text:
The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.

The proto-oncogene product c-Raf-1 is a member of Raf (Sed Thr kinase) family and consists of 648 amino acids (1). The normal Raf is not oncogenic, but once it is mutated, it can cause malignant transformation in mammalian cells; in particular, when it is deprived of the first 305 amino acids, it becomes highly oncogenic (2). This suggests that the N-terminal half potentially could serve as a tumor suppressor. The normal Raf is also a latent protein kinase but can be activated by several distinct mitogenic growth factors such as epidermal growth Research Council of Australia and the Anti-Cancer Council of Victoria.

* This work was supported in part by National Health and Medical
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8 The first three authors contributed eauallv to this work.
Fax: 61-3-347-1938. O TO whom correspondence should be addressed. Tel.: 61-3-347-3155; factor and platelet-derived growth factor through a chain reaction involving Grb-2, Sos and Ras.GTP complex (3). The Ras.GTP complex recruits Raf to plasma membrane-associated cytoskeleton where the activation of Raf appears to take place (4, 5). Raf in turn phosphorylates and activates a SerPThrPryr kinase called MAP' kinase kinase WAF"Ap or MEK) (3). This Ras-Raf-MEK signal cascade eventually leads to malignant transformation via phosphorylation of the elongation factor 4E a t Ser53 (6), or the transcription factors c-Jun and c-Myc at their N-terminal regulatory domains (7,8). The kinase Raf also can be activated by mutations, in particular by deletion of its N-terminal half (Raf257, residues 1-2571, indicating that the Raf kinase activity resides in its C-terminal half, and that the domain Raf257 serves as an intra-molecular kinase suppressor (9).
Interestingly, Raf257 alone can suppress the transcription of specific genes activated by oncogenic mutants of Ras such as v-Ha-Ras but not by an oncogenic mutant of Raf (101, suggesting that Raf257 competes the normal full-length Raf for binding to a Ras.GTP-dependent upstream activator(s) of the kinase Raf. It was shown recently that Raf257, but not the fulllength Raf, binds tightly the Ras. , clearly indicating that (i) Raf is an immediate effector of Ras.GTP and that (ii) the Ras binding of Raf257 is weakened or blocked by the C-terminal kinase domain of Raf. However, it still remains to identify a third protein(s) that allow the Ras.GTP complex to bind tightly the full-length Raf, and also to clarify how the C-terminal kinase domain of Raf is released from its N-terminal domain to be activated on the cytoskeleton.
To determine how closely the NF1-binding and Raf-binding sites (if they differ) on the same Ras molecule are situated in three dimensions, and to further identify the key Ras-binding residues of Raf by x-ray crystallography, NMR analysis, or sitedirected mutagenesis, it is important to produce the smallest Ras-binding fragment of Raf. We have demonstrated previously that a 78-amino acid fragment of NF1 (NF78, residues 1441-1518) is the minimal GTPase-activating peptide (20). Furthermore, we have shown that a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) can suppress v-Ha-Ras-induced malignant transformation (20). However, so far nobody has demonstrated that Ras-binding fragments of Raf can suppress v-Ha-Ras-induced malignant transformation. In this paper, using a series of N-and C-terminal deletion mutants of Raf257, we demonstrate that its 81-amino acid frag-MAP kinase kinase; GAP, GTPase-activating protein; NF, neurofibro-The abbreviations used are: M A P , mitogen-activated protein; MEK, matosis; GST, glutathione S-transferase; PCR, polymerase chain reaction; kb, kilobase pairk).

30105
Ras BindinglAnti-Ras Action of Raf and NF1 Fragments ment Ram1 (residues 51-131) has the highest affinity for the Ras-GTP complex and its overexpression indeed can suppress v-Ha-Ras-induced malignant phenotype, i.e. anchorage-independent cell growth. In addition we demonstrate that the smaller NF1 fragments such as NF78 and NF56 (residues 1441-1496) still can suppress v-Ha-Ras-induced malignancy, although their affinity for Ras is extremely low i n vitro, suggesting that a third protein may be involved in stabilizing the Ras-NF1 interaction i n uiuo.

EXPERIMENTAL PROCEDURES
Preparation of Recombinant c-Raf-1 and NFI Mutants and Ras GTPases-The normal c-Ha-Ras and oncogenic v-Ha-Ras GTPases were purified from Escherichia coli according to the procedure of Gibbs et al. (25). To produce a series of human c-Raf-1 and NF1 fragments as GST (glutathione S-transferase) fusion proteins in the bacteria, we have subcloned the corresponding polymerase chain reaction DNA fragments into the bacterial expression vector pGEX-2TH (21). Each GST fusion protein was then affinity-purified from the bacterial extract as described previously (20,21, 23, 24).
In Vitro Assay for the Binding ofRas.GTP Complex to c-Raf-1 or NFl Mutants-The purified v-Ha-Ras GTPase (25 pg) was incubated with [y3,P1GTP at 37 "C for 15 min in 1 ml of a GDP.GTP exchange buffer containing 50 mM Tris-HC1, pH 7.5, 2.5 mM EDTA, 3 mM ATP, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. To stop the GDP.GTP exchange, 15 mM MgC1, and 20 p~ ZnCI, were then added to the reaction mixture. For the binding assay, an aliquot of the [y-32P]GTP~v-Ha-Ras complex (100 pl) was then incubated at 25 "C for 15 min with GST fusion proteins containing c-Raf-1 or NF1 mutants that were immobilized on GSH-agarose beads. The mixture was transferred onto a glass microfiber filter (Whatman GF/C), and the filter was washed four times with 1 ml of a binding buffer (cold) that was identical to the GDP.GTP exchange buffer except that 2.5 mM EDTA was replaced by 10 mM MgC1, and 20 p~ ZnC1,. The radioactivity of [Y-~~PIGTP.V-Ha-Ras complex left on the filter was measured in a scintillation liquid. Since the oncogenic v-Ha-Ras GTPase is never activated by either NF1 or Raf, the GAP activity of NF1 or Raf fragments (if any) does not interfere with this binding assay.
Construction of the Plasmids Expressing Rafsl, NF78, or NF56 in Mammalian Cells-An EcoRI DNA fragment of 0.3 kilobases, containing at the 5' end a Kozak sequence (GCC GCC ACC ATG) followed by codons 51-131 ofhuman e-Raf-1 (RaBl) and at the 3' end a termination codon (TGA) and HindIII linker (AAGCTT), was prepared by polymerase chain reaction (PCR) and subcloned into the retroviral vector pMV7 expressing also a neomycin resistance selectable marker (20). The orientation of the insert was determined by HindIII digestion, as one HindIII site is located 100 base pairs downstream of the EcoRI insertion site and the other right after the termination codon. The resultant plasmid (RaBl/pMV7) was purified by CsCl density gradient centrifugation and used for transfection. The plasmids NF78/pMV7 and NF56/ pMV7, which encode the human NF1 residues 1441-1518 (NF78) and residues 1441-1496 (NF56), respectively, were also prepared in a similar manner.
Assay for Colony-forming Ability in Soft Agar of u-Ha-Ras-transformed Cells Expressing Rafsl, NF78, or NF56-v-Ha-Ras-transformed NIW3T3 cells (21) were transfected with 1 vg of the plasmids RaB1/ pMV7, NF78/pMV7, NF56/pMV7, or pMV7 alone (as a control) as complexes with liposomes (35 pg) as described previously (23). The resultant G418-resistant transfectants were cloned in the presence of 400 pg/ml G418 (a neomycin analogue) as described previously (23). Colonyforming ability of the parental clone and each transfectant was examined in soft agar by incubating 1000 cells/plate at 37 "C for 2 weeks under the standard culture conditions (21). The colonies were stained with crystal violet and counted.
Reverse Danscriptase PCR Analysis of Raf81 and u-Ha-Ras Expression-Total RNAs from each transfected clone were isolated by the method of Chomczynski and Sacchi (22). 5 pg of each RNA preparation was then used as a template to synthesize cDNA by avian myeloblastosis virus reverse transcriptase (Promega) and a Riboclone cDNA synthesis kit (Promega). The same aliquots of all cDNA preparations were used as templates for PCR to amplify selectively human Ram1 and v-Ha-Ras DNA sequences using the corresponding pairs of sense and antisense primers as described previously (21,23). Under these PCR conditions, the amount of PCR products is linearly proportional to the initial amount of cDNA used as template and is far below the saturation point. Standard titration curves were drawn based on the incorporation of [a-32PldGTP into the PCR bands, using both cloned v-Ha-Ras and c-Raf-1 DNAs at various concentrations (0.1-10 pg) as PCR templates (data not shown). Northern Blot Analysis of NF78, NF56, and u-Ha-Ras Expression-5 pg of poly(A) tail-containing mRNAs, affinity-purified on an oligo(dT)cellulose column from each clone, was separated by electrophoresis on 1.2% agarose gel and transferred onto a nitrocellulose filter paper a s described previously (23). Blotting the filter paper with 32P-labeled NF78, NF56, or v-Ha-Ras DNAs as a probe, the Northern hybridization was then performed as described previously (20).

RESULTS AND DISCUSSION
Ras-binding Domain of c-Raf-I-The fragment Raf257, consisting of the first 257 amino acids of human c-Raf-1, has been shown previously to be sufficient t o bind the Ras.GTP complex directly i n vitro (11,12). Furthermore, using a two-hybrid system in yeast, Votjek and colleagues (26) have provided indirect evidence suggesting, if not proving, that either the first 50 amino acids of c-Raf-1 or any residues downstream of position 132 in c-Raf-1 are not required for its Ras binding. To further these findings and identify the smallest Ras-binding domain of the Raf, we have affinity-purified a series of Nand C-terminal deletion mutants of the Raf as GST fusion proteins from E. coli (see Fig. L4) and examined in uitro the direct binding of the V -H~-R~S , [~-~~P ] G T P complex to these Raf.GST fusion proteins, which are linked t o GSH-agarose beads (see Fig. m). In this assay GST alone does not bind to the v-Ha-Ras.GTP complex. First we have confirmed that the 81-amino acid fragment

Anchorage-independent growth of Rafsl transfectants derived from v-Ha-Ras transformants 1000 cells of each transfectant were plated in soft agar, and after 2
weeks the number of colonies formed in each dish was counted. The size of colony: Large, more than 100 cells; Medium, around 30 cells; Small, less than 10 cells/colony. Each presented value was the average of the data from three independent experiments, and the standard deviation in each case was less than 5%.

Clones
Large  (27). The removal of the first 6 amino acids from Raf8l reduces significantly, but does not abolish, its binding to Ras (Fig. 1B). The resultant 75-amino acid fragment called Ran5 has been shown to consist of a single helix (residues 79-89), five anti-paralleled 0-sheets and five loops (28): 0-sheets I (57-611, I1 (67-71), I11 (96-1011, IV (110-112), and V (125-130). We found that the deletion of either the first 0-sheetAoop or the last two p-sheets/ loops abolishes completely the Ras binding; neither Raf66 (residues 66-1311, Raf52 (residues 51-102), nor Raf37 (residues 66-102) binds to Ras (Fig. 1B). These observations indicate that (i) so far Rat75 (residues 57-131) is the smallest fragment that still binds Ras, although its affinity for Ras appears 3-fold lower than that of Raf81, and that (ii) some residues in both P-sheets I and IV-V are absolutely essential for its binding to Ras. We are currently identifying the key Ras-binding residues of Rat75 by further systematic deletiodpoint mutations. So far Leu6' in the first loop and two basic residues in the helix (Lys8* and AI$') were found to be absolutely essential for Ras binding.' Anti-Ras Action of Rafsl in VivoSince the Raf257 has been shown to suppress the v-Ha-Ras-induced transcription of specific genes in NIW3T3 cells (lo), and among the Raf fragments Raf81 shows the highest affinity for Ras, its potential anti-Ras action was also examined by overexpressing Raf8l in the v-Ha-Ras-transformed NIW3T3 cells under the control of a strong LTR promoter of the retroviral vector pMV7. As shown in Table  I, the Raf81-expressing cells exhibit a much lower colony-forming ability than the Ras-transformed parental cells in a soft agar, indicating that Raf81 also effectively interacts with v-Ha-Ras in vivo. These Raf81-transfected cells express both v-Ha-Ras and Ram1 as revealed by a semiquantitative reverse transcriptase PCR analysis (see Fig. 2 A ) . In an attempt to establish the correlation between the in vitro Ras binding and the in vivo anti-Ras action of the Raf fragments, we are currently examining the anti-oncogenicity of Rat75 and several other Raf fragments, which are smaller than Raf8l but larger than the non-Ras-binding fragment Raf37.
Anti-Ras Action of NF78 and NF56 in Vivo-We have shown previously (20) that (i) the NF1 fragments of 91 and 78 amino acids (NF91, residues 1441-1531 and NF78, residues 1441-1518) can still activate normal Ras GTPases, but the NF56 (residues 1441-1496) does not, and that (  strongly v-Ha-Ras-induced malignant transformation. In an attempt to identify further the minimal NF1 fragment capable of suppressing the oncogenicity of v-Ha-Ras, we have examined the possible anti-Ras action of both the GTPase activating NF78 and the non-GTPase activating NF56 by overexpressing these NF1 fragments in the v-Ha-Ras-transformed cells. As shown in Table 11, out of 20 randomly picked transfectants (G418-resistant cells), 7 NF56 transfectants (35%), and 12 NF78 transfectants (60%) showed the soft agar colony forming ability less than 10% of that of the parental Ras transformants that were transfected with the vector alone. These observations suggest that both NF78 and NF56 are still anti-oncogenic as RaBl and NF91 (20). Further Northern blot analysis has confirmed that the reduction of anchorage-independent growth of v-Ha-Ras transformants by either NF56 or NF78 transfections is due not to any loss of v-Ha-Ras gene expression, but to overexpression of the NF1 fragments (see Fig. 2B ).
Ras Binding Activity of NF78 and NF56-Under the in vitro conditions where both Raf8l and Ran5 form a stable complex with v-Ha-Ras.[y3'P]GTP, no stable complex formation of either NF78 or NF56 was detected with the Ras.GTP (see Fig.  1B). Since NF78 was shown previously to activate normal Ras GTPases (201, it is clear that NF78 binds Ras, but its affinity for Ras must be much lower than that of Raf81 or Ran5 in vitro. Under different assay conditions, however, NF56 still can Despite the great difference in the affinity for Ras between NF78/NF56 and Raf81 in vitro, their anti-oncogenicity (anti-Ras action in uiuo) appears to be quite similar. These observations suggest that a third protein is involved in the stabilization of the Ras.GTP complex with these NF1 fragments in uiuo. Since the microtubule-binding domain and GTPase-activatingmas-binding domain of NF1 overlap each other (29,30), it is possible that the third protein may be microtubule. Alternatively, either Ras or the NF1 fragments are posttranslationally modified, perhaps phosphorylated, to form a tight complex. We are currently identifying the protein(s) in NIW3T3 cell extracts that are involved in the stabilization of the NF78.v-Ha-Ras complex in vitro.
It is of interest to note that the two distinct Ras.GTP-binding fragments, i.e. NF78 (residues 1441-1518) and Raf75 (residues 57-131), of a similar molecular size appear to share little sequence homology, if any. This might suggest that the NF78binding spot probably differs from the Raf7 5-binding spot even if the two fragments appear to bind rather competitively the same general area (or domain) of the Ras molecule. X-ray crystallographic analysis of both NF78.v-Ha-Ras.GTP and Raf75.v-Ha-Ras.GTP complexes in three dimensions is now under way to identify the key Ras residues as well as the residues of Raf75 and NF78 that are responsible for their unique interactions. This would provide valuable information not only for our better understanding of the Raf-Ras or NF1-Ras interaction, but also for further designing a highly specific anti-Ras chemical drug, by a computer-aided molecular modeling, that functionally mimics either Raf75 or NF78 and can be delivered efficiently into the cytoplasm. Such a drug would be potentially useful for the treatments of Ras-associated cancers, which represent about 30% of total human carcinomas, notably more than 90% of pancreas carcinomas and 50% of colon carcinomas (31). C. A. Bradham and S. Campbell-Burk, manuscript in preparation.
Acknowledgments-We are grateful to Drs. Peter Klinken and Shane Colley (University of Western Australia) for the generous gift of human c-Raf-1 cDNA, Dr. Hang J i for preparation of the Raf257.GST fusion protein construct, and Dr. Tony Burgess for consistent encouragement and support as well as invaluable advice and comment throughout this work.
Addendum-After the submission of this manuscript, two reports appeared which confirm our observation that Ram1 is the minimal Ras-binding fragment of c- Raf-1 (27, 28). The latter report (28) further shows an NMR solution structure of this Raf fragment. 14.


extfieldsofstudy: Biology Medicine

text:
Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP- bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

T I~Ma~ phases of the eukaryotic cell cycle (G1, S, G2, and must occur in a defined order if daughter cells to receive a full complement of cellular components. In particular, DNA replication must be complete before the onset of mitosis if chromosome fragmentation and consequent unequal distribution of the genome are to be avoided. This temporal coordination of the cell cycle could potentially occur in one of two ways. If the products of completed DNA replication were essential substrates for the mitotic machinery, then mitosis would not be able to occur if replication were incomplete. Alternatively, regulatory controis could exist which monitor the state of replication of the chromosomes and prevent the onset of mitosis prior to completion of replication. There is considerable evidence for the existence of such checkpoint (or feedback) controls (Hartwell and Weinert, 1989). Fusion of cells in S phase to cells in G2 delays the G2 cell nuclei from entering mitosis until the S phase nuclei have completed replication (Rao and Johnson, 1970). Similarly, arrest of cells in S phase by addition of drugs which block complete replication, such as hydroxyurea or aphidicolin, inhibits entry into mitosis until the drugs are removed. Addition of caffeine (Schlegel and Pardee, 1986) or okadaic acid (Schlegel and Paradee, 1987;Smythe and Newport, 1992) can override such blocks, allowing entry into mitosis, suggesting that checkpoint controls are in effect despite the fact that the mitotic machinery is potentially completely functional.
Mutations that appear to disrupt the feedback pathway which links DNA replication to entry into mitosis have been identified in several organisms. In AspergiUus nidulans, a temperature-sensitive mutation of the BimE gene allows chromosome condensation and mitotic spindle formation even when cells are blocked in S phase by drug treatment or mutation of other cell cycle control genes (Osmani et al., 1988). Similarly, BHK cells carrying a temperature-sensitive mutation in the RCC1 gene (ts BN2 cells) prematurely enter mitosis when the temperature is raised, even if S phase has not yet been completed (Nishimoto et al., 1978;Ohtsubo et al., 1987;Uchida et al., 1990).
The coupling of the onset of mitosis to the completion of DNA replication can be viewed as a signaling pathway from the incompletely replicated DNA to the cellular machinery which controls the onset of mitosis. Entry into mitosis in eukaryotic cells is triggered by maturation-promoting factor (MPF) t composed of a 34-kD serine/threonine protein kinase (the product of the cdc2 gene) and cyclin B, which oscillates in abundance throughout the cell cycle, peaking at the G2-M transition (Nurse, 1990). Binding of cyclin is essential for the mitotic activation of the cdc2 protein kinase. Once cyclin is bound to p34 '~a this complex is held in an inactive state by inhibitory phosphorylation of tyrosine and threonine residues present in the ATP-binding site of p34 ~-2 (Dunphy and Newport, 1989;Gould and Nurse, 1989;Solomon et al., 1990). In both Schizosaccharomyces pombe and Xenopus /aev/s, the control of entry into mitosis by incomplete DNA replication is largely exerted through modulation of this tyrosine phosphorylation of p34~-2/cyclin. In S. Pombe, the mikl and weel kinases appear to carry out the tyrosine phosphorylation of p34 ~2 (Featherstone and Russell, 1991;Lundgren et al., 1991;Parker et al., 1992). Cdc2 protein is not phosphorylated on tyrosine in m/k/weel double mutants at the restrictive temperature, and these mutants can enter mitosis even when DNA replication is inhibited by hydroxyurea (Lundgren et al., 1991). The tyrosine dephosphorylation of p34 ~2 is under the control of the tyrosine phosphatase cdc25 (Dunphy and Kumagai, 1991;Gautier et al., 1991;Millar et al., 1991). Overexpression of cdc25 protein can, like inhibition of weel and mild, override a hydroxyurea arrest (Enoch and Nurse, 1990).
A critical role for tyrosine phosphorylation and dephosphorylation of p34 ~d~ in the regulation of entry into mitosis has been established in cell-free extracts of Xenopus eggs which can oscillate between S and M phases of the cell cycle even in the absence of nuclei (Murray and Kirschner, 1989;Dasso and Newport, 1990;Solomon et al., 1990). If chromatin is added to these "cycling extracts, ~ nuclei are assembled during S phase, replicate their DNA, then become disassembled as the extract enters mitosis. Entry into mitosis can be monitored either microscopically by the observation of nuclear envelope breakdown and chromosome condensation or biochemically by assaying cdc2 kinase activity. Addition of aphidicolin to the extract causes interphase nuclei to be unable to replicate DNA and one observes a consequent inhibition of entry into mitosis (Dasso and Newport, 1990). Thus the feedback system linking completion of replication to entry into mitosis is active in this in vitro system. A similar feedback effect blocking entry into mitosis can be achieved by adding very large numbers of nuclei to the extract (greater than 2,000 nuclei/ul), presumably because the depletion of cellular components necessary for replication in the extract results in incomplete replication (Dasso and Newport, 1990). Overexpression of cdc25 protein in these extracts can override this feedback and promote entry into mitosis CKumagal and Dunphy, 1991). With respect to the molecular target of feedback, we have found that the tyrosine kinase(s) which phosphorylates p34 ~2 is activated when aphidicolin and nuclei are added to the extracts. This indicates that the feedback system linking replication and mitosis operates, at least in part, by increasing the rate of tyrosine phosphorylation of p34 c~2, thereby suppressing its mitotic kinase activity and preventing entry into mitosis (Smythe and Newport, 1992).
A signafing system which inhibits entry into mitosis prior to completion of replication requires components which can monitor the replicative status of the DNA and propagate a signal to the factors such as weel, mikl, and cdc25, which modulate p34 ¢d~2 activity. One attractive candidate for a participant in the monitoring system is the RCC1 protein mentioned above. In both mammalian cells and in S. Pombe, RCC1 mutants enter mitosis prematurely and can override a hydroxyurea arrest (Nishimoto et al., 1978, Matsumoto and. In Xenopus, RCC1 has been shown to be an abundant chromatin-binding protein present at approximately one molecule per nucleosome (Dasso et al., 1992).
Nuclei assembled in extracts depleted of RCC1 are altered in nuclear structure and defective for replication (Dasso et al., 1992). These data suggest that RCC1 acts in some manner to couple DNA replication to the regulation of entry into mitosis. In S. Pombe, RCC1 mutants (called piml) are rescued by overexpression of a gene known as spil, suggesting that spil and RCC1 may interact. Spil appears to be the S.
Pombe homologue of a previously isolated ms-related G protein known as TC4/Ran (Drivas et al., 1990;Matsumoto and Beach, 1991). It has been recently demonstrated that RCC1 is a guanine nucleotide exchange protein for TC4/Ran in vitro (Bischoff and Ponstingl, 1991a), supporting the idea that these proteins functionally interact in vivo.
Given the demonstrated role of ms-like GTP-binding proreins in a variety of signaling pathways and the involvement of RCC1 in feedback control, it seemed possible that TC4 might be a component of the signaling system that modulates the activity of p34 ~d~. We now show, using mutant TC4 proteins, that TC4 has independent distinct effects on nuclear structure and cell cycle progression. Specifically, a TC4 mutant predicted to bind GDP with a much greater affinity than GTP disrupts nuclear structure and replication in interphase Xenopus egg extracts. Moreover, in cycling extracts without added nuclei (lacking the substrate for the signaling pathway) this mutant TC4 prevents p34 ~'2 activation. This inhibition occurs through phosphorylation of p34 ~2. Therefore, we provide direct biochemical evidence for a role of TC4 in the regulation of p34 ~2.

Preparation of Extracts from X. ~ Eggs
Cycling extracts were prepared as described by Murray and Kirschner (1989). All incubations were at room temperature. For visual monitoring of nuclear envelope breakdown and chromosome condensation, sufficient sperm chromafin prepared as described by Lohl~ andMnsni (1983, 1984); (W'flson and ) was a dd~ to form 500 nuclei per ~1 of extract. Throughout the incubation, 2-/d samples were withdrawn at 10-min intervals and diluted 1:1 with 10 #g of Hoechst 33258 per ml in 37% formaldehyde and examined by phase contrast and fluorescence microscopy. In some experiments, aphidicolin (0.05 mg/ml) or caffeine (5 mM) were added at the start of the room temperature incubation. Olmdnic acid was added to the extract 70 min after the start of the room temperature incubation to a final concentration of 1 ~M. Interphnse extracts were prepared as described previously (W'dson and Newport, 1988). Samples were monitored by fluorescence microscopy as for the cycling extracts.

Production of Vdld-D2pe and Mutant TC4 Proteins
Two mutants of human TCA protein were constructed, one changing the 19 th amino acid residue from a glycine to a valine, the other altering the 24 th residue from a threonine to an asperagine. The human TC4 clone was provided to us as an EcoRI fragment in pUCI9 (the kind gift of Dr. P. D'Eustachio, New York University Medical Center, NY). This EcoRI fragment was subcloned into Ml3mpl9 for mutagenesis. Using the Amersham Corp. (Arlington Heights, IL) in vitro mutagenesis kit, we first introduced an NcoI site at the initiator codon of the TC4 clone with an oligouocieotide of sequence 5' AAC C-CC GCC ATG GCT GCG 3'. Using phage which contained this NcoI site, we introduced mutations into the coding sequence with an additional step of mutagenesis using oligonucleotidns of sequence 5' GTT GGT CAT G_TT GGT ,aCT GGA 3' (for producing the G to V mutation) or 5' ACT GGA AAA AAC ACC TTC GTG 3' (for producing the T to N mutation). The altered bases are underlined. The success of each step of mutagenesis was confirmed by sequencing of the resultant phage. The mutagenized EcoRI fragments were excised from M13 and subcloned into pBluescript SK (Stratagene Inc., La Jolla, CA) at the EcoRI site. TC4-containing bluescript plasmids were then digested with NcoI and Hindl~ (present in the polylinker of bluescript, 3' of the TC4 termination codon). The insert was isolated and subcloned into pGEX KG (kindly provided by Jack Dixon, University of Michigan Medical School, Ann Arbor, MI) and digested with NcoI and HindIII for production of glutathione S transferase fusion protein. Alternatively, the RF DNA from the mutagenized phase was restricted with NcoI and BamHI and subcloned into the vector pET 8C for protein produetion.

Isolation of TC4 Protein and GST-sea Urchin Fusion Protein
The bacterial strain DH5~ was transformed with the Gex-TC4 or Gex-sea urchin cyclm B constructs. The recombinant fusion proteins were expressed and purified as described (Solomon et al., 1990). For cleavage of the resultant GST-TC4 fusion proteins with thrombin, ,x,1 nag of fusion protein was incubated with 5/~g of thrombin at room temperature for 45 rain in thrombin cleavage buffer (50 mM Tris 8.0, 150 mM NaC], 2.5 told CaCl2, and 0.1% ~-mercaptoethanol). These proteins were then diluted into XB buffer (I00 mM KCL, 0.I M CaCI2, 1 mM MgCl2, 10 mM K-Hepes, pH 7.7, and 50 mM sucrose) and reconcentrated by centrifugation in a centricon I0 microconcentrator. Proteins were aliquoted and stored at -70°C.

Preparation of Antibodies Against the Human TC4 Protein
In order to raise antibodies against the TC4 protein, a pETSC-derived plasmid containing the wild-type TCA sequence was transformed into LysS strain E. coll. Expression of the protein was induced with HrrG for 3 h, after which the bacteria were pelleted by centrifngation, washed with 0.9% NaC1, repelleted, and frozen at -70°C. The TC4 protein expressed by induced bacteria fractionated to the inclusion bodies and it was purified as follows: the cells from 500 ml of bacterial culture were thawed and resuspended in 12.5 ml of buffer A (10 mM Tris HC1, pH 7.5, 50 mM NaC1, and 1 mM EDTA) plus 0.5% NP40 and allowed to sit on ice for 15 rain. They were then sonicated for 2 rain and the inclusion bodies were pelleted for 15 rain at 12,000 rpm in a Sorvall SS34 rotor. The pellets were washed with 5 ml of buffer B (10 mM Tris HC1, pH 7.5, 500 mM NaC1, and 1 mM EDTA) and pelleted as above. The inclusion bodies were then resuspended in buffer A plus 6 M urea and incubated on ice for 5 rain, after which insoluble material was pelleted for 15 rain at 12,000 rpm in a Sorvall SS34 rotor. The supematant containing the TC4 protein was then electrophoresed on a preparative SDS-polyacrylamide gel. The protein band containing TC4 was cut from the gel and used to generate antibodies by standard methods (Harlow and Lane, 1988).

Replication Assays
For continuous labeling of DNA, extracts containing sperm nuclei were incubated at room temperature in the presence of 0.1 /~Ci of ~[32p]dCTP (3,000 Ci/mM) per/~1.8-~tl samples were withdrawn into 8 ~d of replication sample buffer containing 80 mM Tris-HC1, pH 8.0, 8 mM EDTA, 0.13% phosphoric acid, 10% ficoll, 5% SDS, and 0.2% bromophenol blue. These samples were digested with 1 mg/ml proteinase K for 2 h at room temperature and eleetrophoresed on 1% agarose gels. Gels were dried and exposed to x-ray film at -70°C with an intensifying screen.

Histone H1 Kinase Assays
For assay of histone HI kinage activity, 2-/~1 aliquots of extracts were withdrawn at 10-min intervals into 2 p.l of EB buffer (80 mM ~-glycerophospbate, pH7.3, 20 mM EGTA, and 15 mM MgC12), frozen immediately in liquid nitrogen and stored at -70°C. Thawed samples were diluted 120-fold in EB buffer and were then added 1:1 to H1 essay cocktail (40 mM Hepes, pH 7.3, 10 mM EGTA, 20 mM MgCI2, 0.2 mg of historic Hl/ml, 0.2 mM ATE 5/~Ci 7132PlATP, and 10 ~tM cyclin AMP-dependent protein kinase inhibitor peptide. [Sigma Chem. Co., St. Louis, MO]). After 10 min of incubation at room temperature, protein sample buffer for SDS-PAGE was added to each sample to stop the reactions. Samples were resolved by SDS-PAGE. Gels were dried and exposed to x-ray film for 1 h.

Translation Assays and Binding to p13 Sepharose
[35S]mcthionine (0.04 Ci/ml) was added to extracts at the beginning of the incubation. Samples were removed, diluted 1:1 in protein sample buffer, boiled, and then electropboresed on 10% SDS-polyacrylamide gels. After electrophoresis, gels soaked in I M sodium saiicylate were exposed to x-ray film for autoradiography. For isolation of p34'xl~2--cyclin complexes on p13 Sepharose, p13 was purified as described by (Solomon et al., 1990) and coupled to Sepharose as described by (Dunphy and Newport, 1989). Extracts were incubated 45 rain in the presence of [35S]methionine and then diluted twofold with buffer I (80 mM ~-glycerophosphate, 5ram EDTA, 2 mM sodium orthovanadate, 0.1% NP-40, and 0.5 M NaC1). p13 Sepharose was added to each ~mple and incubated at 4°C with agitation for 45 mill. Beads were washed once in buffer I, once in buffer H (buffer I without NaC1 or NP-40) and once in buffer HI (10 mM Hepes, pH 7.5, I mM dithiothreitol, and 2 mM sodium orthovanadate), p13 beads were then boiled in an equal volume of sample buffer (5 mM EDTA, 5 mM sodium orthovanadate, 20 mM sodium pyropbosphate, 30 mM sodium fluoride, 1% SDS, 80 mM Tris, pH 6.8, 10% glycerol, and 0.1 M dithiothreitol) and pelleted. The supernatants were electrophoresed on 10% SDS-polyacrylamide gels for autoradiography.
For analysis of the state of pbosphorylation of edc2 protein on Tyr 15 and Thr 14, aliquots of extract were resolved by SDS-PAGE, transferred to polyvinyidifluoride membrane, and then immunoblotted with anti-cdc2 sera.

Nuclear Import
For nuclear import assays, a conjugate of TRITC-buman serum albumin and a synthetic peptide containing the nuclear-localization signal of SV-40 T antigen was added to extracts (Newmeyer et al., 1986). Transport substrate/per nucleus was measured using a linearly calibrated charge-coupled device camera and National Institutes of Health Image 1.49 software.

Mutations in Human TC4 Protein
The potential importance of RCC1 in the functioning of the feedback pathway linking completion of DNA replication to entry into mitosis and the interaction of RCC1 with TC4 in several systems suggested that TC4 might be an important component of the feedback signal in Xenopus. To test this, mutant forms of TC4 were produced. Oligonucleotidedirected mutagenesis was performed on a human TC4 cDNA clone (generously provided by P. D'Eustachio) (see Fig. 1 A). The amino acid residues we chose to alter (residues 19 and 24 of TC4) are conserved between ms and TC4 proteins and mutations at these residues in ms have been extensively studied. Mutagenesis of residue 19 from glycine to valine (hereafter referred to as the G19V mutant) was intended to Figure 1. Mutagenesis of human TC4. (,4) To produce the G19V mutant, the codon GGT encoding a valine was changed to GTT. Threonine 24 (ACG) was changed to an asparagine (AAC). Both mutants were made by oligonucleotide mutagenesis in M13 and were subcloned into vectors for expression of glutathione-Stransferase fusion proteins. (B) Aliquots of egg extract in the absence (-) or presence (+) of •150/zg/rni of recombinant human TC4 protein (which had been cleaved with thrombin to separate the TC4 and GST portions of the fusion protein) were resolved by SDS-PAGE and immunoblotted with anti-TC4 sera. (C) GTP binding by bacterially synthesized TC4 proteins. T24N, G19V, and wild-type TC4 proteins were resolved by SDS-PAGE, blotted onto nitrocellulose and processed for binding to a[32P]GTP as described in Materials and Methods. Samples were visualized by autoradiography with an intensifying screen. Exposure was for 1 h at -70°C.
provide a form of TC4 defective in GTP hydrolysis. The expression in tissue culture cells of human ms proteins which contain this mutation results in a transformed phenotype, presumably because this protein remains locked in an activated state through inability to hydrolyze GTP (McGrath et al., 1984). Similarly, mutation of residue 24 from threonine to asparagine (hereafter referred to as T24N) would allow production of a form of TC4 protein which would remain predominantly in a GDP-bound form since the corresponding mutants in ras H have a 20-40-fold decreased affinity for GTP in vitro (Feig and Cooper, 1988). Expression of this ras mutant acts in a dominant negative fashion to inhibit cell growth, presumably by interfering with the normal functioning of the endogenous wild-type protein (Feig and Cooper, 1988).
To demonstrate that the bacterially produced TC4 proteins behaved as might be expected with respect to nucleotide binding, we resolved some of each of the bacterial protein preparations by SDS-PAGE, transferred them to nitrocellulose and blotted with a[32P]GTP ( Fig. 1 C). The wild-type and G19V proteins bounded GTP at similar levels, while the T24N protein had greatly reduced GTP binding (faintly visible on a 12-h exposure). This is similar to the ras Asn 17 mutant (Feig and Cooper, 1988). It is not yet clear whether GTP hydrolysis is affected by the G19V mutation.
To use these mutant proteins to examine TC4 functions in Xenopus egg extracts, we cloned the TC4-coding sequences into a vector (gex KG) capable of producing glutathiune S transferase (GST) fusion proteins in E. coli. The fusion proteins produced were purified on glutathione Sepharose and cleaved with thrombin, removing the GST portion of the protein and leaving intact human TC4 proteins. Complete cleav-age was confirmed by Western blotting with antibodies directed against human TC4 protein (data not shown).
We wished to add the recombinant TC4 proteins to Xenopus extracts at levels roughly equivalent to the levels of Xenopus TC4 found endogenously. Based on estimates of TC4 abundance in other cell types (Bishoff and Ponstingl, 1991b), we predicted that TC4 would be present at •100 ng/ul in the extract. To determine if this was a correct estimate, we added between 100 and 200 ng/ul of E. coilexpressed TC4 protein (cleaved with thrombin to remove the GST portion of the fusion protein) to an extract. Aliquots of extract (with or without supplemented recombinant TC4) were resolved by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted with anti-human TC4 sera which cross-reacts with Xenopus protein (Fig. 1 B). Since Xenopus and human TC4 are separable by SDS-PAGE, it is possible to see that extracts containing the recombinant TC4 protein had approximately equivalent amounts of Xenopus TC4 ( Fig.  1 B, lower band) and bacterially produced human TC4 protein ( Fig. 1 B, upper band). Since the antisera was prepared against human TC4, it seems unlikely that it would recognize Xenopus TC4 more efficiently. Therefore, if anything, we added less recombinant TC4 protein than that present endogenously. For all subsequent experiments, equal amounts of wild-type and mutant TC4 proteins were added to extracts.

Effects on Nuclear Morphology
When demembranated sperm chromatin is added to crude extracts of Xenopus eggs, the condensed chromatin serves as a template for the formation of nuclei. Previously, we have shown that depletion of RCC1 protein from such a nuclear assembly extract significantly limits the nuclear growth around sperm chromatin templates. In RCCl-depleted extracts, nuclear size, as measured by nuclear volume, is reduced •10-fold relative to nuclei assembled in control extracts (Dasso et al., 1992;Dasso, M., unpublished observations). Because RCC1 acts as a nucleotide exchange factor for TC4 we investigated whether the addition of either the T24N or G19V mutants of TC4 caused any visible defect in nuclear assembly around sperm chromatin added to egg extracts. As shown in Fig. 2 A, addition of either wild-type or G19V mutant TC4 proteins had no effect on nuclear formation relative to controls. Under these conditions, an initial nuclear envelope formed around the sperm chromatin and then proceeded to grow rapidly (7-10-fold), coincident with decondensation of the initially highly condensed chromatin. In contrast to this, addition of the T24N mutant TC4 protein to assembly extracts severely restricted nuclear growth. In the presence of T24N protein, the initially elongated sperm chromatin rounded-up into a small sphere encapsulated within a nuclear envelope and then remained in this state for the duration of the experiment (3 h) (Fig. 2 A). This observation demonstrates that, like depletion of RCC1 protein, the addition of the T24N mutant TC4 protein to extracts inhibits a process required for nuclear formation and/or growth.
It has been shown that efficient replication of sperm chromatin added to Xenopus egg extracts occurs only after a nuclear envelope has assembled around the chromatin (Newport, 1987;Sheehan et al., 1988). Moreover, it has also been shown that depletion of RCC1 protein from extracts prevents replication of sperm chromatin which has been assembled Interphase extracts were incubated at room temperature in the presence of sperm chromatin and the indicated "1"124 protein variant. DNA was visualized by fluorescent microscopy following staining with Hoechst 33258. Photographs were taken at 60 min. (B) Interphase extracts were incubated in the presence of 1,000 sperm/#l and 0.2 #Ci//~l a[32P]dCTP. Continuously labeled samples were withdrawn at 30, 60, and 90 rain, treated with proteinase K, and resolved by agarose gel eleetrophoresis on 1% gels. Gels were dried and visualized by autoradiograpby at -70"C with intensifying screen.
into nuclei (Dasso et al., 1992). Because TC4 interacts with RCC1 protein (Bischoff and Ponstingl, 1991b) and the T24N TC4 mutant inhibits proper nuclear assembly, we investigated how different TC4 mutations might effect DNA replication. To do this, we measured the incorporation of 3:p_ labeled dCTP into sperm DNA incubated in egg extracts. In extracts containing either no addition or wild-type TC4 protein the rate of DNA replication was identical. When the G19V TC4 was added, there was a slight (twofold) decrease in replication rate. However, when the T24N TC4 protein was added to extracts, DNA replication was completely (>95%) inhibited over the entire 90-rain period of the experiment (Fig. 2 B). These results show that addition of T24N TC4 blocks both nuclear growth and DNA replication.

T24N TC4 Does Not Block DNA Decondensation
The re, assembly of an intact nuclear structure around a chromatin template following mitosis is a complex process in- volving a large number of steps (Newport, 1987). Membrane vesicles bind to the surface of the chromatin and fuse to form an intact envelope (Wilson and Newport, 1988;Pfaller et al., 1991;Newport and Dunphy, 1992), nuclear pores are incorporated, (Newmeyer et al., 1986;Shechan et al., 1988), and the initially condensed DNA begins to decondense. Although further growth of the nucleus beyond this point has not been well characterized, it is likely to re-quire incorporation of additional membrane vesicles, selective transport of proteins into the nuclear interior (Newport et al., 1991), assembly of the transported proteins into macromolecular structures which can interact with both the envelope and DNA, and further decondensation of the DNA.
In order to address which processes might be blocked by the T?.4N mutant protein, the process of chromatin decondensation was examined first. When demembranated sperm chromatin is added to egg extracts which have been depleted of membrane components by centrifugation, the sperm chromatin decondenses (Fig. 3, A-C). This decondensation process occurs in two stages. The initial stage is rapid (less than 5 mitt) and involves nucleoplasmin-dependent exchange of protamines for histones (Pfaller et al., 1991;Philpott et al., 1991;Newport and Dunphy, 1992). As a result of this exchange, the chromatin volume increases 10-20-fold compared to the size of the original sperm. Following this initial decondensation stage, the DNA continues to decondense slowly over the next 30 rain (Fig. 3, B and C). This second stage of decondensation is dependent on ATP hydrolysis and is reversed if the extract is induced to enter mitosis by addition of cyclin B protein (Yan H., and J. Newport, unpubfished data). Moreover, the kinetics of sperm chromatin decondensation observed in the absence of membrane appears similar, if not identical, to the rate of decondensation of sperm chromatin assembled into nuclei in complete extracts containing membranes. Based on these criteria, the decondensation observed in the absence of membranes appears to be mechanistically similar to the decondensation process which occurs within nuclei. When T24N TC4 protein was added to extracts which had been depleted of membrane (Fig. 3, D-F), both the rate and extent of sperm chromatin decondensation appeared to be identical to the decondensation observed in control extracts (no addition) (Fig. 3, A-C). Thus, the defect in nuclear growth observed with T24N TC4 is unlikely to be due to a failure of the DNA to decondense.

Effects of T24N TC4 on Nuclear Transport
Once an intact envelope containing nuclear pores has formed around chromatin, further assembly of the nucleus is dependent on selective transport Of nuclear proteins through nuclear pores (Newport, 1987). Thus, inhibition of nuclear growth observed in the presence of the T24N TC4 mutant could be explained by a block in nuclear transport. To determine whether this was the case, a fluorescently labeled nuclear transport substrate consisting of rhodamine-labeled HSA coupled to the SV-40 nuclear localization signal peptide (Newmeyer and Forbes, 1986) was added to egg extracts containing nuclei assembled in the presence or absence of the T24N mutant TC4 protein. Nuclear transport was quantitated using a linearly calibrated CCD camera to determine the fluorescent intensity (luminance/pixel) of each nucleus, which is proportional to the substrate concentration. If the T24N mutant inhibited nuclear transport, we would expect nuclei assembled in the presence of this protein to accumulate significantly less fluorescent transport substrate than control nuclei. As shown in Fig. 4, this did not appear to be the case. Indeed, after a 2-h incubation, the concentration of fluorescently labeled transport substrate in nuclei assembled in the presence of the T24N mutant protein was significantly higher than the concentration of the same substrate in control nuclei (Fig. 4 A). This suggests that the failure of nuclei to grow in the presence of the T24N mutation is not due to a defect in nuclear transport.
Although nuclei formed in the presence of the T24N mutant accumulated higher concentrations of transport substrate than control nuclei, this cannot be interpreted as demonstrating that nuclear pores of the T24N nuclei trans- Nuclei grown in the presence of wheat germ agglutinin (0.25 mg/ml). T24N mutant TC4 protein was added to extracts along with sperm chromatin. After nuclear formation, rhodaminelabeled SV-40 T antigen-human serum albumin conjugate was added to the extracts and fluorescent micrographs were taken of the imported substrate. port more efficiently than those of the control nuclei. This is because the volume of the T24N nuclei is much smaller than that of control nuclei. Therefore, a smaller absolute amount of substrate must be transported to achieve higher concentrations within the nuclei. In order to eliminate the effects of differences in nuclear size between control and T24N-treated extracts, we have modified the transport assay. In the above experiment, nuclei were assembled in the presence of wild-type or mutant TC4 and then tested for transport. We instead tested for an effect of mutant TC4 added after the assembly of nuclei. For this, chromatin was added to an extract and nuclei were allowed to grow for 40 min to a relatively large size. The T24N mutant TC4 protein was then added, the reaction incubated for 20 min, and fluorescent transport substrate was added. Quantitation of these assays at several time points revealed a very mild (less than twofold) defect in nuclear transport when T24N mutant TC4 protein was present (data not shown). In addition, the T24N mutant TC4 had less than a twofold effect on nuclear import when added to an in vitro assay system using digitonin-permeabilized tissue culture cells supplemented with Xenopus egg cytosol (similar to the assay used in Moore andBlobel, 1993 andMelchior et al., 1993) (Powers M., L. Strachan, and O. Forbes, unpublished observations).
The experiment described above indicates that addition of T24N to extracts might have a modest effect (twofold) on nuclear transport. However, because little is known about the relationship between nuclear transport and nuclear growth we could not, based on the experimental evidence presented above, rigorously eliminate the possibility that a twofold reduction in transport rate could account for the complete failure of nuclear growth in T24N-treated extracts. To address this possibility directly, we have used limiting concentrations of WGA, a known inhibitor of nuclear transport (Finlay et al., 1987), to investigate the relationship between nuclear transport and nuclear growth rates. By adding different concentrations of WGA to egg extracts we found that WGA concentrations below 0.1 mg/ml of extract had little effect on the rate of either nuclear growth or transport. Concentrations of WGA above 1.0 mg/ml of extract completely abolished both growth and transport. At this high concentration of WGA, small nuclei formed which were identical in size to nuclei formed in the presence of the T24N mutant TC4 protein (results not shown). However, unlike T24N nuclei, these WGA-treated nuclei did not accumulate measurable amounts of transport substrate. At concentrations of WGA between 0.1-1.0 mg/ml of extract, both nuclear transport and nuclear growth were slowed in a dose-dependent fashion. Therefore, using WGA we could reduce transport in a controlled fashion and determine how this reduction affected nuclear growth. Specifically, at a WGA concentration of 0.25 mg/rni we found that nuclear transport rates were decreased four-to fivefold and two-to threefold relative to nuclei in control and T24N-treated extracts, respectively. However, even though nuclear transport was reduced to levels significantly lower than that found in extracts treated with T24N protein, these nuclei continued to grow (Fig. 4, compare D and E). For example, at 70 min of incubation, nuclei assembled in the presence of WGA were 5.4 times larger in volume than nuclei formed in the presence of T24N despite the fact that the rate of transport by the WGA-treated nuclei was reduced approximately threefold relative to nuclei formed in the presence of the T24N mutant. Together these experiments demonstrate several points. First, the observation that limiting nuclear transport limits nuclear growth demonstrates that nuclear transport is, not surprisingly, required for nuclear growth beyond the initial phase of nuclear envelope assembly. Second, the observation that nuclear transport continues at high rates without nuclear growth in the presence of the T24N TC4 protein demonstrates that nuclear transport is necessary but not sufficient for nuclear growth (i.e., WGA-treated nuclei transported less protein but grew quite well). Third, their ability to accumulate the transport substrate demonstrates that nuclei formed in the presence of TC4 have an intact nuclear envelope. Thus, DNA replication is not blocked due to failure to form an intact envelope. Finally, the results indicate that T24N is not inhibiting nuclear growth as a result of inhibiting transport, since nuclear transport is almost unaltered.

TC4 T24N Protein Prevents Entry into Mitosis
To investigate a possible role for TC4 in the signaling pathway linking DNA replication and mitosis, sperm chromatin (500 nuclei//~l) was added to cycling extracts of Xenopus eggs which can perform multiple cell cycles in vitro, oscillating between S and M phases of the cell cycle (Murray and Kirschner, 1989;Dasso and Newport, 1990). Wild-type, T24N or G19V mutant TC4 protein was then added to these cycling extracts to test whether they affected the normal cycling of the extract. Additionally, we included a sample contaming aphidicolin as a control for the functioning of the feedback system in the extract. Extracts were incubated at room temperature and samples were removed every 10 rain, stained with Hoechst, and observed by fluorescence and phase contrast microscopy. Mitosis, as scored by nuclear envelope breakdown and chromatin condensation, occurred at 65-80 min in cycling extracts to which no TC4 was added. Addition of either wild-type or G19V protein delayed entry into mitosis slightly, if at all (Table 19. This was true for several different preparations of proteins from E. coli. In contrast, addition of any of several preparations of mutant T24N protein significantly delayed or entirely arrested the cell cycle prior to mitosis (Table 19. In several cases (see for example, Experiment 1, Table 19 the arrest with the T24N mutant was even more enduring than that obtained with aphidicolin and nuclei (extract to extract variability in the permanency of the aphidicolin arrest may be due to the lability of some component of the DNA feedback system). These results indicate that the T24N mutant protein, like feedback from DNA replication, can prevent entry into mitosis.

Override of the T24N Arrest
In the experiment described above, it was essential to demonstrate that the T24N cell cycle arrest was due to a defect  in the presence of wild-type T24N mutant TC4 protein were treated with recombinant GST-cyclin protein to promote entry into mitosis. Nuclei were fixed and stained with Hoechst dye for visualization by fluorescence microscopy. (B) Protein translation: cycling extracts containing wild-type or T24N mutant TC4 proteins were incubated in the presence of [~SS]methionine and samples were withdrawn and boiled in SDS loading buffer and resolved by SDS-PAGE to visualize total protein synthesis. Gels were soaked in 1 M sodium salicylate prior to autoradiography. (C) cdc2-cyclin complex formation: samples prepared as in A were withdrawn at 45 min. These samples were processed on p13 Sepharose beads. Proteins were eluted from these beads by boiling in SDS-containing sample buffer and resolved by SDS-PAGE. (D) cdc2 tyrosinethreonine phosphorylation: aliquots were withdrawn at 10-rain intervals from cycling extracts containing either wild-type or T24N mutant protein and were resolved by SDS-PAGE. Proteins were transferred to PVDF-immobilon and detected with anti-cdc2 sera and HRP anti-rabbit sera in conjunction with the ECL kit from Amersham.
in regulatory pathways controlling entry into mitosis and not simply a nonspecific effect due to the destruction of some cellular component necessary for the execution of mitosis. Moreover, it was essential to establish that it would be possible to accurately identify the time of mitosis as monitored by nuclear envelope breakdown and mitotic chromatin condensation in the small aberrant nuclei produced in the pres-ence of the T24N mutant. To do this, we have taken advantage of the fact that bacterially produced recombinant GST-sea urchin cyclin B induces mitosis when added to Xenopus egg extracts (Murray and Kirschner, 1989). In addition, it appears that p34~2-cyclin complexes formed with this fusion cyclin protein are not sensitive to inhibition by signals from incompletely replicated DNA, since addition of this protein to extracts arrested with aphidicolin drives these extracts into mitosis (Smythe and Newl~rt, 1992). Using this fusion protein we found that addition of GST-cyclin B to cycling extracts arrested with the T24N mutant protein caused the extracts to enter mitosis within 20 rain of addition. Furthermore, mitosis was easily identifiable in the T24N mutant-containing extract, since nuclear envelope disassembly was observed by phase contrast microscopy and the condensed mitotic chromosomes were quite similar to those seen in wild4ype extracts (taking into account differences in fine structure resulting from the fact that the T24N nuclei did not replicate prior to condensation) (Fig. 5 A). The override of the T24N-induced arrest by the creation of active MPF in the extract indicates that the block to entry into mitosis is upstream of MPF, as has been shown previously for feedback generated by incompletely replicated DNA in Xenopus and other systems (Dasso and Newport, 1990;Enoch and Nurse, 1990). Although creation of fresh MPF did override the T24Ninduced cell cycle arrest, it remained possible that the failure to enter mitosis in the presence of T24N TC4 resulted from an inability of the extract to produce potentially functional endogenous MPF activity. Such a defect might result from a failure to synthesize adequate cyclin protein or an inability of newly translated cyclin to bind to p34 ~. To test these possibilities, we incubated cycling extracts containing either wild-type or T24N mutant TC4 proteins with [35S]methionine and withdrew samples at 15-rain intervals into sample buffer for SDS-PAGE, allowing us to look at both total and cyclin-specific protein synthesis in the extracts. The amount of protein synthesized by these two samples was approximately equivalent (Fig. 5 B). In the wild-type sample which went into mitosis at •80 rain, the cyclin protein was degraded and could not be seen at the 90-min time point. In contrast, in the T24N mutant-treated extracts, cyclin protein continued to accumulate during the entire 90-rain time period. The sucl protein of S. Pombe (p13) binds to p34 ~k2 (and other members of the cdc2 protein family) and can, when coupled to Sepharose beads, be used to precipitate cdc2-comaining protein complexes out of extracts. To determine if newly synthesized cyclin was associating with cdc2, extracts treated with either wild-type or T24N mutant TO4 protein were labeled with [3sS]methionine for 45 rain and then the endogenous odc2 was precipitated with p13 Sepharose beads. After several buffer washes, beads were boiled in protein sample buffer and the released labeled proteins were resolved by SDS-PAGE. Results from this experiment showed that the presence of the T24N TC4 protein had no adverse effect on the binding of p34 ~2 to cyclin protein (Fig. 5 C). In addition, it is clear that similar amounts of p34 ~-2 and cyclin proteins were precipitable from the extracts at 45 min. From these experiments, it can be concluded that addition of the T24N mutant protein to extracts does not inhibit cyclin synthesis or its association with cdc2 protein.
Inhibition of p34 ~2 by feedback from DNA replication operates largely through stimulation of the tyrosine kinase which phosphorylates and thereby inhibits p34 ~w. In the presence of nuclei and aphidicolin, the activity of this kinase is stimulated approximately 5-10-fold over background levels (Smythe and Newport, 1992). In addition, phosphorylation of a threonine at residue 14 of cdc2, which also inhibits the kinase activity of p34 c~2, is elevated when the feedback system is active (Kornbluth et al., 1994). Therefore, it is possible to monitor the presumed last step in the feedback pathway (phosphorylation of Tyr 15 and Thr 14 of p34 ~a) by observing changes in the phospborylation state of cdc2 prorein throughout the cell cycle. It has been shown previously that cdc2 migrates as three bands on SDS-polyacrylamide gels. The fastest migrating form is not phosphorylated at either I~yr 15 or Tar 14 (both negative regulatory sites) (Solomon et al., 1992). The slowest migrating form is phosphorylated at both of these residues and the band between these two is singly phosphorylated at one or the other of these sites. When wild-type TC4 protein was added to a cycling extract, the extract entered mitosis at 75 rain (monitored microscopically) and the cdc2 protein which had been phosphorylated prior to mitosis was returned to a predominantly dephosphorylated state (80 min; Fig. 5 D). The phosphorylated forms of p34~2 began to accumulate again as the extract entered the next cell cycle (90-110 rain). In contrast, addition of equivalent amounts of the T24N mutant protein to cycling extracts led to the progressive accumulation of the singly and double phosphorylated forms of cdc2 with an almost quantitative conversion to the doubly phosphorylated form by 110 min. This result demonstrates that T24N protein promoted the accumulation of the phosphorylated forms of cdc2 in a manner similar to that of feedback from DNA replication, thereby inhibiting MPF activation and preventing entry into mitosis~ In order to test whether the endogenous cdc2-cyclin cornplexes formed in the presence of the T24N mutant were capable of full activation, we added okadaic acid to the extracts. Okadaic acid can uncouple mitosis from the completion of S phase in BHK cells and in Xenopus egg extracts (Lorca et al., 1991;Schiegel and Pardee, 1987). Smythe and Newport (1992) have reported that the ability of okadaic acid to overcome cell cycle arrest is due, at least in pan, to suppression of the tyrosine kinase which normally phosphoryiates p34 ~a, keeping it inactive during DNA replication. In addition, Kumagai and Dunphy (1992) have shown that okadaic acid can stimulate the cdc2-specific tyrosine phosphatase activity of cdc25 protein found in Xenopus extracts, thereby accelerating tyrosine dephosphorylation of p34 ~-2 and, consequently, MPF activation. In Experiment 2 shown in Table I, we added okadaic acid to the TC4 T24N protein-arrested extract at 73 rain and these extracts entered mitosis by 80 rain. This result demonstrates that there was endogenous MPF in the extract poised for activation but that this MPF was suppressed in activity by the added T24N mutant TC4 protein.
Interestingly, it has been shown in several systems that caffeine can override the cell cycle arrest induced by inhibition of DNA replication (Schlegel and Pardee, 1986;Dasso and Newport, 1990). In our experiments, caffeine was able to override the arrest induced by nuclei and aphidicolin but could not override the TC4 T24N protein-induced inhibition of entry into mitosis (data not shown). Therefore, if TC4 is a component of the DNA feedback system, it may well be that the site of action of caffeine is either upstream of or at the level of the TC4 protein.

The TC4 T24N Induced Inhibition of Mitosis Does Not Require DNA
In the experiments described above, the progress of the ex-tracts through the cell cycle was monitored by microscopic observation of the nuclei formed in the extract. It might be argued that TC4 T24N protein could inlu'bit entry into mitosis under these conditions by blocking replication of the sperm nuclei and acting, in effect, like aphidicolin. In other words, these incompletely replicated nuclei would be responsible for generating the feedback signal. To test whether the TC4 mutant protein has an independent effect on the cell cycle, we added wild-type TC4 protein or T24N TC4 protein to cycling extracts in the absence of any sperm chromatin. Aliquots were withdrawn at 10-rain intervals and cdc2/cyclin kinase activity was assayed by measuring in vitro phosphorylation of the substrate historic HI (Arion et al., 1988;Dunphy and Newport, 1989;Labbe et al., 1989). In the presence of added wild-type TC4 protein, the histone HI kinase activity peaked at 90 min and then dropped abruptly, indicating entry into mitosis and subsequent degradation of cyclin protein and inactivation of MPF (Fig. 6). In contrast, in the presence of added T24N mutant protein, the histone H1 kinase activity initially increased and then reached a steady level which was insufficient to induce mitosis (as shown by a visual observation of a parallel sample which contained nuclei and showed an identical suppression of histone H1 kinase activation in the presence of the T24N TC4 protein). This profile ofhistone HI activity can also be seen in extracts where DNA replication feedback is created by addition of nuclei and aphidicolin (Dasso and Newport, 1990; Kornbluth, S., personal observation). The suppression of histone H1 kinase activity by the T24N protein in the absence of nuclei indicates that MPF activity is negatively regulated by this mutant TC4 protein even in the absence of any template DNA to generate a signal.
In support of this conclusion, we found that the phosphorylation of p34 ~2 on Tyr 15 and Thr 14 promoted by the T24N mutant protein in the presence of nuclei (Fig. 5 D) was reproduced in the absence of any nuclei (Fig. 7). Samples of a cycling extract were incubated with either wild-type (Fig. 7, B and D) or T24N (Fig. 7, A and C) TC4 protein in the presence (Fig. 7, C and D) or absence (Fig. 7, A and B) of nuclei. Aliquots of the extracts were withdrawn at 10-rain intervals, resolved by SDS-PAGE, and immunoblotted with anti-cdc2 sera. When T24N TC4 protein was added to the cycling extract, cdc2 became increasingly phosphorylated with time. This phosphorylation occurred even in the complete absence of nuclei (Fig. 7 A). In contrast, in the presence of wild-type TC4 protein, the phosphorylated forms of p34 ~da accumulated only transiently prior to the onset of mitosis both in the presence (Fig. 7 D) and absence (Fig. 7  B) of nuclei, showing that these extracts had entered mitosis. Taken together, these data show that TC4 protein has direct effects on the cell cycle which are distinct and separable from effects on nuclear structure and nuclear import.

Discussion
The mutant T24N TC4 protein described in this study had multiple effects when added to extracts of Xenopus eggs. Upon addition of demembranated sperm chromatin to interphase extracts, this mutant inhibited nuclear growth and DNA replication but did not interfere with nuclear envelope assembly or nuclear transport. In cycling extracts, the mutant protein prevented activation of MPF and hence entry Figure 6. Suppression of histone H1 kinase activity by the T24N mutant of TC4. Cycling extracts were incubated at room temperature in the presence of wild-type TC4 protein or T24N protein. Samples were withdrawn every 10 rain and frozen in liquid nitrogen. Frozen samples were rapidly thawed and assayed for histone HI kinase activity. Assays were boiled in loading buffer for SDS-PAGE and resolved on 10% polyacrylamide gels. Dried gels were exposed to x-ray film for autoradiography for 1 h. into mitosis. This effect was completely independent of its effect on nuclear functions since activation of cdc2 kinase activity was inhibited even in the absence of nuclei. Together, these observations implicate TC4 as functioning both in an essential process for nuclear growth and in regulation of the initiation of mitosis.

Effects of T24N Mutant on Nuclear Morphology
In this report, we found that addition of the TC4-T24N mutant protein to nuclear assembly extracts had profound effects on nuclear growth. Specifically, although sperm chromatin could act as a template for the initial formation of a nuclear envelope containing nuclear pores when mutant T24N TC4 was present, further growth of the nuclei beyond this minimal size did not occur. This observation suggests that TC4 may normally participate in a critical step in nuclear formation at the end of mitosis and/or in maintaining essential nuclear structures during interphase. Using Xenopus extracts, several steps in nuclear formation have been well characterized (Newport, 1987;Wilson and Newport, 1988;Pfaller et al., 1991;Boman et al., 1992;Newport and Dunphy, 1992). The first steps in nuclear formation involve binding of membrane vesicles to ehromatin followed by fusion of these vesicles to each other to form an intact nuclear envelope immediately around the condensed chromatin. Subsequent to or during this fusion step, nuclear pores assemble in the envelope. Our results demonstrate that the TC4-T24N mutation does not inhibit these initial stages of the assembly process. Growth of the nucleus beyond this initial stage is not well characterized but clearly must be contingent upon several processes including: (a) decondensation of chromatin, (b) continued fusion of new membrane to the existing envelope, (c) selective transport of proteins through the nuclear pores, and (d) assembly of these newly transported proteins into essential structural elements within the nucleus. Inhibition of one or more of these processes by the T24N mutant protein would inhibit further nuclear growth.
Our results strongly suggest that the T24N mutant protein does not block chromatin decondensation. In support of this conclusion, we have shown that when highly condensed sperm chromatin is added to egg extracts containing the T24N protein but lacking nuclear membrane components, the sperm chromatin decondenses to the same extent and at the same rate as chromatin added to identical extracts lacking the T24N protein (Fig. 3). Moreover, at the level of light microscopy the extent and rate of decondensation in these membrane-depleted extracts appears to be similar to the decondensation rate observed for chromatin assembled into normal nuclei. Therefore, the condensed appearance of chromatin within nuclei formed in the presence of the T24N mutant of TC4 is likely due to the fact that the chromatin is packaged at a very high density within the minimally sized nuclear envelope. We could find no evidence that the T24N mutant blocks fusion of membrane vesicles at the nuclear envelope. This conclusion is based on the observation that the initial binding and fusion of membrane vesicles to sperm chromatin is the same in control and T24N-treated extracts (data not shown) but, more importantly, on the finding that the nuclei formed accumulate transport substrate and thus clearly have an intact nuclear envelope. Once this initial minimal envelope is formed, further growth of the envelope would normally be dependent on the fusion of vesicles to the outer membrane of the nuclear envelope. This newly inserted membrane would then diffuse around the nuclear pores to distribute itself in both the inner and outer nuclear membrane. Although the T24N mutation could inhibit this later fusion event, preliminary studies indicate that the fusion of nonnuclear-associated endoplasmic reticulum vesicles (the major component of the outer nuclear envelope) is normal in the presence of T24N mutant TC4 protein (data not shown) (for assay details see Newport and Dunphy, 1992). Based on these results, we conclude that the T24N mutant of TC4 does not directly inhibit either chromatin decondensation or nuclear membrane growth.
Once an intact nuclear envelope has completely encapsulated chromatin, further growth of the nucleus is dependent on selective transport of proteins through the nuclear pores. However, by measuring uptake of a fluorescently labeled nuclear transport substrate into nuclei in the presence and absence of T24N we have found that nuclear transport was depressed at most twofold by the T24N mutant protein. In fact, because nuclei do not grow in the presence of the T24N mutation, transport substrate actually accumulates to higher concentrations within nuclei formed in the presence of T24N than within controlled nuclei (Fig. 4, A-C). Using WGA to limit nuclear transport we have investigated the relationship between nuclear growth and transport. These results demon-strated that WGA-treated nuclei which transported less efficiently and accumulated less total transport substrate than T24N-treated nuclei, were still able to grow. Together these observations indicate that the T24N mutation of TC4 does not restrict nuclear growth by inhibiting nuclear transport. Further they show that nuclear transport is necessary but not sufficient for nuclear growth.
Moore and Blobe10993) have recently presented evidence that TC4 is an important factor for nuclear import of proteins in a permeabilized cell system. Similarly, Melchior et al. (1993) find that nuclear transport is GTP~/S sensitive and that this sensitivity can be mediated by TC4. However, it is im.portant to note that both these permeabilized cell assays were performed with dilute cytosol (2.5-10 mg/ml). Indeed, Melchior et al. (1993) found that when physiological concentrations of cytosol were used, GTP3,S caused no TC4-mediated inhibition of nuclear transport. In this study, we used physiological concentrations (~40 mg/ml) of Xenopus cytosol in a system which closely approximates an in vivo situation in that the extract repeatedly cycles through the cell cycle. When we added equimolar (or less) amounts of mutant T24N TC4 to that present in the extract, very tittle effect on transport was observed, while major effects on nuclear growth were seen. While the high cytosol concentration in our assay system might account for the absence of any observed effect of TC4 on nuclear import, it should be noted that the T24N mutant TC4 protein had no marked effects on nuclear import even in the permeabilized tissue culture assay used by Moore and Blobel (1993) and Melchior et al. (1993).
Taken together, our results indicate that in the presence of the T24N mutant of TC4 an intact nuclear envelope containing functional nuclear pores can form. This suggests that the T24N mutation is inhibiting a process which is required for nuclear growth beyond this point. The observation that nuclei formed in extracts depleted of RCC1 protein arrest at a similar point in the assembly pathway (Dasso et al., 1992;Dasso, M., unpublished observations) strongly suggests that TC4 normally interacts with RCC1 to facilitate further nuclear growth beyond this initial stage. Thus, it is quite likely that the T24N mutant acts in a dominant negative fashion, either blocking interaction between wild-type TC4 and RCC1 or inhibiting interaction of endogenous 1"124 protein with other cellular proteins, thereby inhibiting further nuclear growth. At present, relatively little is known about the structural dynamics which drive nuclear growth. Clearly, once a functional nuclear envelope has formed, new proteins will be selectively transported into nuclei where they will accumulate. It is likely that many of these proteins will be assembled into structural elements that both drive nuclear growth and maintain nuclear organization during interphase. For example, it has been shown that at the end of mitosis nuclear lamins, a major structural element of the inner nuclear envelope, enter the nucleus after initial envelope formation and then assemble at the nuclear periphery (Newport, 1987;Newport et al., 1990;Meier et al., 1991;Chaudhary et al., 1993;Foisner and Gerace, 1993). In lamin-depleted extracts, nuclei form a nuclear envelope but grow very slowly. These lamin-depleted nuclei are also unable to carry out DNA replication. The observation that nuclei formed in the presence of T24N are able to accumulate proteins via transport yet do not grow suggests that the T24N mutant may be blocking the assembly of nuclear proteins which, like the lamin proteins, are required for envelope growth and establishing the structural environment needed to carry out basic nuclear functions such as DNA replication.

Effects of T24N on Regulating the Initiation of Mitosis
We have found separately that addition of the T24N mutant of TC4 protein to cycling extracts prevents these extracts from entering mitosis. Moreover, because this arrest can occur in the absence of nuclei it is independent of the effect of T24N on nuclear growth. Arrest of the cell cycle by T24N TC4 is consistent with a role for TC4 as a part of a checkpoint control system which regulates entry into mitosis. Consistent with this, we have shown that addition of the T24N protcin to cycling extracts inhibits the mitotic regulator cdc2 kinase. This inhibition is not due to nonspecific effects such as the inhibition of cyclin synthesis or its association with cdc2 kinase. Rather we have shown that T24N causes cdc2 to become stably phosphorylated on the negative regulatory sites located at Thr 14 and Tyr 15. This strongly suggests that T24N is modulating the activities of the kinase/phosphatase system(s) which control the level of phosphorylation at these regulatory sites (Smythe and Newport, 1992). We have further found that the inhibitory effect of T24N on cell cycle progression is reversible. Specificaliy, addition of the phosphatase inhibitor okadaic acid to extracts arrested in interphase by the T24N mutant protein causes the cell cycle to progress into mitosis.
In all of the many instances studied to date, mutations in ras=related proteins equivalent to the T24N mutation in the TC4 protein generate a protein which acts in a dominant negative manner to suppress signaling by the wild-type protein. Our observations show that the addition of the T24N TC4 mutant protein to cycling extracts prevents im'tiation of mitosis. We would propose two possible models: a somewhat heretical explanation of this data is that the GDP-bound form of TC4 protein is an active intermediary in the signal transduction pathway inhibiting entry into mitosis prior to the completion of replication. This interpretation would imply that the TC4 protein was "active" in negatively regulating p34 ~da when in its GDP-bound conformation and that ongoing DNA replication might generate GDP-TC4 (Fig. 8, Model 2). According to this model, GDP-TC4 would serve (directly or indirectly) as an activator for the Tyr 15 and Thr 14 kinases negatively regulating cdc2. Perhaps a more direct interpretation of the observation that the T24N mutant protein inhibits entry into mitosis is that wild-type TC4 is normally part of a positive signaling system which promotes entry into mitosis when certain premitotic processes are complete (Fig. 8, Model 1). According to this model, completion of some premitotic process(es) would promote exchange of GDP for GTP on TC4 by the RCC1 protein. The GTP-bound form of TC4 would then promote cdc2 activation. While it is possible that the functions of TC4 in maintaining proper nuclear structure and in regulating the cell cycle are unrelated, it is tempting to speculate that nuclear structural rearrangements which are prerequisites for entry into mitosis (such as adequate nuclear growth, completion of DNA replication or rearrangements of chromatin structure) are monitored by RCC1/TC4. Because RCC1 is a highly abundant DNA-binding protein, the protein may play a fundamental role in maintaining chromatin structure. A dual function for the RCC1/TC4 system in model, chromatin-bound RCC1, acting as a guanine nucleotidereleasing protein, catalyzes the exchange of bound GDP on TC4 for GTE The GTP-bound form of TC4 could then act to stimulate entry into mitosis by positively regulating the activity of cdc2-cyclin complexes, perhaps by inactivating the tyrosine and threonine kinases which negatively regulate cdc2. This pathway involved in the positive regulation of cdc2 activity would act in opposition to the negative regulatory pathway inhibiting cdc2 activation prior to completion of DNA replication. The nuclear function(s) that are monitored such that RCCI is stimulated to generate the GTP-bound form of TC4 and promote entry into mitosis are currently unknown. (Model 2) In this model, GDP-TC4 would be generated during replication and this GDP-TC4 could negatively regulate cdc2 activation, perhaps by stimulating the tyrosine and threonine kinases regulating cdc2.
both maintaining chromatin structure and in monitoring certain functions associated with this structural arrangement would be consistent with our observations that addition of the T24N mutant of TC4 inhibits both nuclear growth and progression into mitosis. Regardless of the precise premitotic cellular function being monitored by TC4, our observation that the T24N dominant negative mutant appears to be blocking entry into mitosis by maintaining cdc2 in an inactive state strongly suggests that the wild-type TC4 signaling system normally promotes mitosis by modulating the activities of the kinase/phosphatase system(s) which regulates cdc2.
A role for the RCC1/TC4 system as a positive regulator of mitosis, although consistent with our data, might at first appear to be in direct conflict with previously published observations describing the effects of RCC1 mutations on cell cycle progression. In particular, mutations which result in a quantitative degradation of RCC1 protein enter mitosis even though DNA replication has not been completed (Nishimoto et al., 1978). Importantly, this premature entry into mitosis is a slow process and requires ongoing protein synthesis. Based on the model presented above, we would expect that in the absence of RCC1 protein the positive TC4 signal would not be generated. As such, the model predicts that cells would remain in S phase rather than enter mitosis. However, it should be remembered that in the absence of RCC1, DNA replication also stops. Therefore, in the absence of RCC1, we would expect both the postulated TC4-dependent positive regulatory system, as well as the replication-dependent negative regulatory system, to be inactivated. Under these conditions regulation of ode2 kinase activity would depend on both the basal activities of the kinase/phosphatase system which regulate phosphorylation of cdc2 and the concentration of newly synthesized cyclin protein. At low levels of cdc2-cyclin complex the kinase/phosphatase system will keep cdc2 inactive. However, above a certain cdc2-cyclin concentration, the capacity of the unregulated kinase/phosphatase system to maintain all of the cdc2-cyclin substrate inactive will be exceeded and a small amount of active cdc2 will accumulate. Due to the ability of cdc2 to activate itself in an autocatalytic manner (Solomon et al., 1990), this small amount of active edc2 would rapidly activate the remaining inactive cdc2 and the ceils would enter mitosis spontaneously. The spontaneous oscillations between S phase and mitosis which occur even in the absence of nuclei during the early Xenopus embryonic ceil cycle is an example of such an unregulated cell cycle. Therefore, when potential effects on both positive and negative checkpoint systems are taken into consideration, the observed entry into mitosis of cells lacking RCC1 could be reconciled with a model in which TC4 acts as part of a system positively regulating entry into mitosis.