Human Molecular Genetics, 2002, Vol. 11, No. 13 1497-1504
© 2002 Oxford University Press
Growth arrest by the LKB1 tumor suppressor: induction of p21WAF1/CIP1
Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, PO Box 63, 00014 University of Helsinki, Finland
Received January 31, 2002; Accepted April 17, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Germline mutations of the LKB1 tumor suppressor gene lead to Peutz–Jeghers syndrome (PJS), with a predisposition to cancer. LKB1 encodes for a nuclear and cytoplasmic serine/threonine kinase, which is inactivated by mutations observed in PJS patients. Restoring LKB1 activity into cancer cell lines defective for its expression results in a G1 cell cycle arrest. Here we have investigated molecular mechanisms leading to this arrest. Reintroduced active LKB1 was cytoplasmic and nuclear, whereas most kinase-defective PJS mutants of LKB1 localized predominantly to the nucleus. Moreover, when LKB1 was forced to remain cytoplasmic through disruption of the nuclear localization signal, it retained full growth suppression activity in a kinase-dependent manner. LKB1-mediated G1 arrest was found to be bypassed by co-expression of the G1 cyclins cyclin D1 and cyclin E. In addition, the protein levels of the CDK inhibitor p21WAF1/CIP1 and p21 promoter activity were specifically upregulated in LKB1-transfected cells. Both the growth arrest and the induction of the p21 promoter were found to be p53-dependent. These results suggest that growth suppression by LKB1 is mediated through signaling of cytoplasmic LKB1 to induce p21 through a p53-dependent mechanism.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Germline mutations of the LKB1 gene underlie Peutz–Jeghers syndrome (PJS), which is characterized by mucocutaneous melanin pigmentation, gastrointestinal polyposis and a 15-fold increased risk of cancer (1). LKB1 encodes a serine/threonine kinase, and mutations in PJS patients are loss-of-function mutations of LKB1 (2,3). This, together with the observed loss of heterozygosity of the remaining wild-type allele of LKB1 in a subset of PJS tumors (4,5), indicates that LKB1 is a tumor suppressor gene.
LKB1 homologues include mouse Lkb1 (6), Xenopus XEEK1 (7), and Caenorhabditis elegans par-4 (8). Genetic inactivation of Lkb1 in mice leads to embryonic lethality at midgestation, indicating that Lkb1 is essential for development (9). The developmental defects of Lkb1-/- embryos could be at least partly due to deregulated expression of vascular endothelial growth factor (VEGF), suggesting that Lkb1 regulates the VEGF pathway. XEEK1 is expressed in oocytes and fertilized eggs and phosphorylated on a cAMP-dependent protein kinase (PKA) consensus site (7). Similarly to XEEK1, mouse and human LKB1 are in vitro substrates for PKA on S431 (10) and S428 (our unpublished data), respectively. Phosphorylation of S431 is mediated in vivo by PKA or the related p90RSK kinase (10).
Reintroduction of LKB1 into human cancer cells with impaired LKB1 activity results in growth suppression (10,11), further supporting the role of LKB1 an a tumor suppressor. The growth inhibition of G361 melanoma cells is due to a G1 cell cycle arrest and is dependent on LKB1 kinase activity (11). Phosphorylation of Lkb1 on S431 may be required for this arrest, since an S431A mutant was compromised in its ability to mediate an arrest (10). Recently, it was discovered that LKB1 associates with Brg1, an essential component of chromatin remodeling complexes (12). It was suggested that LKB1 cooperates with Brg1 in inducing flat cell morphology indicative of cell cycle arrest and senescence. LKB1 was also reported to mediate programmed cell death in cells with functional p53 (13), although in some cell lines with wild-type p53, apoptosis has not been observed (11 and our unpublished data).
Studies on the subcellular localization of LKB1 have indicated a wide variety of localization patterns. The C. elegans PAR-4 and Xenopus XEEK1 were detected exclusively in the cytoplasm (7,8). Despite this, XEEK1 shares a conserved nuclear localization signal (NLS) with the human LKB1 and mouse Lkb1 (6). Mouse Lkb1 was reported to be predominantly nuclear, but shifted to the cytoplasm following co-expression of LIP1, a LKB1-interacting protein identified in a yeast two-hybrid screen (14). Human LKB1 has been detected to be both nuclear and cytoplasmic in several cell types (11,13,15). A minor fraction of LKB1 has also been found on the membranes (10) and in mitochondria (13).
Thus, LKB1 has been identified in a variety of subcellular compartments, but it has remained unclear which fraction mediates the cell cycle arrest. Here we have characterized the subcellular localization of LKB1 with respect to its ability to induce growth suppression. Moreover, we have further studied which molecules are involved in executing the LKB1-induced G1 arrest.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Kinase-deficient LKB1 mutants are predominantly nuclear
To investigate the subcellular localization of LKB1, we initially performed immunofluorescence analysis of several variants of LKB1 (mouse Lkb1, human LKB1, LKB1 mutants from PJS patients and engineered mutants) transfected into a number of cell lines. The analyses were performed with either N- or C-terminal epitope-tagged or untagged LKB1. These studies consistently indicated that the subcellular localization of transfected human LKB1 and mouse Lkb1 were indistinguishable from each other with three categories of staining: predominantly cytoplasmic, cytoplasmic and nuclear, and predominantly nuclear (Fig. 1A, Lkb1 and LKB1 expression in mouse myoblast C2C12 cell line; Fig. 1B LKB1 in U2OS osteosarcoma cell line). LKB1 localization was found not to correlate with a specific phase in the cell cycle based on hydroxyurea block-release studies of transfected U2OS cells (data not shown). In contrast, the kinase-deficient LKB1 mutants from PJS patients [SL26 (del303–306), SL8 (stop 308) and SL31 (fs277, stop 283)] or sporadic cancer (G163D) as well as an engineered kinase-deficient mutant (LKB-KD, deletion of amino acids 192–195) demonstrated a dramatic increase in nuclear localization (81–91% nuclear versus 31% in wild-type) in several cell types, including U2OS osteosarcoma cells (Fig. 1B,C) and COS-7 monkey kidney epithelial cells (data not shown). The predominantly nuclear localization was a feature common to all analyzed kinase-deficient mutants, with the exception of LKB1-D176N, which displayed a wild-type subcellular localization (Fig. 1C) consistent with a previous report (15).
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The observation that most analyzed kinase-deficient LKB1 mutants are predominantly nuclear prompted us to compare the kinase activity of wild-type LKB1 in nuclear and cytoplasmic fractions of COS-7 cells (Fig. 1D), in which LKB1 displays a similar subcellular distribution to U2OS cells (data not shown). Interestingly, the results indicate that transfected LKB1 activity is readily detectable in both nuclear and cytoplasmic fractions. The slightly higher activity in the cytoplasmic fraction correlated with LKB1 levels (Fig. 1D, western) indicating similar specific activities in these fractions. Endogenous LKB1 activity was also detected in both cytoplasmic and nuclear fractions, and no clear difference in the specific activities could be detected when comparing with protein levels (Fig. 1E). As LKB1 levels were comparable in the two fractions, the minor cytoplasmic contaminant (compare 14-3-3ß in lanes C and N in Fig. 1E) is unlikely to significantly contribute to LKB1 levels or activity.
One possible reason for the variable localization pattern of LKB1 would involve shuttling between the cytoplasm and the nucleus. Therefore, we investigated whether LKB1 would have a nuclear export signal (NES) in addition to the previously characterized nuclear localization signal (NLS) (6). A putative NES was identified in LKB1 between amino acids 190 and 197 (LKISDLGV), which conforms to the NES consensus. However, as this sequence overlaps with the ATP-binding site of the kinase, its mutation renders LKB1 kinase-deficient (LKB1-KD, Fig. 1C). As most kinase inactive mutants localize predominantly to the nucleus, this mutant is therefore not suitable for investigating whether the NES signal is functional. Therefore we investigated potential nuclear export of LKB1 using leptomycin B, an inhibitor of the CRM1-dependent nuclear export (16) in U2OS cells transfected with HA-tagged LKB1. Immunofluorescence analysis of the subcellular localization of the transfected LKB1 at several time points did not indicate a dramatic increase in nuclear LKB1 (Fig. 1F), whereas endogenous cyclin B1 efficiently accumulated in the nucleus as previously described (17). This suggests that CRM1-dependent nuclear export does not significantly contribute to the noted subcellular localization of LKB1.
Taken together, these results demonstrate a correlation between loss of kinase activity and nuclear accumulation of LKB1. As such, the results would suggest that cytoplasmic localization required kinase activity. This could be through, for example, binding to a cytoplasmic anchor such as the recently described LIP1 (14). However, as the kinase-deficient LKB1-D176N displayed a localization pattern indistinguishable from that of wild-type LKB1 (Fig. 1C) (15), it appears that the regulation of subcellular localization is not solely mediated by the kinase activity.
LKB1-induced growth arrest is mediated by cytoplasmic LKB1
To address from which intracellular compartment LKB1 mediates its growth-suppression function, we generated an LKB1 mutant lacking the nuclear localization signal (NLS) previously characterized in mouse Lkb1 (6). To this end, we deleted the NLS of LKB1 by replacing three conserved residues with alanine: PRRKRA (amino acids 38–43) to AAAKRA. Following transient transfection, 90–98% of LKB1-
NLS-expressing cells displayed predominantly cytoplasmic staining, compared with the 42% of wild-type LKB1 (Fig. 2A). The LKB1-NLS mutant also retained kinase activity (Fig. 2B), although the specific activity was slightly decreased.
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The almost exclusively cytoplasmic localization of LKB1-
NLS prompted us to compare the growth-suppressing activities of LKB1-NLS and wild-type LKB1. For this purpose, we used the ability of LKB1 to induce G1 arrest in G361 melanoma cells readily detectable following a transient transfection and nocodazole block (11). Interestingly, the ability of LKB1-NLS to induce G1 growth arrest was indistinguishable from that of wild-type LKB1, as determined by analyzing transfected G361 cells by FACS analysis (Fig. 2C). The cell cycle arrest is not likely to be mediated by a membrane-bound pool of LKB1, since it represents a minor fraction of total cytoplasmic LKB1 and was previously demonstrated not to be required for growth suppression (10).
The ability of LKB1-NLS to induce G1 arrest comparable to that induced by wild-type LKB1 suggested that the signaling leading to G1 arrest is initiated by cytoplasmic LKB1. As most kinase-deficient LKB1 mutants are predominantly nuclear, this raised the possibility that cytoplasmic localization – regardless of kinase activity – might suffice to induce G1 arrest. To investigate this possibility, we created a kinase-deficient mutant that also lacked the nuclear localization signal, LKB1-NLS-KD. This led to a dramatic shift to an almost exclusively cytoplasmic localization of this kinase-deficient mutant (Fig. 2A). Despite this cytoplasmic localization, LKB1-NLS-KD was unable to induce G1 arrest (Fig. 2C). Consistent with this, the LKB1-D176N mutant, which also localized partly to the cytoplasm despite being kinase-deficient, also was unable to induce G1 arrest (Fig. 2C). These results indicate that cytoplasmic LKB1 is sufficient to induce G1 arrest – presumably by initiating a signal cascade impinging on the cell cycle machinery. This induction requires the kinase acitivity of LKB1.
LKB1 arrest is overcome by co-expression of cyclin D1 or cyclin E
To further elucidate the molecular mechanisms involved in LKB1-induced G1 arrest, we examined whether positive cell cycle regulators could overcome the arrest. G361 cells were co-transfected with LKB1 and the G1 cyclins cyclin D1 or cyclin E followed by analysis of the cell cycle distribution of transfected cells by FACS as described above. Co-expression of cyclin D1 or cyclin E resulted in the release of LKB1-induced G1 arrest, diminishing the proportion of cells in G1 from 51% to 29% and 23%, respectively (Fig. 3). This result suggests that LKB1 signaling leads to a decrease in the activity of endogenous G1 cyclin-dependent kinase (CDK)–cyclin complexes in G361 cells.
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LKB1 induces the expression of p21WAF1/CIP1
Induction of CDK inhibitors (CKIs) is a well-characterized mechanism to inhibit G1 CDK activity (18). To investigate whether LKB1 could mediate cell cycle arrest by regulating expression of CKIs, we analyzed the expression levels of p21 and p27 in G361 cells transiently transfected with LKB1. As a control we used G361 cells arrested in G1 by expression of the forkhead family transcription factor AFX (19). The transfected cells were treated with nocodazole as in the previous analyses (Figs 2 and 3), and separated using a magnetic cell sorting system (see Materials and Methods) followed by western blotting analysis.
The results presented in Figure 4A indicate a specific increase in p21 levels in LKB1-transfected cells. This was not noted in vector-transfected cells or in AFX-transfected cells, which instead demonstrated elevated p27 as previously reported (19). AFX arrests cells in G1 specifically by inducing p27 (19). There is a slight increase in the level of p27 in LKB1-transfected cells; however, unlike p21 levels, the level of p27 is not appreciably increased in LKB1-transfected cells when compared with non-nocodazole-treated control cells (data not shown).
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As the cells analyzed in Figure 4A were treated with nocodazole in accordance with our previous analyses (Figs 2 and 3), we wanted to exclude the possibility that this treatment lead to the p21 induction in LKB1-transfected cells. To this end, we analyzed p21 expression in G361 cells not treated with nocodazole (Fig. 4B), and, as before, p21 levels were elevated specifically in LKB1-expressing cell extracts, but not in G361 cells expressing p16 (and thus arrested in G1) (11). Moreover, the PJS-patient-derived LKB1-SL8 kinase-deficient mutant was unable to increase the levels of p21, demonstrating that this function of LKB1 is dependent on its kinase activity (Fig. 4B). p21 expression was observed to be nuclear in the LKB1-transfected cells, as analyzed by immunofluorescence (Fig. 4C), which is consistent with cell cycle arrest (20).
The elevated levels and nuclear localization of p21 in LKB1-expressing cells are likely to result in an increase in the amount of p21 bound to cyclin E–CDK2 complexes and hence inhibition of cell cycle progression (21). This is consistent with the observation that overexpression of cyclin E can overcome the LKB1 arrest. It is also consistent with the observation that cyclin D1 can overcome the LKB1 arrest, since overexpression of cyclin D1 can lead to sequestering of p21 to cyclin D1–CDK4 complexes and the formation of active cyclin E–CDK2 complexes (22).
The results presented above strongly implicate increased p21 protein levels as the mechanism leading to LKB1-mediated G1 arrest. To investigate how LKB1 expression leads to elevated p21 levels, we explored the possibility that LKB1 expression would specifically induce transcription of p21. To this end, we took advantage of a p21 promoter–luciferase reporter p21P–Luc (23), which was co-transfected into G361 and U2OS cells with wild-type LKB1 or control plasmids. Co-transfection of LKB1 resulted in a robust induction of luciferase activity compared with the vector transfection in G361 cells (Fig. 5A). Importantly, LKB1-mediated p21P–Luc induction was not noted in U2OS cells (Fig. 5A), where LKB1 is unable to mediate cell cycle arrest or function as a growth suppressor (11 and data not shown). p21P–Luc induction was also mediated by the cytoplasmic but active LKB1-NLS in G361 cells. On the contrary, the kinase inactive predominantly nuclear mutants LKB1-SL8 and LKB1-SL26, as well as the kinase-inactive predominantly cytoplasmic mutant LKB1-NLS-KD and D176N with both nuclear and cyoplasmic localization, were unable to induce the reporter (Fig. 5B). These results indicate that LKB1 expression activates transcription from the p21 promoter and that this induction is correlated with the ability of LKB1 to induce growth suppression, further supporting the role of p21 as a mediator of LKB1-induced growth suppression.
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LKB1 has recently been reported to associate with p53 (13). As p53 can induce the transcription of p21 (24), we investigated whether inhibition of p53 would affect LKB1-mediated p21-reporter induction. We co-transfected LKB1 with a plasmid expressing a dominant-negative form of p53, p53dn (25). Expression of p53dn in G361 cells with wild-type endogenous p53 (26) completely abolished the induction of p21 by LKB1 (Fig. 5C). Moreover, co-transfection of p53dn also inhibited LKB1-mediated G1 arrest demonstrated by FACS analysis as described above (Fig. 5D). These results suggest that both LKB1-mediated induction of p21 and LKB1-mediated growth arrest require functional p53.
In summary, our results presented here suggest that the LKB1 tumor suppressor kinase initiates its negative regulation of cell growth in the cytoplasm. This would imply that nuclear associations of LKB1, such as possible cooperation with Brg1 (12), would represent distinct functions. Although the immediate substrates and signaling pathway downstream of LKB1 remain to be characterized, the results presented in this study strongly implicate p21 as a direct mediator of LKB1-mediated cell cycle arrest. Moreover, p21 induction appears to be p53-dependent.
The mechanisms involved in the tumor suppressor function of LKB1 in PJS remain largely uncharacterized. Limited studies on p53 mutations in PJS tumors suggest that p53 mutations are rarely observed in PJS polyps prior to further progression of the polyps to carcinomas (4,5,27). This suggests that p53 mutations do not provide an advantage at early stages of polyp formation in a LKB1 heterozygous background, which is consistent with the notion that LKB1 and p53 would be on the same pathway.
One report indicates decreased apoptosis in PJS polyps compared with the tips of villi in the adjacent normal mucosa (13). As LKB1 was also detected in apoptotic cells, the study suggested that one of the roles of LKB1 would be to participate in this apoptosis (13). Additionally, the same study noted expression of LKB1 throughout the villus, with a gradient of cytoplasmic LKB1 increasing from the crypt upwards, suggesting that LKB1 may also play a role during the differentiation process. Considering our observation of LKB1-mediated p21 induction, it is interesting to note that p21 expression is not noted in terminally differentiated epithelial cells, but appears abruptly in epithelial cells migrating from the crypts, concomitantly with cessation of proliferation (28). It will be interesting to determine whether this induction of p21 is LKB1-dependent.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture and transfections
G361, U2OS, C2C12 and COS-7 cells (all from ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, L-glutamine and penicillin/streptomycin. The cells were transfected using the calcium phosphate transfection method as described (29), or with FUGENE6 transfection reagent (Roche).
Expression vectors
The following expression vectors were used: pCI-Neo (Promega), Lkb1/pAHC, LKB1/pCI-Neo, LKB1/pAHC, LKB1-G163D/pAMC, LKB1-SL8/pAHC, LKB1-SL26/pAHC and LKB1-SL31/pAHC (3) and Rc-cycD1-HA (30). HA–AFX/pMT2 (31) and pEGFP–spectrin (32) were kindly provided by Dr Rene Medema, HAp53CT/pCDNA3 (33) by Dr Giovanni Blandino, p21P–Luc (23) by Dr Marikki Laiho and cyclin E/pCMX by Dr Giulia Piaggio. pMACS Kk was purchased from Miltenyi Biotech and pRLTK–luc from Promega. LKB1-NLS/pAHC and LKB1-KD/pAHC were constructed with the Gene Editor Mutagenesis System (Promega) using primers with the following sequences: 5'-GGT CAT CTA CCA GGC GGC CGC CAA GCG GGC G-3' and 5'-ACC GGT GGC ACC CTC AAA GGC GTG GCC GAG GCA CTG-3'. The LKB1-NLS-KD/pAHC was constructed by ligating an EcoRI-BamHI fragment of LKB1-NLS/pAHC and a BamHI-SalI-fragment of LKB1-KD/pAHC to an empty pAHC vector. The D176N mutant was created by PCR using a forward primer 5'-ATT GTG CAC AAG AAT ATT AAG CCG GGG-3' containing the mutation and a reverse primer 5'-GGG TCG ACG CCT CAC TGC TGC TTG CA-3'. The PCR fragment was digested with ApaLI and SalI and ligated together with an EcoRI–ApaLI fragment from LKB1/pAHC to an empty pAHC vector.
Immunofluorescence
Cells were seeded on coverslips and fixed with 3.5% paraformaldehyde 48 hours post transfection. Immunofluorescence was performed with monoclonal anti-HA (Babco), polyclonal anti-p21 (Santa Cruz Biotechnology, Inc.) and monoclonal anti-cyclin B1 (Santa Cruz Biotechnology, Inc.). Signals were detected with A488-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies (Molecular Probes and Boehringer Mannheim, respectively). The nuclei were visualized with Hoechst 33342 (0.5 µg/ml) and the immunostainings were viewed and documented using a Zeiss Axiophot microscope. At least 200 cells were analyzed from each sample.
Nuclear export inhibition assay
U2OS cells were transfected with HA-tagged LKB1 and treated with leptomycin B 50 ng/ml (a kind gift from Dr M. Yoshida) or solvent (ethanol) for 1, 6 and 12 hours. At each time point, cells were fixed and subjected to anti-HA or anti-cyclin B immunofluorescence, and the subcellular localization of transfected LKB1 and endogenous cyclin B was calculated from 200 cells each.
Flow-cytometric analyses
G361 cells were co-transfected with pEGFP–spectrin and indicated plasmids as described above for 38 hours. The cells were treated with nocodazole (50 ng/ml, Sigma) for an additional 22 hours to induce a G2/M phase block. Subsequently, cells were detached and fixed with 80% ethanol. Propidium iodide (50 µg/ml, Sigma) was used to stain the nuclei. The cell cycle distribution of EGFP-positive and -negative cells was analyzed with a Coulter EPICS flow cytometer (Coulter Electronics). Percentages of cells in G1, S and G2/M phases were determined with the CellFIT cell cycle analysis program (Becton Dickinson).
Cell fractionation
COS-7 cells were treated with a hypotonic buffer containing 10 mM Hepes, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, 1 mM DTT and 5 mM EDTA plus protease and phosphatase inhibitors. After 5 minutes of swelling, the cells were homogenized with a glass Dounce homogenizer and centrifuged. The nuclear pellet was resuspended in ELB lysis buffer (3), and the cytoplasmic fraction was adjusted to correspond the ELB buffer by adding detergent and salt. Total cell extract was prepared by lysing the cells in ELB buffer.
Cell sorting
G361 cells were co-transfected with pMACS Kk (Miltenyi Biotec) and indicated plasmids for 38 hours and treated with nocodazole as described above. The cells were detached with trypsin and incubated with MACS select Kk microbeads (Miltenyi Biotec). pMACS-positive transfected cells were separated according to the manufacturer's instructions, washed with PBS and lysed to ELB buffer.
Western blotting
Twenty-five micrograms of total proteins were analyzed by 10% SDS–PAGE and blotted according to standard protocols (29). The filters were blotted with the following antibodies: monoclonal anti-HA (12CA5, Babco), rabbit anti-LKB1 (11), sheep anti-LKB1 (Upstate Biotechnologies), rabbit anti-p21 (Santa Cruz Biotechnology, Inc.), monoclonal anti-p27 (Santa Cruz Biotechnology, Inc.), monoclonal anti-CDK7 (Zymed laboratories Inc.) and polyclonal 14-3-3ß (Santa Cruz Biotechnology, Inc.), and the signals were detected by enhanced chemiluminescence (Pierce).
Immunoprecipitation-kinase assay
COS-7 and U2OS cells transfected with the indicated expression plasmids were lysed in ELB buffer. Anti-HA, anti-Myc and anti-LKB1 were used to immunoprecipitate tagged and non-tagged proteins, respectively. The kinase assay was performed using a kinase buffer consisting of 20 mM Tris–HCl, pH 7.4, 50 µg/ml BSA, 7 mM MnCl2, 5 mM MgCl2 and [
-32P]ATP.
Luciferase assays
G361 cells were co-transfected with p21P–Luc, RLTK–luc and the indicated plasmids. Luciferase activity was measured after 48 hours transfection using the Dual Luciferase Reporter Assay system (Promega). The firefly luciferase activity was normalized to constitutively expressed Renilla luciferase activity in each sample, and fold induction was calculated with respect to the vector transfection.
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ACKNOWLEDGEMENTS |
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We thank M. Schoulz for help in FACS analyses and B. Tjäder and S. Räsänen for technical assistance. Drs R. Medema, G. Blandino, M. Laiho and G. Piaggio are acknowledged for providing DNA constructs, Dr M. Yoshida for leptomycin B, and Drs R. Medema and D. Rossi for comments on the manuscript. This study was supported by the Finnish Cancer Organization, Sigrid Juselius Foundation and Academy of Finland. M.T. is a recipient of a grant from the Finnish Cancer Research Foundation and A.Y. is a student of the Helsinki Biomedical Graduate School.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +358 9 19125555; Fax: +358 9 19125554; Email: tomi.makela{at}helsinki.fi
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