incubated with GST or GST-Spg1Q69L beads as indicated. After incubation, beads were washed, and bound proteins were eluted with GST elution buffer. Portions of the beads (b) and the eluate (e) were run on a SDS-polyacrylamide gel and blotted with anti-Myc to detect proteins bound to Spg1 and with anti-GST to detect recombinant proteins, as indicated. The remaining eluate was subject to immunoprecipitation with anti-HA antibodies (IP-HA) and immunoblotted with anti-Myc and anti-HA antibodies (lower panel). *, indicates Cdc7-Myc13 (as in A). D, native lysates were prepared from cdc7-myc13 (KGY3842) cells and fractionated over a sucrose gradient. Cdc7-Myc13 was immunoprecipitated from gradient fractions using anti-Myc antibody 9E10 and analyzed by SDS-PAGE. Molecular size standards were run in parallel on an identical sucrose gradient. The relative intensity of the bands in the fractions is represented in the histogram. E, Cdc7 bait plasmids were co-transformed with various cdc7 or control prey plasmids. Transformants were grown on medium lacking histidine and adenine to score for positive interactions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cdc7 is a protein kinase essential for cytokinesis in fission yeast. It is an effector for the Ras superfamily GTPase Spg1, the activation of which is central to determining the timing of septation and cytokinesis in fission yeast. In the current model for SIN activation and signaling, Spg1 is converted to its GTP-bound state, which enables association of Cdc7 to the SPB. Hence it has been suggested that the regulation of Cdc7 kinase activity is mediated by its localization to a signaling competent SPB. In this study we demonstrate an association between Spg1 and a small fraction of Cdc7 during mitosis. Given that Spg1 associates with the SPB dynamically during both interphase and mitosis (16), it is not surprising that we could detect only a small fraction of Cdc7 associating with Spg1. The mitosis-specific association of Cdc7 with Spg1 provides a means for Cdc7 to access its relevant targets at the SPB, and hence regulating that association would be a key step in regulating SIN signaling. To elucidate Cdc7 regulation, we have identified various functional domains within Cdc7. We have shown that a region adjacent to the kinase domain is responsible for association with Spg1 but insufficient for SPB localization. In addition we found that Cdc7 self-associates, adding another layer of complexity to SIN organization.

A region just adjacent to the kinase domain of Cdc7, Cdc7-(250–535), acts as an effector domain and interacts with a G-protein, in this case Spg1, in a nucleotide-dependent manner. It has been shown previously that Cdc7 fails to localize to SPBs in germinating spg1 null spores or spg1–8 cells at restrictive temperature (33). Although these data suggest that Spg1 is required for association of Cdc7 to the SPB, we found that amino acids Cdc7-(250–535) are capable of associating with Spg1 both in vitro and in vivo but fail to localize to the SPB. This suggests that additional residues and mechanisms exist that promote or maintain Cdc7 SPB localization. Indeed, the smallest region that could localize to the SPB, Cdc7-(360–870), is significantly larger than the Spg1-interacting region, although they overlap. Embedded within the Spg1 and SPB binding domain is a coiled-coil region; however, residues within this region are not sufficient to support binding to Spg1 or to localize to the SPB.³ Cdc7 has been shown to associate with the SIN complex anchor Cdc11. However, we were unable to detect any direct interaction between Cdc11 and the SPB binding domain of Cdc7.³ Interestingly, within Cdc15, the S. cerevisiae equivalent of Cdc7, the Tem1 binding domain, is also not sufficient for SPB localization. In fact, the relative organization of the Spg1 binding domain being adjacent to and including residues that are a part of a larger SPB binding domain is reminiscent of the domain structure found for Cdc15 (34).

In testing various fragments of Cdc7 for Spg1 and SPB interaction, we found that a GFP fusion of Cdc7-(1–900) localized efficiently to the SPB when produced at low level (Fig. 3A). This result is inconsistent with a previous report suggesting that the C-terminal 162 amino acids of Cdc7 are essential for SPB targeting (35). It is possible that by using the strong nmt1 promoter to drive expression at high levels, Lu et al. (35) were unable to discriminate specific SPB staining from background.

In the course of our studies, we found that Cdc7 self-associates. Through yeast two-hybrid analysis we find that this association is between the N and C termini with the most likely conformation involving an intermolecular interaction. A number of studies in the recent years have shown that homodimerization of kinases plays a critical role in regulating their functions (36–38). However, our evidence thus far suggests that Cdc7 dimerization is constitutive. First, a monomeric form of Cdc7 was not observed in sedimentation analyses. Second, Spg1 itself does not self-associate, and overproduction of Spg1 in a cdc7-HA₃/ cdc7-myc₁₃ diploid strain was not sufficient to disrupt Cdc7 self-association.³ Finally, bacterially produced Spg1 associated with a oligomer, presumably a dimer, of Cdc7. These data taken together suggest that dimerization is obligate for Spg1 association. Although we did not observe any changes in Cdc7 dimerization at steady state levels, there may still be localized regulation of Cdc7 at the SPB. It is also likely that Spg1 association may still influence Cdc7 kinase activity at the pole. The latter possibility could be best tested in an in vitro system using purified components.

Given that Cdc7 is a protein kinase with a variety of modular domains, understanding how Cdc7 is regulated through these domains to influence cytokinesis and septation in fission yeast is a critical next step. This will require in-depth structural analysis of Cdc7 in complex with Spg1. Such an analysis will help elucidate at the molecular level how Spg1 interacts with Cdc7 and whether changes in Spg1 nucleotide status influence Cdc7 conformation. Furthermore, identifying specific residues within Cdc7 that are responsible for contacting Spg1 and for Cdc7 self-association will enable the generation of specific point mutants of Cdc7 that are defective in Spg1 association, but not self-association, and vice versa and serve as useful tools to investigate Cdc7 regulation.

	FOOTNOTES

 
^* This work was supported by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Investigator of the Howard Hughes Medical Institute (HHMI). To whom correspondence should be addressed: HHMI and Dept. of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-9502; Fax: 615-343-0723; E-mail: kathy.gould{at}vanderbilt.edu.

² The abbreviations used are: SIN, septation initiation network; GFP, green fluorescent protein; GST, glutathione S-transferase; SPB, spindle pole body; HA, hemagglutinin; MEKK, mitogen-activated protein kinase kinase; bis-Tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid.

³ S. Mehta and K. L. Gould, unpublished data.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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