Cloning and functional expression of a ... - Semantic Scholar

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Biochem. J. (2001) 353, 51–57 (Printed in Great Britain)

Cloning and functional expression of a degradation-resistant novel isoform of p27Kip1 Katsuya HIRANO*, Mayumi HIRANO*, Ying ZENG*, Junji NISHIMURA*, Keiichi HARA†, Koichiro MUTA†, Hajime NAWATA† and Hideo KANAIDE*1 *Department of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and †3rd Department of Internal Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

p27Kip" is an inhibitor of cyclin-dependent kinases. It has been implicated as having a role in the induction of growth arrest at the G phase of the cell cycle in response to anti-mitogenic signals " such as cell contact and serum starvation. Proteasome-mediated degradation plays an important role in the rapid inactivation of p27Kip", causing quiescent cells to re-enter the cell cycle. Although the existence of a second isoform has been suggested, no such isoform was isolated. Through screening of a cDNA library derived from growth-arrested confluent porcine endothelial cells, we obtained clones for a novel isoform of p27Kip" in addition to the original isoform. The novel isoform differed from the original isoform at the C-terminus. The tissue-specific expression of the original and novel isoforms was demonstrated at the mRNA and protein levels. An in Šitro degradation assay demonstrated this

novel isoform to be resistant to proteasome-mediated destruction. The expression as a fusion protein with green fluorescent protein revealed this isoform to be targeted to the nucleus by a bipartite nuclear-localization signal with a C-terminal part different from that of the original isoform. The expression of the novel isoform caused the growth arrest of HeLa cells and an accumulation of cells in the G \G phase, and this effect was similar to that seen ! " with the original isoform. The present study suggests that the novel isoform functions as a negative regulator of the cell cycle, and may play a distinct role. The novel isoform was named p27Kip"R because of its resistance to degradation.

INTRODUCTION

quiescent cultured endothelial cells from porcine aorta. In addition to the original isoform, clones encoding a novel isoform of p27Kip" were obtained. It has a unique C-terminus which lacks a part of the nuclear-localization signal (NLS) and the CDKphosphorylation site seen in the original isoform. We confirmed the expression of mRNA for the novel isoform in porcine tissues. Importantly, the novel isoform was found to be resistant to proteasome-mediated degradation. We also examined the effect of the novel isoform by expressing it as a fusion protein with green fluorescent protein (GFP).

p27Kip" is an inhibitor of cyclin-dependent kinases (CDKs) [1,2]. It was suggested that p27Kip" induces growth arrest at the G " phase of the cell cycle by inhibiting the G cyclin–CDK complexes " [3,4]. The expression of p27Kip" is thus regulated during cell-cycle progression, at the transcriptional, post-transcriptional or posttranslational steps [5–7]. The ubiquitin-proteasome pathway plays an important role in the rapid degradation and resultant inactivation of p27Kip" during the G \S transition [6,8]. It was " shown that phosphorylation at threonine-187 by CDK is essential to initiate the degradation process, and the deletion of this phosphorylation site made p27Kip" resistant to degradation [8–10]. Since the tumour that developed in p27Kip"-deficient (j\k) heterozygous mice had no mutation of the wild-type allele, p27Kip" was reported to be a haplo-insufficient tumour suppressor, which is unusual for a tumour-suppressor gene [11]. The existence of other isoform(s) might therefore provide an explanation for the apparent haplo-insufficiency. Toyoshima and Hunter [2] noticed a second smaller band in addition to a 2.4 kb band in a Northern-blot analysis, which suggested the existence of a second isoform. However, no such isoform has been reported, although there have been several reports of N- or C-terminally truncated forms of p27Kip" [12,13]. We performed homologous screening for a possible isoform of p27Kip" by using a cDNA library derived from contact-induced

Key words : cyclin-dependent kinase inhibitor, endothelial cells, splice isoform.

EXPERIMENTAL Library screening The construction of a cDNA library of growth-arrested confluent porcine aortic endothelial cells in primary culture was described previously [14]. A probe for screening was obtained by amplifying a cDNA fragment encoding amino acids 1–173 of p27Kip" [1,2] from total RNA of cultured porcine aortic endothelial cells with reverse transcriptase (RT)-PCR. The total RNA was isolated as described previously [15]. Library screening was performed using the standard plaque-hybridization method [16]. Nucleotide sequences were determined by the dideoxy chain-termination method [17] on an ABI Prism 310 Genetic Analyser (Perkin-Elmer, Foster City, CA, U.S.A.).

Abbreviations used : CDK, cyclin-dependent kinase ; RT, reverse transcriptase ; GFP, green fluorescent protein ; p-APMSF, 4-aminidophenylmethanesulphonyl fluoride ; NLS, nuclear-localization signal. 1 To whom correspondence should be addressed (e-mail kanaide!molcar.med.kyushu-u.ac.jp). The nucleotide sequence data reported have been submitted to the DDBJ/EMBL/GenBank2/GSDB Nucleotide Sequence Databases under accession numbers AB031955 (clone K1), AB031956 (K2), AB031957 (K3) and AB031958 (K4). # 2001 Biochemical Society

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Plasmid constructs All cDNA fragments for the inserts were obtained by PCR amplification of clones K1 or K4 (see Figure 1). The nucleotide sequences of the constructs were determined to have no unintended mutations. pQE32 (Qiagen, Hilden, Germany) was used for the expression of the (His) -tagged proteins. pEGFP-N1 ' (Clontech, Palo Alto, CA, U.S.A.) was used for the expression of GFP fusion proteins in mammalian cells.

Immunoblot analyses Adult male Wistar rats were sacrificed according to the guidelines for animal experiments in the Faculty of Medicine, Kyushu University, Fukuoka, Japan. The tissue specimens, as indicated in Figure 2, were isolated immediately and homogenized by a Polytron homogenizer in 50 mM Hepes\150 mM NaCl\1 mM EDTA\0.5 % Nonidet P-40\0.5 mM sodium orthovanadate\ 1 mM dithiothreitol\1 µM 4-aminidophenylmethanesulphonyl fluoride (p-APMSF)\10 µg\ml leupeptin. After 15 min on ice, the homogenates were clarified by centrifugation at 10 000 g, 4 mC, for 15 min. The endothelium of porcine aorta (obtained at a local slaughterhouse) was scraped off with a scalpel blade and homogenized as described above for rat tissues. Protein concentrations were determined by the Bradford method [18] with BSA as the standard. Total proteins (100 µg) of the homogenates were subjected to SDS\PAGE (18 % gel) [19] in duplicate, and transferred on to PVDF membranes (Bio-Rad, Hercules, CA, U.S.A.) [20]. One membrane was treated with a monoclonal anti-p27Kip" antibody (TDL ; Transduction Laboratories, Lexington, KY, U.S.A.), whereas the other membrane was treated with a polyclonal anti-p27Kip" antibody (C-19 ; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The primary antibodies were detected using appropriate secondary antibodies conjugated with horseradish peroxidase, followed by detection with enhanced chemiluminescence (Amersham, Piscataway, NJ, U.S.A.) on X-OMAT AR film (Kodak, Rochester, NY, U.S.A.).

amplification step (35 cycles for p27Kip" and p27Kip"R, and 30 cycles for β-actin) consisting of 1 min of denaturation at 94 mC, 1 min of annealing at 55 mC and 1 min of extension at 72 mC. The RT-PCR products were analysed by 3 % agarose gel electrophoresis, and visualized by ethidium bromide staining. The fluorescence intensity of the band was determined on NIH Image version 1.61 after obtaining the fluorescence image with a CCD camera (Atto, Tokyo, Japan).

In vitro degradation assay Endothelial cells were synchronized at the G \S boundary of the " cell cycle by harvesting cells at 15 h after seeding the growtharrested confluent endothelial cells at a 50 % confluent density. HeLa cells were synchronized at the S phase by harvesting cells 3 h after the release of the 24 h block with 2 mM hydroxyurea [9]. The hypotonic cell extracts were prepared as described in [9,22]. The (His) -tagged proteins were expressed and purified as ' substrates according to the manufacturer’s instructions (Qiagen). Assays were performed at 30 mC in the mixtures consisting of 10 µl of cell extract supplemented with 25 mM phosphocreatine, 10 µg\ml creatine kinase, 30 ng of substrate and 1 mM ATP, unless otherwise specified. The reaction was terminated by adding an equal volume of Laemmli sample buffer [19] and boiling for 1 min. The samples were then separated by SDS\ PAGE (15 % gel), and subjected to immunoblot detection with anti-p27Kip" (TDL) as described above under immunoblot analyses.

Expression of GFP fusion proteins in mammalian cells The cells were transfected with the expression plasmid by a 5 h incubation in serum-free medium containing 5–10 µg\ml plasmid DNA and 10–20 µg\ml AMINE (Life Technologies). After incubation in 10 % serum-containing growth medium for about 24 h, the fluorescence image was observed under a laser scanning microscope (LSM GB200, Olympus, Tokyo, Japan).

RT-PCR analysis of the expression of p27Kip1 and p27Kip1R in porcine tissues

Analysis of cell growth

Porcine heart (ventricular muscle), liver and aorta were obtained fresh at a local slaughterhouse, and transported to the laboratory in ice-cold PBS (136.9 mM NaCl\2.7 mM KCl\8.1 mM Na HPO \1.47 mM K HPO , pH 7.4). The total RNA was # % # % isolated as described in [15]. Total RNA (2 µg) was subjected to RT reaction with Moloney murine leukaemia virus RT (Life Technologies, Rockville, MD, U.S.A.) in a 20 µl reaction mixture as described previously [21]. The RT primer (5h-ACT GAT GAA TAG TTT AAC TG-3h) for p27 is located in the coding region of segment IV (Figure 1). The RT reaction mixture also contained an RT primer for porcine β-actin [21]. The RT product (2 µl) was subjected to three separate PCRs in 10 µl of reaction mixture. One of the PCR reactions was to amplify the coding region of p27Kip", and another reaction was to amplify the coding region of p27Kip"R. These two PCR reactions used a common upper (sense) primer (5h-GGC AGG AAT TCG AAA AGG GCA GCT TGC3h), in combination with a specific lower (antisense) primer (5h-ATT TTC TTC TGT TCT GTT GG-3h for p27Kip" ; 5h-TAC ATT CCA ATT TTA AGG GA-3h for p27Kip"R). The upper primer is located in the coding region of segment I (Figure 1). The lower primers are located in the coding regions of segments II (for p27Kip") and IV (for p27Kip"R ; Figure 1), respectively. The other PCR was to detect β-actin as a control [21]. Each PCR reaction involved initial denaturation at 94 mC for 2 min and a subsequent

HeLa cells (1.0i10& cells\60 mm-diameter dish) were cultured overnight and then transfected by a 5 h incubation in serum-free medium containing 1 µg\ml plasmid DNA and 10 µg\ml AMINE. The transfection mixture then was removed and the cells were cultured in 10 % serum-containing growth medium. At each time point (1, 2, 3 and 5 days after removing the transfection mixture), the cells were harvested by treatment with trypsin in PBS containing 0.2 % (w\v) EDTA. The total cell number was counted on a haemocytometer, and the transfection rate (the proportion of GFP fluorescence-positive cells as a percentage of the total cells) was determined with a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA, U.S.A.). According to this transfection rate, the numbers of the GFP fluorescence-positive and -negative cells were estimated from the total cell number. To analyse the effect of p27Kip" and p27Kip"R on cell-cycle progression, HeLa cells (2i10' cells\100 mm-diameter dish) were cultured for 2 days, and then were transfected as described above. The GFP fluorescence-positive and -negative cells were separated with a cell sorter (Epics Elite, Beckman Coulter, Fullerton, CA, U.S.A.) and the phase of the cell cycle was analysed with the flow cytometer after staining with propidium iodide (Sigma). The fraction of each phase of the cell cycle was analysed using the ModFit LT program, version 2.0 (Verity Software House, Topsham, ME, U.S.A.).

# 2001 Biochemical Society

A degradation-resistant isoform of p27Kip1

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Analysis of apoptosis of cells expressing p27Kip1 and p27Kip1R Possible apoptosis of the transfected cells was investigated on day 2 after transfection, as described in [23]. In brief, the cells were harvested by trypsin treatment and then examined for the transfection rate with a flow-cytometric analysis of GFP fluorescence, as described above. The cells were then fixed in 70 % ethanol at k20 mC for 24 h, extracted in 0.2 M Na HPO \0.1 M citric acid, pH 7.8, for 30 min at room tem# % perature and stained with propidium iodide. The cells were then subjected to a flow-cytometric analysis of propidium iodide fluorescence, and the fraction of the sub-G population was " determined as an indicator of apoptotic cells [23]. The basal apoptosis of non-transfected cells was determined in cells treated with 10 µg\ml AMINE alone. According to the basal rate of apoptosis in the non-transfected cells (A ), trans! fection rate (a) and the fraction of the sub-G population of " Kip "and p27Kip"Rthe total cells obtained in the GFP-, p27 transfection experiments (AT), and by assuming the rate of apoptosis in the untransfected cells obtained in the transfection experiments to be same as the basal rate of apoptosis, the rates of apoptosis in the cells expressing GFP, p27Kip" and p27Kip"R (A) were estimated using the following formula : A ( %) l A j[100i(ATkA )\a] ! !

Statistical analysis Unpaired Student’s t test was used to determine statistical significance. P 0.05 was considered to be significantly different.

RESULTS Cloning of a novel isoform of p27Kip1 We screened a cDNA library derived from contact-induced quiescent porcine aortic endothelial cells [14] for the porcine homologue and possible isoform of p27Kip". From 5i10& primary plaques, 13 positive clones were obtained. Analyses of the inserts with PCR and restriction digestion resulted in the categorization of the 13 clones into four groups. The clones K1 (four clones), K2 (two clones), K3 (one clone) and K4 (six clones) are representative for each group (Figure 1a). K1 (1958 bp) contained the sequences found in the other three clones, except for short 3h extensions seen in clones K3 and K4. Alignment of all clones divided the sequence of K1 into five segments (Figure 1a). K1, K2 and K3 encoded the porcine homologue of p27Kip", which was 91.9 % identical to its human counterpart [1]. The protein-coding region resides on segments I and II, divided at codon 163. This is consistent with the genomic structure determined in humans [24] and mouse [25], although their coding regions are divided at codon 159. On the other hand, K4 contained a 5h-terminusdeficient segment I connected directly to segment IV. Alignment of the nucleotide sequences suggested that K4 encodes the polypeptides identical to residues 9–162 of the K1-encoded p27Kip", followed by 18 unique residues (Figure 1b). Collectively, K4 was suggested to encode a splice isoform of p27Kip". The two isoforms become divergent at amino acid 163 and possess different C-termini (Figure 1b). The N-terminal region of the original isoform, which is necessary and sufficient to inhibit the cyclin-CDK activity [26,27], is conserved in K4 (Figure 1c). In the C-terminal region, the novel isoform lacked the two structural characteristics seen in the original isoform (Figure 1c) ; a major CDK-phosphorylation site (Thr-187) [1,8–10,28] and part of a putative bipartite NLS (residues 152–169 of the original isoform)

Figure 1 p27Kip1

Comparison between a novel isoform and the original isoform of

(a) Comparison of the four representative clones obtained from a cDNA library of porcine endothelial cells. The identical regions between the different clones were aligned. The nucleotide sequence of clone K1 is divided into five segments (I–V). Striped boxes, coding regions ; open boxes, non-coding regions ; >, poly(A)+ signal ; polyA, poly(A)+ tail. The nucleotide length (bp) is shown at the top. The nucleotide sequences of clones K1, K2, K3 and K4 have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB031955, AB031956, AB031957 and AB031958, respectively. (b) Comparison of the deduced amino acid sequences of clones K1 and K4. The amino acid numbering is based on the sequence of clone K1. (c) A schematic presentation of the protein structure of p27Kip1 and a comparison with its novel isoform, p27Kip1R. The structure of p27Kip1 is based on the references [26,37].

[1,2]. The most important implication of our results is that the novel isoform, by virtue of the C-terminal differences, may be resistant to proteasome-mediated degradation.

Tissue-specific expression of p27Kip1 isoforms We examined the expression of two isoforms of p27Kip" in rat tissues and porcine aortic endothelium by immunoblot analyses using two antibodies against different epitopes. The epitope for anti-p27Kip" (TDL) was located to a region around residue 60 [29]. Anti-p27Kip" (C-19) was raised against a synthetic peptide corresponding to the C-terminus of human p27Kip". # 2001 Biochemical Society

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Figure 2

K. Hirano and others

Tissue distribution of p27Kip1 and p27Kip1R

Immunoblot detection of p27Kip1 in various rat tissues (lanes 1–12) and porcine aortic endothelium (lane 13). (His)6-tagged recombinant proteins (10 ng) were loaded as a positive control (lanes 14–16). Two different antibodies were used as the primary antibody ; anti-p27Kip1 (TDL) and anti-p27Kip1 (C-19). p27Kip1, (His)6-tagged full-length p27Kip1 ; p27Kip1∆N, (His)6tagged p27Kip1 lacking the N-terminal eight residues ; p27Kip1R, (His)6-tagged p27Kip1R encoded by clone K4. Molecular masses are indicated on the left of the upper panel.

Anti-p27Kip" (TDL) detected all three recombinant proteins ; p27Kip", p27Kip"∆N (which was N-terminally truncated) and p27Kip"R, whereas anti-p27Kip" (C-19) failed to detect p27Kip"R (Figure 2). These observations confirmed the specificity of the antibodies. In rat tissue, anti-p27Kip" (TDL) detected two bands of approx. 28.5 and 25.3 kDa (Figure 2). The difference in mobility between these two bands was similar to that seen between p27Kip"∆N and p27Kip"R. Therefore, the difference in the two bands could be due to the difference in the C-terminus. This was supported by the immunoblot detection by anti-p27Kip" (C-19). The upper band was detected by anti-p27Kip" (C-19), thus suggesting that this band represented p27Kip". However, detection of the lower bands with anti-p27Kip" (C-19) was different. For most tissues, a lower band was not detected, except for cerebrum and cerebellum. In these latter tissues, the lower bands may be similar to the N-terminally truncated form of p27Kip", as reported previously [12]. The lower bands in other tissue specimens, i.e. those detected by anti-p27Kip" (TDL), were suggested to represent isoforms with a different C-terminal region, similar to the novel isoform encoded by K4. The lower band represented the major isoform in oesophagus, small intestine, liver, kidney, spleen and aorta in the rat (Figure 2). On the other hand, p27Kip" is the major isoform in the cerebrum, cerebellum and skeletal muscle. In the rat lung and heart, the expression of the two isoforms appeared to be equal. In porcine aortic endothelial cells in situ, p27Kip" was the major isoform, while the lower band was scarcely detected. The expression of mRNA for the novel isoform was confirmed in the porcine liver, heart and aortic endothelial cells by RT-PCR analysis (results not shown). The ratio of the novel to the original isoform was higher in liver (3.54p0.85) than in heart (0.52p0.19) or endothelium (0.3p0.02). The tissue-specific expression pattern according to the RT-PCR analysis thus correlated well with that obtained by the Western-blot analysis (Figure 2). # 2001 Biochemical Society

Figure 3

In vitro degradation assay of p27Kip1 and p27Kip1R

(a) The time courses of degradation in vitro of the (His)6-tagged recombinant proteins by the extracts of endothelial cells at the G1/S boundary. p27Kip1, p27Kip1∆N and p27Kip1R are the same as in Figure 2. p27Kip1T187A is p27Kip1 with the substitution of Thr-187 by Ala. Images shown are representative of three independent experiments. (b) The effects of protease inhibitors on the degradation of p27Kip1. p27Kip1 was incubated for 1 h with endothelial-cell extracts in the absence and presence of MG132, MG115, calpain inhibitor I, leupeptin or p-APMSF. Input indicates the sample obtained at time 0. 0 ATP, degradation with no supplementation of ATP. Adenosine 5h-[γ-thio]triphosphate (ATPγS) was added at the indicated concentrations in place of ATP.

Resistance to proteasome-mediated degradation To determine the functional significance related to the unique Cterminus of the novel isoform, we first performed an in Šitro degradation assay with the cell extracts obtained from endothelial cells synchronized at the G \S boundary. The cell extracts " degraded p27Kip" rapidly (Figure 3a). The proteolytic activity was inhibited completely by three proteasome inhibitors, MG132, MG115 and calpain inhibitor I, and also by leupeptin (Figure 3b). Serine protease inhibitor, p-APMSF, had no effect even at 100 µM. The cell extracts in the absence of ATP degraded p27Kip" to the same extent as in the presence of 1 mM ATP. However, the addition of adenosine 5h-[γ-thio]triphosphate, a non-hydrolysable analogue of ATP, in place of ATP, antagonized the degradation in a concentration-dependent manner (Figure 3b). These findings suggested strongly that the degradation of p27Kip" by these cell extracts was mediated by proteasomes. In contrast, p27Kip"R was resistant to this degradation activity (Figure 3a). Since the truncation of the N-terminus (p27Kip"∆N) had little effect on

A degradation-resistant isoform of p27Kip1 Table 1

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Effects of p27Kip1 and p27Kip1R on cell-cycle progression

Presented is a summary of the cell-cycle profiles obtained in three independent experiments as shown in Figure 4(b). The percentage of cells in each cell-cycle phase was estimated based on propidium iodide fluorescence by the ModFit LT program version 2.0. Data are the meanspS.E.M. (n l 3) ; *P 0.05 for value in GFP fluorescence-positive cells (j) as compared with the corresponding value obtained in the GFP fluorescence-negative cells (k). Cells in phase (%) p27Kip1

GFP j G0/G1 S G2/M

Figure 4

Nuclear localization of p27Kip1 isoforms and growth inhibition

(a) p27Kip1, p27Kip1R and their truncated mutants tagged with GFP at their C-termini were expressed in the cells as indicated. GFP, GFP alone ; p27Kip1, full-length p27Kip1 ; p27Kip1∆N, p27Kip1 starting with the second methionine (M16) ; p27Kip1R, p27Kip1R encoded by clone K4, starting with the methionine corresponding to M16 of p27Kip1 ; p27Kip1/p27Kip1R, a construct containing the region 16–162, common to p27Kip1 and p27Kip1R. Shown are photos representative of three independent experiments. Scale bar, 50 µm. (b) Analysis of cell cycle of the cells expressing (j) and not expressing (k) GFP fluorescence, on day 2 after the transfection with GFP alone (GFP), GFP-tagged p27Kip1 (p27Kip1) and GFP-tagged p27Kip1R (p27Kip1R).

the degradation of p27Kip" (Figure 3a), it is suggested that the difference in the C-terminal region is essential for the resistance of p27Kip"R to degradation. Previous reports [9,10] suggested that the absence of the putative CDK-dependent phosphorylation site in p27Kip"R was associated with its resistance to proteasomemediated degradation. Unexpectedly, p27Kip"T187A was degraded in a manner similar to that of the wild type (Figure 3a). This finding suggests that other differences in the C-terminal region of p27Kip"R may be associated with the resistance to degradation. Similar results were observed with the cell extract obtained from HeLa cells synchronized at the S phase (results not shown).

Nuclear localization and growth inhibition We next examined the nuclear localization of p27Kip" and p27Kip"R, because nuclear localization is considered to be a prerequisite for its function as a CDK inhibitor and cell-cycle regulator. For this purpose, p27Kip" and p27Kip"R were expressed

k

76.8p0.5 70.5p3.5 11.0p0.1 17.6p4.0 12.3p0.4 11.9p2.6

j

p27Kip1R k

j

83.8p4.3* 74.1p4.1 12.7p4.2 13.1p5.8 3.6p3.7* 12.8p2.0

k

84.4p2.0* 69.0p2.0 14.3p2.9* 19.5p0.7 1.4p1.8* 11.5p1.3

as GFP fusion proteins and the subcellular localization was examined in endothelial cells, HeLa cells, NIH 3T3 fibroblasts and COS7 cells (Figure 4). The same results were obtained with all of the cell types examined. GFP alone was distributed homogeneously in both cytosol and nuclei, with a slightly denser accumulation in the nuclei. On the contrary, the p27Kip"–GFP fusion protein was localized exclusively to the nuclei. Deletion of 15 residues from p27Kip" had no effect on this localization (Figure 4, p27Kip"∆N). p27Kip"R also localized to the nuclei. In addition to the dense nuclear fluorescence, a weak cytosolic fluorescence was observed with p27Kip"R. However, the construct containing the region common to p27Kip" and p27Kip"R, namely residues 16–162, showed a fluorescence pattern which was indistinguishable from that observed with GFP alone. The effects of p27Kip" and p27Kip"R on cell growth were determined in HeLa cells by analysing cell growth after transient transfection of the GFP-tagged proteins (results not shown). The number of HeLa cells expressing the GFP tag alone increased by 6.7 times from day 1 to 5, whereas the number of GFP-negative cells increased by 7.3 times. On the other hand, forced expression of p27Kip" and p27Kip"R caused complete growth arrest. There was no increase in the number of cells expressing GFP-tagged p27Kip" and p27Kip"R. However, the number of GFP-negative cells obtained in the same transfection increased by 4.6 and 5.7 times, respectively. The expression of p27Kip" and p27Kip"R caused no change in cell morphology, as observed under phase-contrast (results not shown) and fluorescence (Figure 4a) microscopes. The cell cycles of the p27Kip"- and p27Kip"R-induced growtharrested cells were examined on day 2 by a flow-cytometric analysis of propidium iodide fluorescence after separating the GFP fluorescence-positive and -negative cells on a cell sorter (Figure 4b). The profiles of propidium iodide fluorescence obtained with GFP fluorescence-negative cells in all transfections did not differ significantly from each other (Table 1), and were consistent with the pattern of unsynchronized cells (results not shown). The expression of GFP tag alone had no effect on this fluorescence profile (Figure 4b and Table 1). However, the expression of p27Kip" and p27Kip"R altered the fluorescence profile (Figure 4b), and significantly decreased the fraction of cells in G \M phase and increased the fraction of cells in G \G phase # ! " (Table 1). The fraction of cells in S phase seen with the p27Kip"Rexpressing cells decreased significantly when compared with the non-expressing cells, while that seen in the p27Kip"-expressing cells decreased slightly but not significantly (Table 1). The cellcycle profile seen with the cells expressing p27Kip"R did not differ significantly (P
0.05) from that seen with the cells expressing p27Kip" (Table 1). These findings suggest that p27Kip"R # 2001 Biochemical Society

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and p27Kip" both induced cell-cycle arrest mainly at the G \S " transition, and that there was no significant difference in their inhibitory effects on the cell cycle. The shoulder seen below the peak of the G \G phase in the ! " propidium iodide fluorescence profile obtained with the cells expressing GFP, p27Kip" and p27Kip"R was indicative of apoptosis. Possible apoptosis in the expressing cells was investigated in separate experiments by determining the sub-G population after " staining nuclear DNA with propidium iodide according to the method described by Gong et al. [23]. The apoptosis in the cells with no treatment and those treated with 10 µg\ml AMINE alone was negligible and the amount of apoptosis for these cells was 0.7p0.4 and 0.6p0.2 % (n l 3), respectively. Treatment with AMINE itself had no effect on apoptosis. The amounts of apoptosis in the cells expressing GFP, p27Kip" and p27Kip"R were 4.1p0.9, 22.1p1.4 and 32.4p3.2 % (n l 3), respectively. The apoptosis seen in the p27Kip"- and p27Kip"R-expressing cells was significantly greater than that seen in the GFP-expressing cells, and p27Kip"R induced more apoptosis than p27Kip".

DISCUSSION Here, we report for the first time the cloning of a second isoform of p27Kip". The existence of the transcript for the novel isoform was confirmed in porcine tissues by RT-PCR analysis. The critical point was that the novel isoform was resistant to proteasome-mediated degradation in Šitro. Therefore this isoform was termed p27Kip"R to indicate its resistance to proteasomemediated degradation. We also present evidence for the role of p27Kip"R as a cell-cycle regulator. p27Kip"R was demonstrated to be targeted to the nucleus and to cause growth arrest, mainly at the G \S transition. The differences in the tissue-specific ex" pression patterns and the sensitivity to proteasome-mediated degradation for p27Kip" and p27Kip"R suggest that the novel isoform may play a distinct role in regulation of the cell cycle. The major transcript for p27Kip" was reported to be 2.4–2.5 kb in Northern-blot analyses [1,2]. K1 was the longest clone (about 2.0 kb) obtained in the present study and this terminated in the middle of a putative poly(A)+ signal. The transcription-initiation site of p27Kip" was reported to be located 437 bp upstream from the translation-initiation codon [5]. Collectively, K1 is considered to represent the 2.4–2.5 kb transcript, but lacks the poly(A)+ tail and most of the 5h untranslated region. Thus K1 should contain most, if not all, of the possible exons. One important conclusion from this assignment is the existence of an exon containing the coding region for the p27Kip"R-specific C-terminus, and therefore the novel isoform would be derived from alternative splicing. Our results also suggest that the p27Kip" gene is composed of at least five exons, corresponding to the five segments shown in Figure 1. However, only three exons have been determined in human and mouse, and no exon containing a putative poly(A)+ signal has been reported [24,25,30,31]. Thus the exon containing the coding region specific to p27Kip"R (corresponding to segment IV) and that containing a poly(A)+ signal (corresponding to segment V) remain to be determined. Several reports have demonstrated phosphorylation at Thr-187 to be essential for activation of ubiquitination and the subsequent degradation of p27Kip" [8–10]. Lack of this phosphorylation site in the novel isoform could be one reason for its resistance to degradation. However, the situation is more complex since mutation of Thr-187 to Ala in p27Kip" did not inhibit proteolysis. The degradation of p27Kip" appeared to be independent of phosphorylation under the experimental conditions utilized in # 2001 Biochemical Society

the present study. Other differences in the sequence, other than the lack of phosphorylation, may therefore contribute to the difference in sensitivity towards degradation between the original and novel isoforms. It is noteworthy that the degradation of cyclin D1 in the complex with CDK was dependent on phosphorylation of Thr-286, while the degradation of free cyclin D1 was reported to be phosphorylation-independent [32]. It is thus possible that complex formation of p27Kip" with cyclin and CDK may alter dependence of its degradation on phosphorylation. We demonstrated that p27Kip"R was localized to the nucleus. The putative NLS of the original isoform was reported to be a bipartite form located in the region 152–169 [1,2]. We have recently determined the region 153–166 (KR … KR) to be a minimal requirement for nuclear localization of the original isoform [33]. An N-terminal cluster of two basic amino acids (Lys-Arg) is conserved in the novel isoform, while the C-terminal cluster of basic amino acids is missing from p27Kip"R (Figure 1). However, failure of the truncation mutant p27Kip"\p27Kip"R to localize to the nucleus indicates the requirement for a p27Kip"Rspecific C-terminal region for nuclear localization, and suggests that the NLS of the novel isoform is also in a bipartite form. Accordingly, the C-terminal half of the NLS is different in the two isoforms. The C-terminal region of p27Kip"R contains only one basic amino acid, Lys-168 (Figure 1). The reduced number of basic residues (in p27Kip"R) may reduce the efficiency of the NLS and cause some retention of p27Kip"R in the cytosol (Figure 4a). Mutation of Lys-168 to Ala caused more cytosolic staining than with the wild type, suggesting that this residue may play a critical role in the NLS of p27Kip"R (Y. Zeng, K. Hirano, M. Hirano, J. Nishimura and H. Kanaide, unpublished work). Further delineation of the NLS of the novel isoform is now under investigation. Exactly how the expression of the novel isoform is regulated in ŠiŠo remains to be answered. If it caused a G block, one may " wonder how the cells recover from the cell-cycle arrest. We herein demonstrated the novel isoform to be resistant to degradation by using an in Šitro degradation assay and the cell extract obtained at G \S transition. However, we observed that the " novel isoform was degraded in the endothelial-cell extract obtained at the G phase and at confluence (results not shown). " It is thus suggested that the novel isoform is not an ‘ immortal ’ molecule but that its expression may be regulated in a cellcycle-dependent manner which would be different from that for the original isoform, and it may thus play a distinct role in the regulation of the cell cycle. However, degradation of the novel isoform in ŠiŠo remains to be determined, to elucidate the regulation of the expression of the novel isoform under physiological conditions. In the present study, the overexpression of p27Kip" and p27Kip"R induced apoptosis to a greater extent than that seen with GFP alone. The induction of apoptosis by p27Kip" is controversial and appeared to be conditional. In overexpression experiments similar to those performed in the present study, p27Kip" induced apoptosis in 15 % of transfected breast cancer cells [34]. This rate of apoptosis is consistent with that seen in the present study. On the other hand, the mesangial cells and fibroblasts derived from the p27Kip"V/V mice were reported to have elevated rates of apoptosis, when deprived of growth factors, whereas the restoration of p27Kip" rescued the cells from apoptosis [35]. It was reported that the level of expression of p27Kip" was inversely correlated with apoptosis in Hodgkin’s disease, which suggests that p27Kip" is involved in protection from apoptosis in this disease [36]. The role of p27Kip" in apoptosis in ŠiŠo thus remains to be elucidated. In the present study, p27Kip"R appeared to induce apoptosis more potently than p27Kip". However, since the expression level was

A degradation-resistant isoform of p27Kip1 not normalized in the present study, the greater induction of apoptosis by p27Kip"R than by p27Kip" remains to be confirmed. In conclusion, this study presents the first direct evidence for the existence of a second isoform of p27Kip" derived from alternative splicing. The most important finding was that the novel isoform is resistant to proteasome-mediated degradation. We demonstrated that the novel isoform functions as a negative regulator of the cell cycle. The expression of p27Kip" is reported to be regulated in a cell-cycle-dependent manner at the transcriptional, post-transcriptional and post-translational levels. The existence of a splice isoform will add alternative splicing as one of the regulatory mechanisms in the expression of p27Kip". We thank Dr David J. Hartshorne (University of Arizona, Tucson, AZ, U.S.A.) for comments and help with the manuscript, and Mr B. Quinn for linguistic advice. This study was supported in part by Grants in Aid for Scientific Research (nos. 10557072, 11838013 and 11670687) and for Scientific Research on Priority Areas (no. 12213103) from the Ministry of Education, Science, Sports and Culture, Japan, by a Research Grant for Cardiovascular Diseases (11C-1) from the Ministry of Health and Welfare, Japan, and by grants from the Foundation for the Promotion of Clinical Medicine, the Suzuken Memorial Foundation and KANZAWA Medical Research Foundation.

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Received 12 July 2000/25 September 2000 ; accepted 20 October 2000

# 2001 Biochemical Society