Regulation of Nuclear Transport and Degradation of the Xenopus Cyclin-dependent Kinase Inhibitor, p27Xic1*

Li-Chiou Chuang and P. Renee YewDagger

From the University of Texas Health Science Center at San Antonio, Department of Molecular Medicine, Institute of Biotechnology, San Antonio, Texas 78245-3207

Received for publication, September 29, 2000, and in revised form, October 19, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27Xic1 (Xic1) is a member of the p21Cip1/p27Kip1/p57Kip2 CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The progression of the vertebrate cell cycle is positively regulated by cyclin-dependent kinases (CDKs)1 associated with their cyclin partners and is negatively regulated by cyclin-dependent kinase inhibitors (CKIs) (reviewed in Refs. 1-4). The mammalian cell cycle transition between G1 and S phases requires the activity of CDK2-cyclin E (5-7). The function of this kinase is negatively modulated by the CDK2 inhibitor, p27Kip1 in quiescent cells and in G1 phase cells (2, 8-12). Entry into S phase is accompanied by the ubiquitin-dependent proteolysis of p27Kip1 by a protein complex consisting of Cdc34 and SCFp45Skp2 (13-21). The ubiquitin conjugating enzyme Cdc34 (UBC3) was first identified in budding yeast as a protein essential for the G1 to S phase transition (22, 23). The SCF (Skp1, cullin, F-box) complex is comprised of the F-box binding protein, p19-Skp1; the cullin family member protein, Cul1; the p27Kip1 F-box protein, p45Skp2; and the ring finger protein, Rbx1/Roc1/Hrt1 (reviewed in Refs. 24-27). p27Kip1 must be phosphorylated on threonine 187 by CDK2-cyclin E before binding to the F-box protein p45Skp2 (15, 20). This binding targets p27Kip1 to the Cdc34-SCF complex for polyubiquitination followed by subsequent degradation by the 26S proteasome (15, 20). p27Kip1 can also be degraded upon the ectopic overexpression of the c-Jun and Jun D coactivator protein, Jab1 (28, 29). This mode of p27Kip1 degradation is mediated by the proteasome-dependent export of p27Kip1 from the nucleus and subsequent ubiquitination and degradation in the cytoplasm (28).

In the frog, Xenopus laevis, two CDK2 inhibitors of the Cip/Kip family have been identified that exhibit homology to mammalian p27Kip1, p57Kip2, and p21Cip1. Xenopus inhibitor of CDK (p27Xic1 or Xic1) and kinase inhibitor from Xenopus (p28Kix1 or Kix1) share ~90% amino acid sequence identity with each other and preferentially inhibit the activity of CDK2-cyclin E (30, 31). Xic1 and Kix1 also inhibit chromosomal nuclear DNA synthesis in interphase egg extracts and bind both CDK-cyclin complexes and proliferating cell nuclear antigen (PCNA) (30, 31).

Xic1 is degraded in Xenopus interphase egg extracts in a manner dependent on Cdc34, polyubiquitination, and the proteasome (32, 33). Furthermore, the Cdc34-dependent degradation of Xic1 is only observed in the presence of nuclei and increasing amounts of nuclei result in progressively shorter half-lives of Xic1 (32). The requirement for nuclei in ubiquitin-dependent degradation of Xic1 is intriguing, because mammalian p27Kip1 is readily ubiquitinated and degraded in whole cell extracts without the need for intact nuclei, whereas p21Cip1 is degraded in nuclei by the proteasome but without a need for ubiquitination (15, 19, 20, 34).

To better define the requirement for nuclei in Xic1 degradation, we use Xenopus interphase extracts and transport-competent nuclei to demonstrate that Xic1 ubiquitination and degradation require transport of Xic1 into the nucleus. We also identify the sequences of Xic1 that are required for efficient nuclear transport and show that nuclear localization is mediated by CDK2-cyclin E binding to Xic1 and Xic1 nuclear localization sequences (NLSs). We also show that binding to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation in the nucleus. Finally, we demonstrate that the basic residues spanning amino acids 180-183 are critical for Xic1 degradation and ubiquitination. We propose that this region of Xic1 is essential for either binding the ubiquitin conjugation machinery or interacting with an alternate as yet unidentified regulatory protein.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Xenopus Interphase Egg Extract, Xenopus Demembranated Sperm Chromatin, His6-Ubiquitin, and Methyl-ubiquitin-- X. laevis egg interphase extract (low speed supernatant (LSS)) and demembranated sperm chromatin were prepared as described previously (32, 35, 36). Mouse ubiquitin was cloned into pQE30 (Qiagen) from a 9.5/10.5 days postcoital mouse embryo library with an N-terminal tag of 6 histidine residues (His6-ubiquitin). Mouse ubiquitin protein was expressed in M15 bacterial cells and purified on nickel-nitrilotriacetic acid-agarose (Qiagen) under native conditions. Ubiquitin (Sigma) was methylated as described previously and used at a final concentration of 2 mg/ml (37).

DNA Constructs, Site-directed Mutagenesis, and DNA Sequencing-- Mutations of the Xic1 gene were generated by using the pCS2+/Xic1 plasmid and the QuikChange site-directed mutagenesis kit (Stratagene) (32). Xic1 point mutations were generated by making substitutions of the amino acids indicated: NLS1 = R166A, K167A, and R168A; NLS2 = K180A, K182A, and K183A; NLS3 = R206A, K207A, and K208A; c = R33A and L35A; k = F65A and F67A; ck- = R33A, L35A, F65A, and F67A. Following are the oligonucleotide primers (Integrated DNA Technologies) utilized to generate the specified mutations: NLS1, 5'-CTAACACATCTACACAGCGCGCGGCCGCGGAGATCACCACTCCCATC and 5'-GATGGGAGTGGTGATCTCCGCGGCCGCGCGCTGTGTAGATGTGTTAG; NLS2 (ARAA), 5'-CATCACCGATTATTTCCCTGCCCGGGCAGCGATACTGAGTGCCAAGCC and 5'-GGCTTGGCACTCAGTATCGCTGCCCGGGCAGGGAAATAATCGGTGATG; NLS2 (RRRR), 5'-CACCGATTATTTCCCTAGACGTCGACGGATACTGAGTGCCAAGC and 5'-GCTTGGCACTCAGTATCCGTCGACGTCTAGGGAAATAATCGGTG; NLS3, 5'-CTGGAACAGACCCCCGCGGCAGCGATTCGATGAAACC and 5'-GGTTTCATCGAATCGCTGCCGCGGGGGTCTGTTCCAG; c, 5'-GGAGGGGAGCCTGTGCGAATGCCTTCGGTCCTATCG and 5'-CGATAGGACCGAAGGCATTCGCACAGGCTCCCCTCC; and k, 5'-GTCAGAGGTGGAACGCTGACGCTGAAAGTGGCACCC and 5'-GGGTGCCACTTTCAGCGTCAGCGTTCCACCTCTGAC. All point mutations were confirmed by DNA sequencing using the Amplicycle Sequencing Kit (PerkinElmer Life Sciences). The entire coding sequence of the Xic1-NLS2 (ARAA) subclone was sequenced to confirm that it did not contain any other mutations.

In Vitro Transcription and Translation-- Wild type and mutant Xic1 were in vitro transcribed and translated from the SP6 promoter in pCS2+ using [35S]methionine (New Life Science Products) and the TNT-coupled reticulocyte lysate system (Promega). The amounts of protein for all Xic1 mutants were compared with the Xic1-wild type (WT) by SDS-polyacrylamide gel electrophoresis (PAGE) and PhosphorImager analysis. The amounts of protein for all Xic1 mutants were normalized and added at levels relatively equal to the Xic1-WT level.

Coimmunoprecipitation Assay-- LSS (20 µl) was mixed with 1 µl of [35S]methionine-labeled Xic1-WT and precleared with 7.5 µl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) for 30 min at 23 °C. After preclearing, the supernatant was incubated with anti-cyclin E, anti-CDK2, or normal rabbit serum for 30 min at 23 °C, and then 7.5 µl of protein A-Sepharose beads were added, and the incubation was continued for 1 h at 23 °C. The protein A-Sepharose beads were pelleted by centrifugation and washed four times with EB (80 mM beta -glycerol phosphate, pH 7.4, 20 mM EGTA, and 15 mM MgCl2) containing 0.1% Nonidet P-40, 10 µg/ml leupeptin, chymostatin, pepstatin, and 1 mM phenylmethylsulfonyl fluoride, followed by three washes with HBS (10 mM HEPES, pH 7.4, 150 mM NaCl) at 23 °C (31). The samples were then subjected to SDS-PAGE and quantitation by PhosphorImager analysis using ImageQuant software (Molecular Dynamics).

Nuclei Spin Down Assay-- Nuclei were allowed to form by incubating demembranated sperm chromatin (10 ng/µl) in 25 µl of LSS containing 0.1 µg/µl cycloheximide and an energy regenerating system (60 mM phosphocreatine, 1 mM ATP, 150 µg/ml creatine phosphokinase) at 23 °C for 1 h. Following nuclei formation, 0.5 µl/25 µl [35S]methionine-labeled WT-Xic1 was added for nuclear transport assays, whereas 2 µl/25 µl [35S]methionine-labeled GST-Xic1-WT was added for ubiquitination assays, followed by incubation at 23 °C for 45 min. For nuclear transport and certain ubiquitination experiments, methyl-ubiquitin was included at a final concentration of 2 mg/ml. The samples were then diluted 12-fold with NIB (50 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 0.5 mM spermine 4HCl, 0.15 mM spermidine 3HCl) and overlaid onto a 300-µl cushion of NIB containing 15% sucrose. The samples were then centrifuged in a Sorvall RC-5B at 5000 × g in a HB-6 rotor at 4 °C for 10 min. A 20-µl aliquot of supernatant was collected as the cytoplasmic fraction. The pelleted nuclei were washed and recentrifuged twice with 1 ml of NIB containing 250 mM sucrose at 3300 × g for 8 min at 4 °C. Samples were subjected to SDS-PAGE and quantitated by PhosphorImager analysis. The amounts of [35S]methionine-labeled mutant Xic1 proteins were quantitated by PhosphorImager analysis and added at a volume equal to the relative amount of radioactively labeled Xic1-WT or GST-Xic1-WT protein. In general, all [35S]methionine-labeled samples were used between final dilutions of 1:15 to 1:72. Chromatin spin down assays were conducted by including 0.1% Nonidet P-40 in NIB (38). The percentage of nuclear transport was calculated as the amount of Xic1 in the nucleus divided by the sum of the amount of Xic1 in the nucleus and the cytosol multiplied by 100%. All the samples were normalized to 100% of the WT Xic1 transported to the nucleus. For quantitation analyses, each sample was measured at least two or three times, and the standard error of the mean for each sample was calculated and displayed as error bars.

Degradation Assay and Dephosphorylation-- [35S]Methionine-labeled Xic1 (0.5 µl) was incubated in LSS (8 µl) containing cycloheximide, an energy regenerating system, and ubiquitin (1.25 mg/ml) with or without demembranated sperm chromatin (10 ng/µl) at 23 °C for 0, 1, and 3 h. Aliquots of 1.4 µl were removed from the samples for analysis by SDS-PAGE followed by quantitation by PhosphorImager analysis. The amounts of [35S]methionine-labeled mutant Xic1 proteins were added at a volume normalized to equal the same relative amount of radioactively labeled Xic1-WT protein. Dephosphorylation was performed using calf intestinal phosphatase as described previously (32).

DNA Replication Assay-- DNA replication assays were conducted as described previously with the following modifications (32, 36). [35S]Methionine-labeled Xic1 (0.625 µl) was added to LSS (10 µl) containing cycloheximide, an energy regenerating system, ubiquitin (1.25 mg/ml), and demembranated sperm chromatin (10 ng/µl). DNA replication was normalized to 100% of the control containing unprogrammed rabbit reticulocyte lysate. Each sample was measured three times, and the standard error of the mean for each sample was calculated and displayed as error bars.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ubiquitination and Degradation of Xic1 Occur in the Nucleus-- To begin to understand the requirement for nuclei in Xic1 degradation, we first determined whether Xic1 is transported into nuclei and where ubiquitinated Xic1 is localized. Xenopus interphase egg extracts derived from a low speed centrifugation of eggs (low speed supernatant or LSS) support the formation of transport-competent nuclei as well as a single round of initiation and elongation phases of semi-conservative chromosomal DNA replication (36, 39, 40). Demembranated sperm chromatin was preincubated with LSS, followed by the addition of [35S]methionine-labeled Xic1 and His6-ubiquitin and separation of the nuclear and cytosolic fractions by centrifugation. Our results show that polyubiquitinated Xic1 is found exclusively in the nuclear fraction, whereas Xic1 in the cytosolic fraction remains unmodified (Fig. 1A). We next asked whether Xic1 degradation also occurs in the nucleus. To do this, we incubated [35S]methionine-labeled Xic1 with demembranated sperm chromatin in LSS and then separated the nuclei and cytosol. The isolated nuclei and cytosol were added back separately to fresh LSS and incubated for 0 or 3 h. The sample containing nuclei in fresh LSS was again centrifuged to separate the nuclear and cytosolic fractions, and all the samples were analyzed by SDS-PAGE and PhosphorImager analysis to quantitate the amount of Xic1 remaining. The results show that the nuclear Xic1 pool is 90% degraded after 3 h (Fig. 1B, lanes 1, 2, 6, and 7), whereas Xic1 in the cytosolic fraction remains stable under these conditions (Fig. 1B, lanes 4, 5, 9, and 10). A slower migrating form of Xic1, which most likely represents a mono-ubiquitinated species, was observed in the nucleus (Fig. 1B, lanes 1 and 2). To eliminate the possibility that Xic1 must be exported to the cytoplasm to be degraded, we tested the degradation of Xic1 in the presence of leptomycin B, a specific inhibitor of CRM1/exportin 1 nuclear export (41-43). We did not observe any inhibitory effect of leptomycin B on the efficiency of Xic1 degradation (data not shown), an observation consistent with a recent study of Xic1 ubiquitination (33). Collectively, these results indicate that Xic1 is predominantly ubiquitinated and degraded in the nucleus.



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Fig. 1.   Xic1 is ubiquitinated and degraded in the nucleus. A, [35S]methionine-labeled Xic1 was incubated in LSS with demembranated sperm nuclei and then separated into nuclear (NUC) and cytosolic (CYT) fractions by centrifugation using a chromatin spin down assay. Results were resolved by SDS-PAGE and analyzed by PhosphorImager analysis. Molecular mass markers (lane M) are indicated in kDa. Arrows denote [35S]methionine-labeled Xic1 (XIC1), mono-ubiquitinated Xic1 (MONO-Ub), di-ubiquitinated Xic1 (DI-Ub), and polyubiquitinated Xic1 (POLY-Ub). B, left panel, [35S]methionine-labeled Xic1 was added to LSS and demembranated sperm chromatin, the nuclei and cytosol were separated by nuclei spin down assay (SPIN), and the nuclei (NUC) and cytosol (CYT) were re-incubated in fresh LSS for 0 or 3 h. At 0 or 3 h, the samples were further separated into nuclear (NUCLEI, lanes 1 and 2) and cytosolic (CYT, lane 3) fractions. The samples were subjected to SDS-PAGE and autoradiography. The Xic1 band is indicated by *, and the mono-ubiquitinated Xic1 is represented by **. Right panel, quantitation of results by PhosphorImager analysis where the 0 h time point is normalized to 100% of the Xic1 remaining at the 0 h time point. Black bars represent the nuclear fraction (NUC), NUC CYT represents the cytosol of the second nuclei incubation (lane 8), and gray bars represent the cytosolic fraction (CYT, lanes 9 and 10).

The Xic1 Primary Sequence Contains Two Potential Nuclear Transportation Regulatory Domains-- Because Xic1 appears to be ubiquitinated and degraded in the nucleus, it is important to identify the sequences of Xic1 that regulate its nuclear localization. The Xic1 primary sequence contains two regions that are likely to contribute to nuclear localization and/or nuclear retention. These regions are comprised of the CDK-cyclin binding region within the N terminus and several putative NLSs within the C terminus of Xic1 (30). The residues of mammalian p27Kip1 critical for CDK-cyclin binding have been identified by mutagenesis and by examination of the co-crystal structure of human CDK2 and cyclin A with a fragment of p27Kip1 (14, 16, 44). Because Xic1 exhibits significant sequence homology with human p27Kip1 in the CDK-cyclin binding domain, we identified and mutated residues in Xic1 that we predicted would disrupt binding of Xic1 to CDK2 (Xic1k: F65A and F67A) and to cyclin E (Xic1c: R33A and L35A) (Fig. 2A). These Xic1 mutants were in vitro translated with [35S]methionine, added to LSS, and immunoprecipitated with either CDK2 or cyclin E antibodies. The results indicate that both the WT Xic1 and Xic1c mutant readily form trimeric complexes with endogenous CDK2-cyclin E (Fig. 2B, lanes 1-4), whereas the Xic1k mutant is decreased in its ability to form a trimeric complex with CDK2-cyclin E (Fig. 2B, lanes 1-4). Interestingly, Xic1k protein bands in association with endogenous CDK2-cyclin E were shifted to slower mobility species that could be converted to a faster migrating species of Xic1 by treatment with calf intestinal phosphatase (Fig. 2B, lanes 5-7). These species may represent Xic1k phosphorylated by a catalytic CDK2-cyclin E that is only loosely associated with the Xic1k mutant or by an alternate kinase present in interphase extracts. When both c and k mutations are combined in Xic1ck- (R33A, L35A, F65A, and F67A), all detectable binding to CDK2 and cyclin E is abolished (Fig. 2B, lanes 1-4). However, it remains possible that a small amount of the Xic1ck- mutant remains very loosely bound to CDK2-cyclin E in the extract and is not stable to coimmunoprecipitation.



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Fig. 2.   Xic1 contains two potential nuclear transportation regulatory domains. A, Top panel, schematic of Xic1 showing the CDK binding region (CDK BINDING) and putative NLS sites (NLS). Bottom panel, schematic of Xic1 indicating the sites of point mutation. The numbers designate specific amino acid residues of Xic1 (). The Xic1 mutations are as follows: c, 33RNL to ANA; k, 65FDF to ADA; ck-, 33RNL to ANA and 65FDF to ADA; NLS1, 165RRKR to RAAA; NLS2, 180KRKK to ARAA or RRRR; NLS3, 205PRKK to PAAA. B, [35S]methionine-labeled Xic1-WT and mutants were added to LSS and immunoprecipitated with normal rabbit serum (NRS), anti-CDK2 antibody (alpha -CDK2), or anti-cyclin E antibody (alpha -CycE). One-tenth of the total reaction is shown for each sample in lanes 1 and 8 (10% INPUT). [35S]Methionine-labeled Xic1k was incubated in LSS (+LSS) without (-) or with (+) calf intestinal phosphatase (lanes 5-7).

There are three regions in the C terminus of Xic1 that are rich in arginine and lysine residues (165RRKR, 180KRKK, and 205PRKK), indicating they may be important for nuclear localization of Xic1 through an importin pathway (45). To address how these sequences influence the nuclear localization of Xic1, the residues in each of these three putative NLSs were mutated, resulting in the following mutants: Xic1-NLS1, R166A, K167A, and R168A; Xic1-NLS2 (ARAA), K180A, K182A, and K183A; Xic1-NLS2 (RRRR), K180R, K182R, and K183R; and Xic1-NLS3, R206A, K207A, and K208A (Fig. 2A). Xic1 mutants were also generated that combined all three individual NLS mutations (Xic1-NLS1/2/3) as well as both CDK2-cyclin E binding mutations and all NLS mutations (Xic1ck-&NLS1/2/3). These mutants were tested for their ability to bind to endogenous CDK2 and cyclin E in LSS. The results show that Xic1-NLS2 (ARAA) and Xic1-NLS1/2/3 both bind endogenous CDK2 and cyclin E efficiently, whereas, as expected, the Xic1-ck-&NLS1/2/3 mutant is totally defective for CDK2-cyclin E binding (Fig. 2B, lanes 8-11).

Nuclear Localization of Xic1 Is Mediated by CDK2-Cyclin E Binding and C-terminal NLS Sites-- To examine how either CDK2-cyclin E binding to Xic1 or the putative NLSs contribute to the nuclear localization of Xic1, we tested the Xic1 mutants described in Fig. 2 for their abilities to translocate to the nucleus. Xic1 WT and mutants were [35S]methionine-labeled and individually incubated in LSS containing preformed nuclei and supplemented with methyl-ubiquitin. The addition of methyl-ubiquitin allows mono-ubiquitination of lysine residues while preventing polyubiquitination (37). Because polyubiquitination is largely blocked, degradation by the proteasome is inhibited and mono-ubiquitinated substrate species accumulate. The nuclei were separated from the cytoplasm, and the amount of Xic1 localized to nuclei was determined as a percentage of the total Xic1 in the nucleus and cytosol. The Xic1-WT and mutants Xic1-NLS1, Xic1-NLS2 (ARAA), Xic1-NLS2 (RRRR), and Xic1-NLS3 all translocated efficiently to the nucleus (Fig. 3, lanes 2-6). This indicates that no single putative NLS site alone functions as a prototypic NLS. By contrast, both the Xic1-NLS1/2/3 and Xic1ck- mutants were reduced 2.5-3-fold in nuclear transport proficiency compared with WT Xic1 (Fig. 3, lanes 7 and 8). This suggests that the binding of Xic1 to CDK2-cyclin E and the NLS sequences of Xic1 independently contribute equally to efficient nuclear localization or retention. Importantly, when both the CDK2-cyclin E binding sites and NLS sites are mutated together, the ability of Xic1 to localize to the nucleus is severely reduced to near background levels as determined by the amount of the cytoplasmic protein GST found in the nuclear fraction (Fig. 3, lanes 1 and 9). These results suggest that the amount of Xic1 transported to or retained in the nucleus is regulated through the interactions of Xic1 with CDK2-cyclin E and importin.



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Fig. 3.   Nuclear localization of Xic1 is mediated by binding to CDK2-cyclin E and C-terminal NLSs. [35S]Methionine-labeled GST, Xic1-WT, and Xic1 mutants were added to LSS and preformed nuclei. After 45 min, the amount of protein transported to nuclei was analyzed by SDS-PAGE and quantitated by PhosphorImager analysis. The percent nuclear transport was calculated as the amount of protein in the nucleus divided by the sum of the total amount of protein in the nucleus and cytosol. The percentage of nuclear transport for the Xic1 WT was set at 100%, and the other samples were normalized relative to the WT. The bars represent the mean of two to four experiments with error bars representing the standard error of the mean.

A Stable Trimeric Complex between Xic1 and CDK2-Cyclin E Is Not Required for Xic1 Ubiquitination or Degradation-- Previous studies have shown that Xic1 degradation is sensitive to the purine analog 6-dimethylaminopurine, an inhibitor of CDKs and DNA replication initiation events (32, 46, 47). This suggests that Xic1 degradation is dependent on CDK activity or initiation events but does not address whether Xic1 must form a stable trimeric complex with CDK2-cyclin E for its ubiquitination and degradation. Studies show that mutations that disrupt the binding of mammalian p27Kip1 to CDK2-cyclin E eliminate ubiquitination of p27Kip1, indicating that a stable trimeric complex between CDK2-cyclin E-p27Kip1 is necessary for ubiquitination and degradation (14, 16). We studied the ubiquitination and degradation of Xic1 point mutants defective for binding to CDK2-cyclin E (Fig. 2). We used a GST-Xic1 fusion protein for our ubiquitination studies because it facilitated the detection of the ubiquitinated Xic1 species by allowing Xic1 to be labeled to a higher specific activity (Xic1 contains 2 methionines and GST-Xic1 contains 11 methionines). GST-Xic1-WT is localized to both the nucleus and cytosol but is predominantly ubiquitinated in the nucleus, whereas GST is localized predominantly to the cytosol and is not ubiquitinated (Fig. 4A). Equivalent amounts of [35S]methionine-labeled GST-Xic1-WT or GST-Xic1ck- were added to LSS and nuclei in the presence of methyl-ubiquitin to block polyubiquitination and subsequent degradation and to allow the accumulation of mono-ubiquitinated Xic1 species (37). In the presence of methyl-ubiquitin, both GST-Xic1-WT and GST-Xic1ck- efficiently accumulate mono-ubiquitinated Xic1 species in the nucleus (Fig. 4B). Based on the molecular mass of ubiquitin (~8 kDa) and the estimated molecular masses of the stabilized bands in Fig. 4B, ubiquitination appears to occur at ~3-5 independent sites on Xic1. The amount of ubiquitinated GST-Xic1ck- observed in the nucleus was reduced by ~2-3-fold compared with the GST-Xic1 WT, correlating with the ~2-3-fold reduction in nuclear transport proficiency observed for Xic1ck- compared with Xic1-WT (Fig. 3, lanes 2 and 8). Thus, the inability of Xic1ck- to bind CDK2-cyclin E reduces the efficiency of its nuclear transport by ~2-fold but does not prevent the efficient ubiquitination of Xic1ck- once it is localized to the nucleus.



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Fig. 4.   Xic1 binding to CDK2-cyclin E is not required for Xic1 ubiquitination or degradation. A, [35S]methionine-labeled GST and GST-Xic1-WT were incubated in LSS with preformed nuclei and methyl-ubiquitin, after which the nuclear (NUC) and cytoplasmic (CYT) fractions were separated by nuclei spin down assay and analyzed by PhosphorImager. B, [35S]methionine-labeled GST-Xic1-WT and GST-Xic1ck- were incubated in LSS with preformed nuclei and methyl-ubiquitin. The nuclear fractions were recovered by nuclei spin down assay and analyzed by PhosphorImager. Molecular mass markers are indicated in kDa (M). Arrows denote [35S]methionine-labeled GST-Xic1 (GST-Xic1) and GST-Xic1 mono-ubiquitinated at one site (1-MeUb), two different sites (2-MeUb), three different sites (3-MeUb), or four or five different sites (*). C, [35S]methionine-labeled Xic1 was incubated in LSS with (+) or without (-) nuclei for 0, 1, and 3 h and was resolved by SDS-PAGE. The results of two separate experiments were quantitated, and the mean percentage of Xic1 remaining is shown where the 0 h time point of Xic1 was normalized to 100% of Xic1 remaining for each sample.

To fully understand how Xic1 binding to CDK2-cyclin E modulates its turnover, the c, k, and ck- mutants were analyzed for degradation in LSS containing nuclei. Both the Xic1-WT and Xic1c, which form stable trimeric complexes with CDK2-cyclin E, are efficiently degraded (Fig. 4C, lanes 1-8). The Xic1k mutant, which was reduced for CDK2-cyclin E binding, is still degraded effectively (Fig. 4C, lanes 9-12). The Xic1ck- mutant, which was totally defective for CDK2-cyclin E binding, is also degraded with only slightly reduced efficiency (Fig. 4C, lanes 13-16). The slight reduction in degradation efficiency of Xic1ck- can be attributed to its reduced nuclear transport proficiency. Degradation of all the mutants required the presence of nuclei (Fig. 4C). These results suggest that binding of Xic1 to CDK2-cyclin E or the formation of a stable trimeric complex is dispensable for nuclei-dependent degradation of Xic1.

Nuclear Localization of Xic1 Is Necessary but Not Sufficient for Degradation-- We next asked how the C-terminal NLS sites contribute to the degradation of Xic1 by examining the turnover of the Xic1 NLS mutants. To accomplish this, Xic1 single, double, and triple NLS mutants were tested for Xic1 degradation, and the results are shown in Fig. 5A. The data from two separate experiments were averaged and are shown graphically in Fig. 5B. The results show that greater than 85% of Xic1 WT is degraded after 3 h (Fig. 5, A, lanes 1-3, and B). Xic1-NLS1, Xic1-NLS3, and Xic1-NLS1/3 are also degraded efficiently to levels comparable with the Xic1 WT (Fig. 5, A, lanes 4-6, 10-12, and 16-18, and B). Interestingly, Xic1-NLS2 (ARAA) exhibits a striking defect in degradation with only ~15% of the Xic1-NLS2 (ARAA) degraded after 3 h. This indicates that even though Xic1-NLS2 is transported to the nucleus as efficiently as WT Xic1 (Fig. 3), it is not appreciably degraded (Fig. 5, A, lanes 7-9, and B). As expected, the degradation of the Xic1ck-&NLS1/2/3 mutant that is not efficiently transported to the nucleus as well as other NLS mutant combinations containing NLS2 (ARAA) are not degraded (data not shown and Fig. 5, A, lanes 13-24, and B). The destruction of the degradable NLS mutants occurred in a nuclei-dependent manner (data not shown). These results indicate that Xic1 degradation involves at least two functional requirements, one being nuclear transport and the other involving at least one nuclear event that is defective in the Xic1-NLS2 mutant. Therefore, we conclude that nuclear transport is necessary but not sufficient for Xic1 degradation.



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Fig. 5.   Nuclear localization is necessary, but not sufficient for Xic1 degradation. A, [35S]methionine-labeled Xic1 was incubated in LSS with nuclei for 0, 1, and 3 h and was resolved by SDS-PAGE. B, the results of two separate experiments were quantitated by PhosphorImager analysis, and the mean percentage of Xic1 remaining is shown where the 0 h time point of Xic1 was normalized to 100% of Xic1 remaining for each sample.

Xic1-NLS2 Mutation Does Not Inhibit DNA Replication in the Xic1 Degradation Assay-- One trivial explanation for the stability of Xic1-NLS2 (ARAA) is that this nondegradable mutant inhibits the events of DNA replication initiation in the degradation assay, events that appear to be required for Xic1 degradation (32). To test this possibility, we measured DNA replication and Xic1 degradation in parallel, using the same preparation of LSS and the conditions we use for Xic1 degradation. Xic1-WT and Xic1-NLS2 (ARAA) were [35S]methionine-labeled and were added to Xic1 degradation and DNA replication assays. Under the degradation assay conditions, we found that the added amount of Xic1-WT and Xic1-NLS2 (ARAA) did not appreciably inhibit DNA replication compared with a control sample containing unprogrammed rabbit reticulocyte lysate (Fig. 6). In parallel degradation assays, Xic1-WT was degraded efficiently as expected, whereas Xic1-NLS2 (ARAA) was stable (data not shown). Therefore, the degradation defect of Xic1-NLS2 (ARAA) cannot be attributed to a block in DNA replication initiation by Xic1-NLS2 (ARAA) in the degradation assay.



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Fig. 6.   Xic1-NLS2 (ARAA) does not inhibit DNA replication under conditions used for Xic1 degradation. Semi-conservative DNA replication was measured in LSS using demembranated sperm chromatin as a template and [alpha -32P]dATP. The replication assay was conducted under degradation assay conditions with either unprogrammed rabbit reticulocyte lysate, [35S]methionine-labeled Xic1-WT, or Xic1-NLS2 (ARAA). The results are the mean of three experiments, and the error was calculated as the standard error of the mean. DNA replication for the control rabbit reticulocyte lysate sample was normalized to 100%.

Basic Lysine Residues within NLS2 Are Essential for Xic1 Degradation, but Do Not Comprise the Only Critical Ubiquitination Sites of Xic1-- It is possible that the region of Xic1-NLS2 (ARAA) may comprise the only critical lysine residues that are ubiquitinated in Xic1 or, alternatively, that this region of Xic1 is important for binding a cofactor protein required for its degradation. This putative cofactor might be a component of the ubiquitin conjugation machinery or a regulator required for post-translational modification of Xic1 prior to ubiquitination. To test these possibilities, we engineered an additional Xic1 mutant to carry conservative arginine mutations at the NLS2 residues (K180R, K182R, and K183R). This mutant would prevent ubiquitination at NLS2 residues but should not disrupt the interaction of Xic1 with a regulator that is dependent upon the basic residues of NLS2 (Fig. 2A) for binding. The Xic1-NLS2 (RRRR) mutant localizes to the nucleus as efficiently as Xic1-WT (Fig. 3, lane 5). Equivalent amounts of [35S]methionine-labeled GST-Xic1 proteins were incubated with LSS in the presence of nuclei and methyl-ubiquitin. The results show that GST-Xic1-WT, GST-Xic1-NLS1, GST-Xic1-NLS3, and GST-Xic1-NLS2 (RRRR) are all efficiently mono-ubiquitinated on ~3-5 independent sites in the nucleus (Fig. 7, lanes 1, 2, 4, and 5). In contrast, GST-Xic1-NLS2 (ARAA) and GST-Xic1-NLS1/2/3 are not appreciably ubiquitinated (Fig. 7, lanes 3 and 6). None of the GST-Xic1 derivatives was appreciably ubiquitinated in the cytosol (Fig. 7, lanes 7-12). There was little difference in the ubiquitination pattern of a specific NLS mutant when tested as a GST or a non-GST fusion protein (data not shown). These results indicate that although Xic1-NLS2 (ARAA) is not efficiently ubiquitinated, when the NLS2 lysine residues are converted to arginine residues, NLS2 (RRRR) is now efficiently ubiquitinated. NLS2 (RRRR), like NLS2 (ARAA) cannot be ubiquitinated at lysine residues 180, 182, or 183, indicating that NLS2 (RRRR) must be ubiquitinated at alternative lysine residues. Because NLS2 (RRRR) can be ubiquitinated, this demonstrates that lysines 180, 182, and 183 are not the only critical lysine residues that may be ubiquitinated in Xic1.



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Fig. 7.   Basic lysine residues 180, 182, and 183 of NLS2 are not the only critical ubiquitination sites of Xic1. [35S]Methionine-labeled GST-Xic1 proteins were incubated in LSS with nuclei and methyl-ubiquitin. The nuclear (NUCLEI) and cytoplasmic (CYTOSOL) fractions were then separated and analyzed by PhosphorImager. Molecular mass markers are indicated in kDa (M). Arrows denote [35S]methionine-labeled GST-Xic1 (GST-Xic1) and GST-Xic1 mono-ubiquitinated at one site (1-MeUb), two different sites (2-MeUb), three different sites (3-MeUb), or four or five different sites (*).

To confirm and extend the ubiquitination results in Fig. 7, the GST-Xic1 fusion proteins were tested for degradation. The results indicate that Xic1-WT and GST-Xic1-WT are efficiently degraded, although GST alone is not (Fig. 8A). GST-Xic1-NLS3, GST-Xic1-NLS1, and GST-Xic1-NLS2 (RRRR) are all degraded, whereas GST-Xic1-NLS2 (ARAA) and GST-Xic1-NLS1/2/3 are not degraded (Fig. 8A). Similarly, as non-GST fusion proteins, Xic1-NLS2 (RRRR) is degraded, whereas Xic1-NLS2 (ARAA) is not appreciably degraded (Fig. 8B). Taken together, these results suggest that the basic lysine residues of Xic1 amino acids 180-183 are essential for the efficient ubiquitination and degradation of Xic1. We postulate that this region of Xic1 is critical for binding a mediator of Xic1 degradation.



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Fig. 8.   A Xic1 mutant with conservative arginine substitutions at NLS2 (KRKK to RRRR) is degraded, while NLS2 (KRKK to ARAA) is not degraded. A, [35S]methionine-labeled proteins were added to LSS with (+) or without (-) nuclei and time points were analyzed for degradation after 0, 1, and 3 h. The arrow indicates the full-length product for GST-Xic1. The results from two separate experiments were quantitated, and the averages of the results are shown as the percentage of Xic1 remaining (% Xic1 Remaining) where the 0 h time point was normalized to 100% of the Xic1 remaining for each sample. B, [35S]methionine-labeled Xic1-WT and Xic1-NLS2 (RRRR or ARAA) mutants were added to LSS with (+) or without (-) nuclei and incubated for 0, 1, and 3 h. The results from two separate experiments were quantitated and the averages of the results are shown as the percentage of the Xic1 remaining (% Xic1 remaining). The 0 h time point was normalized to 100% of the Xic1 remaining for each sample.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of Xic1 Nuclear Transport and Additional Events Required for Xic1 Nuclear Degradation-- Our work indicates that Xic1 degradation is dependent upon its transport to the nucleus, a requirement mediated through CDK2-cyclin E binding and three C-terminal NLS sites of Xic1. It is unclear from our studies whether Xic1 binding to CDK2-cyclin E influences its active nuclear transport, nuclear retention, or both. Our results also suggest that the binding of Xic1 to CDK2-cyclin E and importin may be sufficient for its efficient nuclear localization. However, because we do not demonstrate a direct interaction between the C terminus of Xic1 and importin, it is possible that an alternative and as yet unidentified protein binds to the Xic1 C terminus and mediates its nuclear import. Although nuclear transport is necessary, it is not sufficient for Xic1 degradation. There is at least one other nuclear event required for Xic1 degradation that is defective in the Xic1 mutant NLS2 (K180A, K182A, K183A). The ubiquitination and degradation defects of Xic1-NLS2 (ARAA) are not attributable to defective nuclear import or defective CDK2-cyclin E binding. Conservative arginine substitutions at lysine residues 180, 182, and 183 do not prevent ubiquitination of Xic1, indicating that these residues do not comprise the only possible ubiquitination sites of Xic1. However, because the degradation of Xic1-NLS2 (RRRR) is reduced ~2-fold compared with the Xic1-WT, this may indicate that the lysine residues at amino acids 180-183 are important sites of Xic1 ubiquitination and that alternative lysine residues are not used quite as efficiently. Nonetheless, despite the ubiquitination of alternative sites in NLS2 (RRRR), these sites are not ubiquitinated in NLS2 (ARAA). This indicates that the basic residues of Xic1 (amino acids 180-183) more importantly define a region that is important for binding a protein required for efficient nuclear ubiquitination and degradation. This putative regulator is likely to be either a component of the ubiquitination machinery such as an F-box protein or alternatively, a regulator that must modify Xic1 prior to ubiquitination such as a kinase. Substrates of SCF are thought to require specific phosphorylation to bind their designated F-box proteins (reviewed in Ref. 48). The binding of Xic1 to an F-box protein is predicted because it has been shown that Xic1 is degraded in a Cdc34-dependent manner, implying that it will be degraded in a SCF- and F-box-dependent manner (32). However, to date, a requirement for SCF has not been demonstrated, nor has a Xic1 F-box protein been identified and shown to be required for Xic1 degradation. Xic1 has also been shown to bind to PCNA within unspecified C-terminal residues between Xic1 amino acids 97 to 210 (30). Based on weak homology to the PCNA binding regions of p21Cip1 and p57Kip2, we predict that Xic1 may bind to PCNA through amino acids flanking the NLS2 sequence (49, 50). How NLS2 may influence the binding of Xic1 to PCNA is currently under investigation.

Binding of Xic1 to CDK2-Cyclin E Is Dispensable for Nuclear Degradation-- We find that although Xic1 binding to CDK2-cyclin E is required for efficient nuclear transport or nuclear retention of Xic1, it is not required for Xic1 degradation. The Xic1ck- mutant that retains no measurable binding to CDK2 or cyclin E, is still ubiquitinated and degraded efficiently in the nucleus. This finding also implies that the phosphorylation of Xic1 by CDK2-cyclin E may not be required for its nuclear degradation. Our results conflict somewhat with a recent study suggesting that an association with CDK2-cyclin E is required for Xic1 destruction (33). This study demonstrates that immunodepletion of cyclin E inhibits Xic1 destruction (33). Xic1 degradation has previously been shown to be sensitive to the CDK inhibitor, 6-dimethylaminopurine, which inhibits DNA replication initiation (32, 46, 47). In light of our current results, it is likely that Xic1 degradation is dependent on initiation events mediated by CDK2-cyclin E but is not directly dependent on CDK2-cyclin E binding or phosphorylation. In vivo, it is unclear whether Xic1 is a physiological substrate of CDK2-cyclin E or how phosphorylation of Xic1 by CDK2-cyclin E may regulate its function.

Proteolysis of Vertebrate CKIs: Similarities and Differences-- Although a stable trimeric complex of CDK2-cyclin E with mammalian p27Kip1 has been shown to be required for its ubiquitination and degradation, our results indicate that a stable complex of Xic1 with CDK2-cyclin E is not essential for the degradation of Xic1 (14, 16). Additionally, the degradation of p27Kip1 exhibits a strict dependence on CDK2-cyclin E phosphorylation of Thr187, whereas phosphorylation of Thr204 (the residue equivalent to Thr187 of p27Kip1) is not required for Xic1 degradation (14, 16, 20). Two individual mutations of Xic1 at this residue, T204A and T204E, are both degraded in a nuclei-dependent manner with the exact kinetics as the Xic1-WT.2 This is surprising because Thr187 of p27Kip1 and Thr204 of Xic1 are located within highly conserved C-terminal sequences termed the QT domain. Moreover, although both Xic1 and Kix1 are efficiently degraded in a nuclei-dependent manner in Xenopus interphase extracts, neither human nor mouse p27Kip1 are degraded in Xenopus extracts with or without nuclei.3 Considering the high homology of the vertebrate CKIs, why then does Xic1 appear to be degraded so differently compared with p27Kip1? The differences observed to date may be explained by the structural differences that exist between the vertebrate CKI molecules, their possible functional differences or experimental differences. It is likely that Xic1 possesses additional functions compared with p27Kip1 as suggested by the homology of the C-terminal Xic1 and p21Cip1 sequences, as well as the observed binding of Xic1 to PCNA (30, 31). The unique features of Xic1 may result in altered regulation by protein turnover for the Xenopus CKI compared with the mammalian CKI. Additionally, Xic1 may be degraded along more than one pathway as suggested by Swanson et al. (33), one dependent upon and one independent of phosphorylation by CDK2-cyclin E. These alternate degradation pathways may be dictated by the binding status of Xic1 to CDK2-cyclin E. p21Cip1 has been shown to be degraded by the proteasome only in the nucleus, suggesting that the degradation of Xic1 may resemble the degradation of p21Cip1, although p21Cip1 degradation does not require ubiquitination (34). With regard to the methodology used in mammalian versus Xenopus degradation experiments, one chief difference exists that may potentially account for some discrepancies observed between p27Kip1 and Xic1 degradation. The Xenopus extract measures S phase events that occur within early embryonic cell cycles, whereas mammalian extracts measure S phase events that occur largely in differentiated somatic cells. Further studies will be required to clarify the differences observed in vertebrate CKI degradation and to fully understand the mechanisms regulating the events of DNA replication initiation.


    ACKNOWLEDGEMENTS

We are grateful to M. W. Kirschner and T. G. Boyer for their continued support; T. G. Boyer, J. Roberts, and all past and present members of the laboratory for helpful discussions; K. Block for constructing pCS2+/GST and pCS2+/GST-Xic1-WT; P. K. Jackson and M. W. Kirschner for anti-CDK2 and anti-cyclin E antibodies; S. Hollenberg for the 9.5/10.5 days postcoital mouse embryo library; W. Dunphy for the Kix1 plasmid; J. Roberts and B. Amati for human and mouse p27Kip1 plasmids; F. Aparicio-Ting and C. Herrera for technical assistance; H. Yan, W. M. Michael, A. Philpott, T. Civco, and R. Davis for advice on sperm chromatin preparation and frog care; and T. G. Boyer, A. Tomkinson, and P. Sung for critical reading of the manuscript.


    FOOTNOTES

* This work was supported in part by a Leukemia and Lymphoma Society Special Fellowship, a Howard Hughes Medical Institute New Faculty Start Up Award, an UTHSCSA Institutional Research Grant, and an award from the UTHSCSA Competitive Research Enhancement Fund for New Faculty (to P. R. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. E-mail: yew@uthscsa.edu.

Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008896200

2 K. Block and P. R. Yew, unpublished result.

3 P. R. Yew and M. W. Kirschner, unpublished results.


    ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; LSS, low speed supernatant; NLS, nuclear localization sequence; PAGE, polyacrylamide gel electrophoresis; WT, wild type; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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