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J. Biol. Chem., Vol. 279, Issue 44, 45957-45968, October 29, 2004
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From the
Division of Cardiology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611 and the ¶Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229
Received for publication, July 9, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Geminin is an unstable 25-kDa protein that has profound effects on both cell division and cell differentiation (1–4). Geminin is a protein of complex multicellular organisms; it is found universally in vertebrates and in Drosophila but is absent from yeasts and the nematode Caenorhabditis elegans. Several different activities of geminin have been described.
Geminin prevents a second round of DNA replication during S and G2 phase by inhibiting the reassembly of prereplication complex, a collection of essential replication factors that assembles on replication origins before DNA synthesis begins (1). Geminin binds and inhibits the protein Cdt1, an essential component of prereplication complex with an unknown biochemical function (5, 6). Geminin is destroyed by ubiquitin-dependent proteolysis during mitosis at the metaphase/anaphase transition, which allows a new round of DNA synthesis in the succeeding cell cycle (1).
Geminin also induces entry into mitosis by antagonizing the checkpoint kinase Chk1. When geminin is depleted from Xenopus embryos or cultured somatic cells, Chk1 accumulates in its active phosphorylated form (7, 8). Chk1 activation leads to phosphorylation and inhibition of the mitotic protein kinase Cdc2. The mechanism by which geminin influences Chk1 activity is unknown. It might affect Chk1 activity indirectly by preventing replication abnormalities, or it may be part of a regulatory pathway that directly down-regulates the kinase.
Several groups have described specific effects of geminin on the development and differentiation of embryonic cells. In early Xenopus embryos, geminin induces uncommitted ectodermal cells to differentiate into nervous tissue (2). The mechanism of this induction is unknown, but the activity is reproduced by a fragment of the protein consisting of amino acids 38–89. More recently, it has been reported that geminin can inhibit eye formation in medaka fish embryos by binding and inhibiting the transcription factor Six3 (3). Geminin can also perturb the axial segmentation pattern of chick embryos by binding and inhibiting transcription factors in the hox gene family (4). These same workers also reported that geminin binds to Scmh1, a protein in the polycomb gene family. The biological consequences of this interaction were not described, but the association suggests that geminin might modify chromatin structure.
It has been difficult to understand how a small 25-kDa protein can have such diverse biological effects. Geminin is not homologous to any previously characterized protein. Sequence analysis indicates that geminin has an internal coiled-coil domain consisting of at least five heptad repeats (Figs. 1B and 10). A nine-amino acid destruction box located near the amino terminus is required for the ubiquitylation and destruction of geminin during mitosis (1). The region between the coiled-coil and the destruction sequence is rich in basic amino acids that could serve as nuclear localization signals or points of ubiquitin attachment. The carboxyl terminal region is rich in acidic amino acid residues but otherwise poorly conserved among species.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction—For rescue studies, geminin deletion mutants were constructed by PCR amplification of geminin fragments using pCS2-gemininwobble as a template. pCS2-gemininwobble encodes wild-type geminin H with eight wobble mutations that preserve the amino acid sequence but make the RNA refractory to inhibition by anti-geminin morpholino oligonucleotides (7). Fragments amplified from pCS2-gemininwobble were inserted between the EcoRI and XhoI sites of pCS2. Geminin missense mutants were constructed by QuikChange site-directed mutagenesis (Stratagene) using pCS2-gemininwobble as a template. pCS2-geminin
100–117 was constructed by amplifying an EcoRI fragment of pCS2-gemininwobble encoding amino acids 1–100 and inserting it into the EcoRI site of pCS-2-gemininN117. Two extra bases were added during the PCR reaction in order to preserve the correct reading frame. A similar method was used to construct geminin63–80, geminin63–90, and geminin63–100. The sequence of each mutant was confirmed by dideoxy sequencing. pET28-geminin clones were used to express geminin protein in bacteria as described (1). Geminin missense mutants were constructed by the QuikChange site-directed mutagenesis method (Stratagene) using pET28-gemininWT as a template. pCS2-MT-gemininWT, encoding the geminin H gene fused to a Myc tag, was constructed by amplifying the geminin H from the original Xenopus clone (6.42.152) and inserting it between the EcoRI and XhoI sites of pCS2-MT. Geminin deletion mutants were constructed in the same manner. pCS2-MT-gemininWT/NH has unique NdeI and HindIII sites flanking the region encoding amino acids 93–121. It was constructed from pCS2-MT-gemininWT by QuikChange mutagenesis. The NdeI site was generated by changing codon 93 from GCT to GCA, and the HindII site was generated by changing codon 121 from GCA to GCT. Neither mutation changes the amino acid sequence of the encoded protein. The mutation at codon 93 simultaneously destroys a second HindIII site found naturally in Xenopus geminin, making the engineered HindIII site unique. To make the NdeI site unique, we destroyed a second NdeI site found in the pCS-MT vector by QuikChange mutagenesis. Random missense mutations between amino acids 100 and 117 of geminin were constructed using a degenerate oligonucleotide approach (9). We first synthesized a degenerate oligonucleotide that encoded this region flanked by an NdeI site and a HindIII site (CGACGACATATGACCTTATGGTGAAAgaAacAccAacTtgCctTtaCtgGaaGgGgtTgcAgaGgaAcgAagAaaGgcCCTCTATGAAGCTTCA). At each position denoted by a lowercase letter, a mixture of oligonucleotide precursors was used that contained 90% of the correct base and 3.3% of each of the three other bases. The 3' end of the oligonucleotide was self-complementary. The oligonucleotide was hybridized to itself and made double-stranded using the large (Klenow) fragment of DNA polymerase I. It was then digested with NdeI and HindIII and inserted into pCS2-MTgemininWT/NH cut with the same enzymes. Individual mutant clones were screened for their ability to bind recombinant Cdt1 (see "Binding Assays") and transferred to pCS2-gemininwobble and pET28-gemininWT. pCS2-MTCdt1WT, encoding wild-type Xenopus Cdt1 fused to a Myc tag, was constructed by amplifying the Cdt1 gene from a cDNA clone of Xenopus Cdt1 (10) and inserting it between the XbaI and XhoI sites of pCS2-MT.
Protein Purification—Hexahistidine-tagged proteins were expressed in bacterial strain BL21 and purified using Ni2+-NTA-agarose according to standard techniques (Qiagen). For analytical ultracentrifuge analysis and importin-binding assays, the hexahistidine tag was removed by thrombin treatment, and contaminants were removed by passing the digest over Ni2+-NTA-agarose. For analytical ultracentrifuge analysis, geminin was further purified over Q-Sepharose and dialyzed against 50 mM sodium phosphate, pH 7.4, 300 mM NaCl.
Analytical Ultracentrifugation—Sedimentation experiments were performed using a Beckman Optima XL-A centrifuge with an AN 60 Ti rotor. Samples were dissolved in 50 mM sodium phosphate, pH 7.4, 300 mM NaCl. The partial specific volume of geminin was estimated to be 0.7232 ml/g using the method of Cohn and Edsall (11). The molar extinction coefficient was estimated to be 18,140 at 280 nm using the method of Gill and von Hippel (12). Molar extinction coefficients at other wavelengths were determined as previously described (13). Sedimentation velocity experiments were performed at 45,000 rpm at 20 °C. The loading concentration was 
8.6 µM. A total of 150 scans were collected and analyzed using the van Holde-Weischet method and the finite element method (14, 15). Sedimentation equilibrium experiments were performed at 4 °C at three different speeds (20,000, 26,600, and 33,300 rpm). Scans were collected at equilibrium at 230 and 280 nm. Multiple loading concentrations were measured at both wavelengths, and data points exceeding 0.9 OD were discarded. The protein concentration for the analyzed data points ranged between 80 nM and 40 µM. Data were fitted to multiple models. The most appropriate model was chosen based upon visual inspection of the residual run patterns and upon the best statistics. 95% confidence intervals were determined by Monte Carlo analysis. A minimum of 5000 Monte Carlo iterations were performed. All data analyses were performed using Ultrascan version 6.2.2
Binding Assays—Proteins were either expressed in bacteria or produced by in vitro transcription and translation of plasmid DNA in reticulocyte lysate (Promega TNT system). Reactions typically contained 5 ng to 1.5 µg of recombinant protein or 10–25 µl of reticulocyte lysate. Proteins were mixed in a total volume of 25–50 µl and incubated at room temperature for 1 h. After binding, an aliquot was removed to be used as a loading control. Nickel-NTA or antibody-coated beads (2–5 µl of packed beads containing 1 µg of antibody/µl) were added, and the mixture was tumbled at room temperature for 1 h. The beads were recovered and washed with immunoprecipitation buffer (50 mM 
-glycerol phosphate, pH 7.4, 5 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 100–500 mM NaCl, and 10 µg/ml each leupeptin, pepstatin, and chymostatin). For importin binding assays, the wash buffer also contained 10% glycerol and 1 M NaCl, and the dithiothreitol was omitted. Proteins were separated on polyacrylamide gels and visualized by immunoblotting. For in vivo binding assays, Xenopus oocytes were injected with 16 ng of anti-geminin morpholino oligonucleotide and 200–400 pg of RNA encoding geminin or Cdt1. Our previous work has shown that this amount of RNA produces a physiological concentration of geminin (7).
Immunofluorescence—BHK cells were cultured on coverslips in 1x Dulbecco's modified Eagle's medium, 10% calf serum, 10% tryptose phosphate broth. Cells were transiently transfected with plasmids encoding Myc-tagged geminin mutants using LipofectamineTM (Invitrogen). Twenty-four hours after transfection, cells were fixed with 1x phosphate-buffered saline, 3.1% formaldehyde, permeabilized with 1x phosphate-buffered saline, 0.1% Triton X-100, and stained with 9e10 anti-Myc antibody (Zymed Laboratories Inc.) and CY3-conjugated goat anti-mouse antibody (Zymed Laboratories Inc.). Nuclei were counterstained with 1 µg/ml 4',6-diamidino-2-phenylindole in phosphate-buffered saline. To visualize nuclei in Xenopus embryos, two-cell embryos were injected with fluoro-green (Amersham Biosciences) and allowed to develop to stage 9. Confocal images were taken at 10 µM intervals and projected onto a single plane.
DNA Replication Assays—DNA replication assays were performed using cytostatic factor-arrested Xenopus egg extracts and demembranated sperm DNA template as described (1). Proteins were added at a concentration of 50 ng/µl. The negative controls were no template added and no calcium added. The positive controls were no addition and geminin dilution buffer only (10 mM HEPES, pH 7.7, 300 mM NaCl). Percentage replication was normalized to the positive controls. The average of at least two measurements is reported for each protein.
Rescue Assays—Two-cell Xenopus embryos were injected with morpholino anti-geminin oligonucleotide and geminin RNA as described previously (7). For each mutant, a minimum of 18 two-cell embryos were injected on each side (36 injections total). Rescue efficiency was calculated as the percentage of rescue produced by the mutant divided by the percent of rescue produced by wild-type geminin, multiplied by 100%. Injection and scoring were performed blindly to avoid bias in the results.
Degradation Assays—Geminin mutants were transcribed and translated from plasmid DNA using reticulocyte lysate (Promega TNT System) in the presence of [35S]methionine. Translation lysate was mixed with cytostatic factor-arrested Xenopus extract in a 1:4 volume ratio. An aliquot was withdrawn for the t = 0 sample. Degradation was initiated by adding calcium, and a second aliquot was taken after 1 h. The destruction of geminin was visualized by polyacrylamide gel electrophoresis and autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To determine whether oligomerization occurs through the coiled-coil domain, the binding experiment was repeated using full-length His-tagged geminin and a series of Myc-tagged geminin deletion mutants (Fig. 1, B and C). Removal of amino-terminal amino acids up to residue 117 did not affect the interaction between the two tagged proteins (Fig. 1C, lanes 1–4); nor did removal of carboxyl terminal amino acids beyond residue 180 (lanes 7–9). However, deletion of residues between positions 117 and 160 completely abolished the association (lanes 5 and 6). This indicates that the self-association domain lies between residues 120 and 160, which almost exactly corresponds the limits of the coiled-coil (residues 118–152).
To see if geminin oligomerizes in vivo, mature stage VI oocytes were injected with 200 pg of RNA encoding Myc-tagged full-length geminin. Injection of this amount of RNA yields an amount of geminin protein that is similar to the endogenous level (7). The oocytes were then treated with progesterone to induce the translation of both the injected and the endogenous geminin RNA. To see if these two proteins associated, Myc-geminin was precipitated with 9e10 anti-Myc antibody, and the precipitate was blotted with anti-geminin antibody. Both Myc-tagged geminin (asterisk) and endogenous geminin (arrowheads) were detected in the precipitate (Fig. 1D, lane 2). The Myc antibody did not precipitate untagged geminin from uninjected eggs (lane 1). This indicates that geminin forms oligomers in vivo at physiological concentrations. To confirm that the interaction occurred through the coiled-coil, oocytes were injected with different Myc-tagged geminin deletion mutants. Mutants that included the coiled-coil domain associated with the endogenous geminin (lanes 3–6 and 9), and mutants that encroached upon this region did not bind (lanes 7 and 8). In this experiment, gemininC160, consisting of amino acids 1–160, associates less strongly with Myc-geminin than the wild-type. This suggests that the coiled-coil may extend past residue 160.
To determine the number of geminin subunits in the multimer, we performed sedimentation velocity and sedimentation equilibrium analysis of highly purified bacterially expressed geminin. The velocity analysis indicated that 92% of the protein exhibited a sedimentation coefficient of 2.44 S (Fig. 2B). The equilibrium analysis was performed at several different protein concentrations in order to allow for the possibility that several different geminin species may be present in reversible equilibrium with each other. All experimental observations (18 scans total) could be fit to a model in which a single ideal species is present with a molecular mass of 52.69 ± 0.2 kDa. The variance was extremely low at 2.2 x 10–5. A plot of the residuals and overlays for this fit is shown in Fig. 2C. The measured molecular weight is in excellent agreement with that predicted for a geminin dimer based upon the protein sequence (51.31 kDa). Adding additional parameters to account for more than one ideal species (e.g. monomer + dimer or monomer + trimer) did not reduce the variance. We conclude that the data are best described by a single species consisting of geminin dimers. Because no monomer could be detected in the preparation, we estimate that the geminin-geminin association constant is less than 100 nM. Combining the data from the equilibrium and velocity analyses, we estimate that the frictional ratio for geminin (f/fo) is about 2.2. This value suggests that geminin assumes an elongated shape, consistent with the observation that geminin elutes from a gel filtration column at an apparent molecular weight that is markedly higher than the true molecular weight (Fig. 2A).
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Geminin Must Be Degraded to Allow DNA Replication—To evaluate the biological significance of geminin degradation, we injected geminin-depleted embryos with RNA encoding gemininDEL, a nondegradable mutant in which the destruction box is deleted. Embryos expressing gemininDEL had an unusual "roughened" appearance (Fig. 3D). The cells were irregularly sized and had a more uniform pigmentation pattern than normal. To see if this was caused by a replication defect, the embryos were also injected with a fluorescent nucleotide analog to visualize the DNA. We found that gemininDEL-expressing embryos had far fewer nuclei than embryos expressing gemininWT (Fig. 3, compare E and F). The same appearance is seen when embryos are injected with gemininDEL protein (1). We conclude that geminin must be degraded during mitosis in order to allow replication in the next cell cycle.
Geminin Has a Bipartite Nuclear Localization Signal—Several independent studies have established that geminin is a nuclear protein (1, 2, 17). The amino-terminal portion of geminin contains several clusters of basic amino acids that could serve as a nuclear localization signal (NLS). To map the NLS, wild-type geminin and several different amino-terminal deletion mutants were tested to see if they bound to the nuclear transport protein importin-
. Each mutant was purified from bacteria and incubated with recombinant His-tagged importin-. The importin was precipitated with nickel-NTA-agarose beads, and the precipitate was blotted for geminin. Full-length geminin was precipitated by the Ni2+-NTA beads when importin was present but not when importin was omitted from the mixture (Fig. 5A, lane 1, compare top and middle panels). Mutants with deletions of the first 30 or 45 amino acids also bound tightly to importin- (lanes 2 and 3), but mutants with deletions of the first 60 or 80 amino acids did not bind above background (lanes 4 and 5). These results indicate that an importin- binding site lies between amino acids 45 and 60.
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Residues 50–62 of geminin (RtKepvknstKRK) have the sequence of a classic bipartite NLS: two clusters of positively charged residues separated by a short spacer (18). The underlined basic residues are highly conserved among different species from Drosophila to humans. To see if this is the NLS of geminin, two mutants were constructed. In one, the KRK sequence was changed to AAA (gemininKRK), and in the other, all of the basic amino acids were changed to alanines (gemininRTK/KRK) (Fig. 6A). Both mutants accumulated extensively in the cytoplasm when expressed in BHK cells; in each case, about 80% of the cells showed strong or exclusive cytoplasmic staining. These results indicate that amino acids 50–62 constitute a functional bipartite NLS. We believe that the NLS is bipartite because the gemininRTK/KRK shows more extensive cytoplasmic localization that gemininKRK and because the gemininN60 protein actually includes the KRK sequence.
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The results with the NLS mutants led us to examine the possibility that sequences outside the destruction box were required for geminin destruction. We previously showed that a mutant with a deletion of all residues amino-terminal to the destruction box (gemininN30) is degraded normally (1). We constructed several different missense mutants mapping near the destruction box and tested their stability in Xenopus egg extracts (Fig. 6). Two other mutations on the carboxyl-terminal side of the D-box were also nondegradable (gemininSASG and gemininLVG, lanes 11–14), whereas a mutation on the amino-terminal side was degraded normally (gemininLAP, lanes 3 and 4). Geminin-deficient embryos injected with gemininLVG or gemininSASG had the same "roughened" appearance as embryos injected with gemininDEL (not shown). This indicates that the entire region from the destruction box to the nuclear localization signal is required for geminin destruction.
The Neuralizing Domain—Kroll et al. (2) reported that a fragment of geminin consisting of amino acids 38–89 is sufficient to induce uncommitted embryonic cells to differentiate as neurons. Our mapping studies show that this fragment consists of the NLS and sequences between the NLS and the dimerization domain. To see if this region is required for geminin's cell cycle functions, we generated an internal deletion mutant, geminin63–80 that preserves the NLS and the dimerization domain but removes most of the neuralizing domain (Fig. 7A). We also generated several site-directed missense mutants between amino acids 63 and 100, targeting amino acids that are well conserved among species (Fig. 7A). Both the deletion and the missense mutants were able to efficiently rescue the G2 arrest caused by geminin depletion, and all of the missense mutants were able to inhibit replication when expressed in bacteria and added to replication extracts (Fig. 7, B and C, and Table I). Mutations in the neuralizing domain have no effect on the cell cycle activities of geminin.
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We next sought to generate missense mutants that were compromised in their ability to bind Cdt1. We concentrated our efforts on amino acids 100–117, since this portion of the Cdt1 binding domain lies completely outside the dimerization domain. Deletion of this entire region destroys the geminin-Cdt1 interaction in oocytes (Fig. 8C, lane 18). We first constructed three site-directed mutations that targeted amino acids that are highly conserved among geminin orthologs from different species (gemininPTC, gemininYWK, and gemininAEERR). We found that all three of these mutants bind Cdt1 normally in the oocyte assay (Fig. 9B, lanes 11, 13, and 15). To make random missense mutations in the Cdt1 binding domain, we synthesized a degenerate oligonucleotide encoding amino acids 100–117 in which 10% of the bases were randomly mutated. The oligonucleotide was made double-stranded and used to replace the geminin coding sequence in this region. We screened 32 individual clones and recovered one mutant, gemininSAPD, that did not bind Cdt1 in the oocyte assay (Fig. 9B, lane 16).
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101–117) were expressed in bacteria and added to Xenopus egg extract to see if they could inhibit DNA replication (Table I and Fig. 9D, black bars). They had little effect on DNA synthesis, confirming that geminin must bind Cdt1 in order to inhibit replication. The nonbinding mutants were also injected into geminin-depleted Xenopus embryos to see if they could rescue the lethal G2 arrest. Both were completely inactive (Table I and Fig. 9D, gray bars). In summary, our results indicate that the Cdt1 binding region of geminin lies between amino acids 100 and 140 and that this region of geminin is required both to inhibit replication and to rescue the lethal phenotype of geminin-depleted embryos. Separation of DNA Replication Inhibition and G2 Arrest Prevention—Geminin has two activities in early Xenopus embryos: it inhibits DNA replication in vitro and is required for passage into mitosis in vivo. It is not known if these two activities are both manifestations of a single biochemical activity or if geminin is a bifunctional protein. To distinguish between these possibilities, we tested the geminin missense mutants that bound Cdt1 to see if they affected one activity but not the other. The mutations were located in the Cdt1 binding domain, because this region is required for both activities. Each mutant protein was purified from bacteria and added to Xenopus egg extracts to see if it would inhibit replication, and RNA encoding each mutant was injected into geminin-deficient Xenopus to see if it could rescue the G2 arrest phenotype.
Most of the mutants were indistinguishable from wild-type geminin; they bound to Cdt1, inhibited replication in egg extracts, and prevented the G2 arrest when expressed in geminin-depleted embryos (Table I and Fig. 9D, black and gray bars). Three of the mutants that bound Cdt1 were nevertheless compromised in their ability to inhibit replication (gemininYWK, gemininRTGG, and gemininKKFEV). In particular, gemininYWK did not inhibit replication at all. This indicates that the binding of geminin to Cdt1 is necessary but not sufficient for geminin to inhibit replication. Because the YWK mutation changes tyrosine 106 to an alanine, we considered the possibility that geminin must be phosphorylated on this tyrosine in order to be active. To test this, we constructed gemininY106F, which changes tyrosine 106 to phenylalanine. GemininY106F, however, was able to both inhibit DNA replication and prevent the G2 arrest just like gemininWT (Table I and Fig. 9D).
Although they did not inhibit replication well, gemininYWK and gemininKKFEV were able to rescue the lethal phenotype of geminin-depleted embryos to some extent. The background of rescue assay is so low (<5%) that even partial rescue is significant. The phenotype of gemininKKFEV and gemininYWK mutants indicates that the replication inhibition and the mitotic entry functions of geminin can be separately mutated, suggesting that they represent two different activities of the protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this paper, we describe the structural and functional domains of the geminin protein (Fig. 10). We find that geminin forms homodimers in solution and that dimerization occurs through the coiled-coil domain. Geminin dimerization is required for activity. The Cdt1 binding domain is located immediately amino-terminal to the coiled-coil and overlaps it partially. Geminin mutants that do not bind Cdt1 are nonfunctional. Geminin contains a typical bipartite nuclear localization signal in its basic region. The amino-terminal portion of geminin contains the sequences required for its mitosis-specific destruction. The destruction signal includes the nine-amino acid destruction box and sequences immediately C-terminal to it. Interestingly, the nuclear localization signal is required for the geminin destruction. Embryos that express a nondegradable mutant are severely deficient in DNA replication, indicating that geminin destruction is necessary to allow replication after mitosis is complete. Complementation of the lethal G2 arrest of geminin-deficient embryo requires all of the identified domains.
Previous studies have identified the domains of geminin that are required for its developmental effects. A fragment of geminin consisting of amino acids 38–89 is sufficient to induce neural tissue in Xenopus embryos (2). Our mapping studies show that this fragment consists of part of the destruction signal, the nuclear localization signal, and sequences between the NLS and the Cdt1 binding domain. We find that the sequences between the NLS and the Cdt1 binding domain are not required for inhibition of replication or for rescue of a geminin-deficient embryo. The mechanism by which geminin induces neural tissue is unknown; our results suggest that the induction is not secondary to an effect on the cell cycle. Geminin structurally resembles transcription factors in the basic HLH leucine zipper class; it has a dimerization domain, an N-terminal basic region, and a C-terminal tail rich in acidic amino acids. We have found, however, no evidence that geminin activates transcription. The acidic tail and most of the basic region can be deleted without affecting the protein's cell cycle activities. Moreover, we find that geminin cannot activate transcription of a luciferase reporter gene when expressed in NIH3T3 cells (not shown). Two groups have recently reported that geminin affects eye development and embryonic segmentation by binding and inhibiting transcription factors such as Six3 and Hox family members (3, 4). Both groups find that transcription factors compete with Cdt1 for binding to geminin, so presumably both bind to the same site.
Previous observations suggested that geminin forms higher molecular weight multimers in solution. When Xenopus egg extract is fractionated on a gel filtration column, geminin elutes in a broad peak with an apparent molecular mass of about 250 kDa (19). Bacterially expressed geminin elutes at about the same position on gel filtration columns (Fig. 2A), yet when its molecular weight is experimentally determined by analytical ultracentrifugation, it is found to be mostly composed of dimers. Geminin's high frictional coefficient in velocity sedimentation experiments suggests that it has an elongated shape, which may explain its aberrant behavior on gel filtration columns.
Geminin has a classic bipartite NLS consisting of two clusters of basic residues separated by a 7-amino acid spacer (RtKepvknstKRK). The underlined arginine and the KRK sequence have been strongly conserved among vertebrates and Drosophila. Although mutation of the NLS causes geminin to accumulate in the cytoplasm, a significant number of cells still show some nuclear geminin. This may be an artifact of overexpression; geminin is near the size exclusion limit for nuclear pores and may passively diffuse inside. Alternatively, geminin might "piggyback" inside while attached to another protein such as Cdt1. A second conserved cluster of basic amino acids (RRK) is found within the Cdt1 binding domain. We find, however, that a mutant in which two of these residues are changed to alanines (gemininAEERR) (Fig. 9A) has normal nuclear localization (not shown). It has been reported that geminin must be transported into the nucleus in order to be activated as a replication inhibitor (19). We might expect gemininN60 or gemininRTK/KRK to inhibit replication less efficiently than the wild-type protein, because these mutants show defective nuclear import. We cannot, however, demonstrate any such difference; the two mutants inhibit replication at the same concentration as gemininWT (data not shown). In Xenopus extracts, a large fraction of endogenous geminin is bound to Cdt1 before nuclear assembly occurs, but very little is bound after assembly (19). Perhaps only unbound geminin is active as a replication inhibitor, and the effect of nuclear assembly is to free geminin from Cdt1.
We find that the destruction signal required for geminin's mitotic degradation is larger than the canonical destruction box and includes the nuclear localization signal. One explanation for this result is that geminin must be transported into the nucleus in order to be degraded. We disfavor this hypothesis, however, because geminin is degraded in egg extracts even when no nuclei are present (Fig. 8B). Furthermore, we cannot demonstrate any difference in the degradation rate of geminin in extracts containing different concentrations of nuclei (data not shown). It is also possible that gemininRTK/KRK is stabilized because lysines required for covalent ubiquitin attachment are lost. An exhaustive analysis of cyclin destruction, however, showed that there was no lysine or group of lysines that were absolutely required for ubiquitylation of this protein (20).
We previously showed that a fragment of geminin consisting of amino acids 87–160 is sufficient to inhibit DNA replication in Xenopus egg extracts (1). This region is here shown to consist of the Cdt1 binding domain and the dimerization domain. Although these two domains overlap slightly, they are clearly distinct; three mutants that do not bind Cdt1 lie completely outside the dimerization domain (gemininSAPD, gemininN117, and geminin100–117), and the deletion mutant gemininC140 does not dimerize yet still binds Cdt1.
Our results have implications for the mechanism by which geminin inhibits Cdt1. Four mutants that failed to bind Cdt1 (gemininC120, gemininN120, gemininSAPD, and geminin100–117) also did not inhibit replication (Table II), confirming that geminin affects replication by binding and inhibiting Cdt1. We isolated several mutants that bound Cdt1 yet inhibited replication poorly or not at all (gemininC140, gemininKKFEV, gemininRTGG, and gemininYWK), indicating that Cdt1 binding is necessary but not sufficient for this activity (Table II). These results argue strongly that geminin does not simply bind and sequester Cdt1. GemininC140 does not dimerize, indicating that dimerization is also required for replication inhibition. The dimerization domain may recruit a third protein to the geminin-Cdt1 complex, or it may occlude an active site on Cdt1. GemininYWK both dimerizes and binds Cdt1 yet does not inhibit replication at all. We postulate that the highly conserved YWK sequence blocks an active site on Cdt1.
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FOOTNOTES |
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These authors contributed equally to this work.
|| To whom correspondence should be addressed: Tarry 15-759, 303 E. Chicago Avenue, Chicago, IL 60611. Tel.: 312-503-4386; Fax: 312-908-9032. E-mail: t-mcgarry{at}northwestern.edu.
1 The abbreviations used are: NLS, nuclear localization sequence; WT, wild type; NTA, nitrilotriacetic acid.
2 B. Demeler, University of Texas Health Science Center at San Antonio, Department of Biochemistry.
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ACKNOWLEDGMENTS |
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and Anne Strohecker for help with transfections. We thankfully acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University (available on the World Wide Web at www.biochem.northwestern.edu/Keck/keckmain.html). |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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