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Address correspondence to C. Liang, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. Tel.: 852-23587296. Fax.: 852-23581552. email: bccliang{at}ust.hk
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Abstract |
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Key Words: CDK; CDK inhibitor; p97; VCP; ubiquitinproteosome proteolysis
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Introduction |
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Ubiquitin-mediated proteolysis plays critical roles in the cell cycle by regulating cyclin-dependent kinase (CDK) activities through degradation of CDK activators or inhibitors, thus promoting cell cycle transitions. After target proteins are multi-ubiquitinated, they are transported to the 26S proteasome for degradation. Recently, the crucial role of a chaperone-like Cdc48Ufd1Npl4 complex has been identified in the recognition and transport of polyubiquitin-tagged proteins, bringing them to the 26S proteasome for degradation (Meyer et al., 2000, 2002; Dai and Li, 2001; Ye et al., 2001). These studies suggest that Cdc48p regulates many cellular processes by this mechanism.
Besides mitosis, the G1 phase of the cell cycle is also controlled in part by ubiquitin-mediated proteolysis. One of the critical control points in G1 phase is the Start in yeast, which is equivalent to the restriction point in mammalian cells. After yeast cells have passed through Start, they not only initiate DNA replication but also form buds and duplicate their spindle pole. Activation of Cdc28 protein kinase by G1-specific cyclins is necessary for all of these Start events. However, Cdc48p has not been implicated in G1 control.
The previous conditional cdc48 mutants are quite "leaky," as indicated by several cell doublings before eventual G2/M arrest at the restrictive temperatures (Moir et al., 1982; Frohlich et al., 1991), which has led to the idea that Cdc48p plays an essential role only during mitosis. By using a "tight" temperature-sensitive degron (td) mutant in CDC48, we found that Cdc48p is required for Start in G1 phase, as well as for mitosis. Furthermore, Cdc48p is essential for Start execution in both mitotic cell cycle and cell cycle reentry after mating pheromone removal; this function is achieved through degradation of the G1-CDK inhibitor Far1p.
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Results and discussion |
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The wild-type control and cdc48-td strains were first synchronized in G1 phase with the mating pheromone factor, and each culture was split into two halves: one half was left at the permissive temperature of 25°C as the control, while the other half was induced to degrade the Cdc48-td protein in cdc48-td cells at 37°C. Next, cells in the two cultures of each strain were released from
factor arrest into fresh medium at 25°C (Fig. 1 A) or 37°C (Fig. 1 B), respectively, and cell cycle progressions were monitored by flow cytometry and budding index counting at various time points after release. Wild-type cells at both 25°C and 37°C and cdc48-td cells at 25°C showed normal kinetics of bud formation and S phase entry and progression (Fig. 1). In contrast, most cdc48-td cells were unable to bud or enter S phase at 37°C (Fig. 1). Because bud formation and S phase entry are two independent cell cycle events that initiate simultaneously at the G1/S transition after cells have traversed Start, our data suggest that Cdc48p is required for the execution of Start.
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Because Cdc48p is known to be required for ubiquitinproteasome proteolysis pathways, we hypothesized that inhibition of Cdc28-Cln kinase activities in cdc48-td resulted from failure to degrade a Cdc28p-Cln inhibitor in cdc48-td cells. One plausible candidate is Far1p (factor arrest), which is required for G1 arrest through inhibition of CLN gene expression and of Cdc28-Cln kinase activities in response to factor (Chang and Herskowitz, 1990; McKinney et al., 1993; Peter et al., 1993; Valdivieso et al., 1993; Peter and Herskowitz, 1994). Far1p is degraded through the ubiquitinproteasome pathway to allow cell cycle reentry after
factor withdrawal (Henchoz et al., 1997; Blondel et al., 2000).
To test if the G1 arrest was attributable to failure to degrade Far1p in cdc48-td cells, we examined the levels of Far1p in factor arrest-and-release experiments with FAR1-Myc strains. As expected, Far1p accumulated in both wild-type and cdc48-td cells in the presence of
factor (Fig. 2 C, 0 min). Also as expected, the Far1p level decreased and ultimately disappeared after wild-type cells were released from
factor arrest (Fig. 2 C). However, Far1p was quite stable in cdc48-td cells after release from
factor arrest (Fig. 2 C). These results support our hypothesis that the inability to reenter the cell cycle resulted from failure of Far1p degradation in cdc48-td cells.
It has been shown that Cdc48p is responsible for bringing ubiquitinated proteins to the 26S proteosome for degradation, but it is not required for ubiquitination of protein substrates (Dai and Li, 2001; Hitchcock et al., 2001; Rape et al., 2001; Ye et al., 2001). To determine if the role of Cdc48p in Far1p degradation was also at a postubiquitination step, we used cdc48-td cells to check the ubiquitination status of Far1p in factor block-and-release experiments. Far1-Myc was immunoprecipitated from yeast cell extracts with an anti-Myc antibody, and precipitated proteins were immunoblotted with an antiubiquitin antiserum. Far1-Myc was found ubiquitinated in cdc48-td cells at 37°C, as evident by the presence of smears (Fig. 2 D). We confirmed that the signals attributed to Far1-Ub were indeed from Far1-Ub, as smear signals could be detected by antiubiquitin immunoblotting in anti-Myc immunoprecipites from the FAR1-Myc tagged, but not untagged, strains (Fig. 2 E). These results suggest that Cdc48p is not required for ubiquitination of Far1p, as in the case of other substrates whose degradation is mediated through Cdc48p.
We have established that cdc48-td cells are defective for Start due to failure of Far1p degradation. However, factor was used to synchronize cells in G1 in these experiments, as in previous Far1p studies. To determine if Cdc48p was also required for degradation of Far1p at Start in the mitotic cell cycle, we examined cell cycle arrest phenotypes of cdc48-td cells in experiments that did not use
factor for cell presynchronization.
Asynchronous wild-type and cdc48-td cells were shifted to 37°C after induction of UBR1 to degrade the Cdc48-td protein, or kept at 25°C as the control, and cell cycle distributions were examined by flow cytometry and bud index counting. Judging from the DNA contents, cdc48-td cells had both G1 and G2/M populations at both 25 and 37°C, as did the wild-type cells (Fig. 3 A). However, only cdc48-td cells at 37°C had most of their budded cells as large budded compared with a more or less equal mixture of small- and large-budded cells in wild-type cells at 25 and 37°C and cdc48-td cells at 25°C (Fig. 3 A). This suggests that some cdc48-td cells arrested in G1 as unbudded cells with 1C DNA, whereas others arrested in G2/M as large-budded cells with 2C DNA at 37°C. To formally rule out the possibility that cdc48-td cells might have no cell cycle arrest at 37°C, nocodazole or factor was added to yeast cell cultures just before they were shifted to 37°C (Fig. 3 A). As expected, wild-type cells at both temperatures, and cdc48-td cells at 25°C, were able to traverse G1 and arrest in G2/M in the nocodazole experiment, and to go through mitosis and arrest in G1 in the
factor experiment. In contrast, at 37°C in the presence of nocodazole or
factor, cdc48-td cells arrested in both G1 and G2/M; i.e., they could not traverse either G1 (in the nocodazole experiment) or mitosis (in the
factor experiment). Together, these results demonstrate that Cdc48p is required for cells to pass through G1, as well as mitosis, in the mitotic cell cycle.
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In addition to deletion of the FAR1 gene, elimination of the Far1p-mediated inhibition of Cdc28p-Cln kinase activities can be achieved by constitutive overexpression of CLN2 (Oehlen and Cross, 1994). By using a cdc48-td/GAL-CLN2 strain in which the chromosomal copy of CLN2 was placed under the control of a galactose inducible promoter, we tested whether GAL-CLN2 could also allow cdc48-td cells to traverse Start at 37°C. Unlike cdc48-td/CLN2 cells, which arrested in both G1 and G2/M at 37°C (Fig. 3, A and C), cdc48-td/GAL-CLN2 cells could bypass the G1 block and only arrested in G2/M at 37°C (Fig. 3 C). Together, the results from Fig. 3 strongly suggest that the essential function of Cdc48p in Start is achieved through degradation of Far1p, leading to activation of CLN expression and of the Cdc28p-Cln kinase.
To determine if the action of Cdc48p in Far1p degradation was through binding to Far1p, we performed reciprocal coimmunoprecipitation (coIP) experiments between Cdc48p and Far1p. Cells were first blocked in G1 with factor, and then released into fresh medium. Cells, harvested 720 min after
factor removal, were pooled for the coIP experiments. Because Far1p degradation occurs during this period, possible interactions between Cdc48p and Far1p are most likely to be detected. Both Far1-Myc and Cdc48p were detected in the anti-Myc immunoprecipitates from the FAR1-Myc, but not the untagged control strains (Fig. 4 A). In the reciprocal coIP, an anti-Cdc48 antiserum also precipitated both Cdc48p and Far1-Myc (Fig. 4 B). To confirm that the smear on the anti-Myc immunoblot after anti-Cdc48 immunoprecipitation (IP; Fig. 4 B, lane 3) represented ubiquitinated Far1-Myc, we performed a two-step IP experiment. First, yeast cell extracts were immunoprecipitated with anti-Cdc48. Next, the precipitated proteins were dissolved, denatured, and reprecipitated with an anti-Myc antibody. The precipitated proteins after the two-step IP were analyzed by immunoblotting using anti-Myc and antiubiquitin antibodies. Far1-Myc and its ubiquitinated forms were detected in the immunoprecipitates from the FAR1-Myc, but not the untagged control strains (Fig. 4 C). Moreover, ubiquitinated Far1p was enriched relative to un-ubiquitinated Far1p (Fig. 4 B, compare lane 3 with lane 1; and Fig. 4 C, compare lane 4 with lane 2), as has been shown for some other proteolysis substrates (Dai et al., 1998; Rape et al., 2001). These results suggest that Cdc48p participates in Far1p degradation by interacting with Far1p.
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The G2/M arrest phenotypes of the previous cdc48-1 mutant had led to the classification of CDC48 as a cell division cycle gene required for mitosis (Moir et al., 1982; Frohlich et al., 1991). We have established that Cdc48p is also essential for activation of the Cdc28-Cln kinase via degradation of Far1p in G1 phase, and that degradation of Far1p is required for Start traverse in the normal mitotic cell cycle, as well as for cell cycle reentry after factor treatment and subsequent release. Our findings demonstrate new functions of Cdc48p; i.e., to be a critical G1 regulator through degradation of Far1p, which is the first CDK inhibitor discovered as a substrate of Cdc48p-mediated proteolysis. It will be of interest and significance to examine if some other CDK inhibitors are also substrates in Cdc48p/p97-mediated proteolysis pathways in yeast and other eukaryotes.
The functions of Far1p have previously been defined only in the context of response to mating pheromone. However, the expression of FAR1 is cell cycle regulated, with a peak at the M/G1 transition (McKinney et al., 1993), suggesting that Far1p may play a role in the normal cell cycle. Consistent with this, Far1p was found in a complex with Cdc28p-Cln in cells not exposed to pheromone, although the amount of Far1p bound to Cdc28p-Cln increased significantly when cells were treated with factor (Peter et al., 1993; Gartner et al., 1998). Now, we conclude that efficient turnover of Far1p is necessary for cell cycle reentry after
factor withdrawal and for normal cell cycle progression, suggesting that Far1p may play a role in the mitotic cell cycle.
In the presence of factor, phosphorylation of Far1p by the pheromone-induced MAP kinase Fus3p inhibits degradation of Far1p, leading to Far1p accumulation and G1 arrest (Peter et al., 1993). We show that if Cdc48p is absent, as in cdc48-td cells at 37°C, accumulation of Far1p, even in the absence of
factor, can result in cell cycle arrest in G1. Therefore, Cdc48p-mediated Far1p degradation is essential for the mitotic cell cycle. Our findings raise the intriguing possibility that Far1p may play an important role in the normal cell cycle; perhaps it ensures genomic stability, a function attributed to the Cdc28p-Clb inhibitor Sic1p (Lengronne and Schwob, 2002) and mammalian CDK inhibitors, such as p21 and p27, whose loss of function can lead to genomic instability and cancer. In such a hypothesis (Fig. 4 E), we propose that yeast cells use Far1p to inhibit the expression of CLN genes and Cdc28p/Cln kinase activities in early G1, until the cells are well prepared to commit to another cell cycle. Far1p is degraded through the ubiquitinCdc48pproteosome proteolysis pathway, and the cells traverse Start, leading to the G1 to S transition.
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Materials and methods |
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Sequences of the primers
Primers used for strain construction were as follows: OL164, 5'-agcagtgcctcatctttaatttcttttggtatgggcaatacccaagtaatacggatccccgggttaattaa-3'; OL165, 5'-ttttggtacgtttggcaaattggcattcatttatcatgaaaagaacaggaagaattcgagctcgtttaaac-3';OL172, 5'-gtctatagatccactggaaagcttcgtgggcgtaagaaggcaatctattacggatccccgggttaattaa-3'; OL173, 5'-aaaggaaaagcaaaagcctcgaaatacgggcctcgattcccgaactagaattcgagctcgtttaaac-3'; OL174, 5'-tggtaaagcagcaaagaattcatcaga-ccctgaagttcccaacctccggatccccgggttaattaa-3'; OL190, 5'-ctctatagctgccaa-ttcattcgcttaccacatcataatttgcatacagaattcgagctcgtttaaac-3'; and OL191, 5'-gatgacgagtcccatacggggtcttggttcagcactagcagccattgtgcactgagcagcgtaatctg-3'.
Cdc28 kinase assay
Soluble cell extracts were prepared by bead beating with lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaF, 5 mM EDTA, 0.1% NP-40, 250 mM NaCl, and 1x "Complete Protease Inhibitors" added just before use; Roche). Each extract (200 µg of total proteins) was added to 15 µl p13Suc1-agarose beads (Upstate Biotechnology) and incubated at 4°C for 2 h to precipitate Cdc28p. The beads were washed four times with lysis buffer. Proteins bound to half of the beads were used in immunoblotting to check the precipitation efficiency of Cdc28p. The other half of the beads was washed twice with H1 kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM DTT). Some 10 µl of kinase assay mixture (10 µCi -[32P] ATP, 2 µg histone H1, and 100 µM ATP in 1x kinase buffer) was added to the beads, and the mixture was incubated at 25°C for 10 min. The reaction was stopped by adding 10 µl of 2x Laemmli's buffer followed by boiling. Samples were resolved by 10% SDS-PAGE gel and subjected to autoradiography.
Two-step IP
Immunoprecipitates of the first (anti-Cdc48; a gift from K. Frohlich, Physiologisch-chemisches Institut, Tubingen, Germany) IP were boiled in 50 µl of lysis/IP buffer (Zhang et al., 2002) containing 1% SDS for 10 min. Next, solublized proteins were diluted with 450 µl of lysis/IP buffer and subjected to the second (anti-Myc) IP.
Cell synchronization, flow cytometry, immunoprecipitation, and immunoblotting
These experiments were performed as described previously (Liang and Stillman, 1997; Zhang et al., 2002).
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Acknowledgments |
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This work was supported by the Hong Kong Research Grants Council (HKUST6193/99M and 6203/00M to C. Liang).
Submitted: 10 July 2003
Accepted: 3 September 2003
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References |
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