Nucleocytoplasmic Localization of P70 S6K1, but Not of Its Isoforms P85 and P31, Is Regulated by TSC2/Mtor

Oncogene (2011) 30, 4509–4522 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Nucleocytoplasmic localization of p70 S6K1, but not of its isoforms p85 and p31, is regulated by TSC2/mTOR

M Rosner and M Hengstschla¨ger

Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria

The tuberous sclerosis complex gene 2 (TSC2)/mamma- activity of the oncogenic kinase Akt (also known as lian target of rapamycin (mTOR) pathway controls many protein kinase B) via PDK1 (phosphoinositide-depen- cellular functions via phosphorylation of ribosomal protein dent kinase-1). Akt-mediated phosphorylation of the S6 kinases (S6Ks). Alternative splicing and translation tuberous sclerosis tumor suppressor protein tuberin generate three S6K1 proteins. Although nuclear and (encoded by tuberous sclerosis complex gene 2 (TSC2)) cytoplasmic S6K targets are known, the nucleocytoplas- causes its functional inactivation and leads to enhanced mic localization of the S6K1 proteins has not been mTOR activity. In mammalian cells two structurally comparatively elucidated so far. We show that in primary and functionally distinct mTOR-containing complexes fibroblasts p85 S6K1 is cytoplasmic, p70 can be found in have been identified. mTORC1 is composed of mTOR, both compartments and p31 is exclusively nuclear. As raptor and mLST8 and mTORC2 contains mTOR, already known for p70 and p85, our data suggest that p31 rictor, mLST8 and sin1 (Yang and Guan, 2007; Wang is also a target of mTOR-mediated phosphorylation. and Proud, 2009; Sengupta et al., 2010). Blocking mTOR kinase activity via rapamycin and Whereas mTORC2 phosphorylates and activates its activation in TSC2À/À cells and via TSC2 small Akt, the best-characterized phosphorylation targets interfering RNAs revealed that it regulates the localiza- and downstream effectors of mTORC1 are the eukar- tion of p70, but not of p85 and p31. The mTOR-dependent yotic initiation factor 4E-binding protein and the phosphorylation of p70 S6K1 at T389 is essential for its ribosomal protein S6 kinases (S6Ks). mTOR is known nuclear localization and exclusively hyperphosphorylated to phosphorylate and thereby activate p70 S6K1 at p70 S6K1 can be found in the nucleus. We further T389 and p85 S6K1 at T412. The mTOR-mediated demonstrate this mTOR-controlled p70 S6K1 localization phosphorylation status of p70 S6K1 T389 has been to be growth factor dependent. During the cell-cycle demonstrated to always correlate with p70 S6K1 kinase phosphorylation and nuclear localization of p70 S6K1 activity (Ruvinsky and Meyuhas, 2006; Shaw, 2008). occur in mid G1 phase. We report that the different S6K1 Mammalian cells express S6K1, encoded by proteins exhibit different nucleocytoplasmic localizations RPS6KB1, and S6K2, encoded by RPS6KB2. Due to and that the TSC2/mTOR cascade not only regulates p70 alternative translation two isoform 1 S6K1 proteins are S6K1 activity, but also its localization. These findings known to exist in mammalian cells: p85 S6K1 and p70 provide new important insights into the temporal and S6K1, which is identical to p85 S6K but lacks its first 23 spatial dynamics of TSC2/mTOR/S6K regulation. amino acids (Meyuhas and Dreazen, 2010). In addition, Oncogene (2011) 30, 4509–4522; doi:10.1038/onc.2011.165; mammalian cells express a second S6K1 isoform published online 23 May 2011 spanning 316 amino acids (p31 S6K1) (Figures 1a and b). Overexpression of the alternative splicing protein Keywords: TSC2; mTOR; S6K1; localization SF2/ASF (also known as SFRS1) triggers predominant expression of p31 S6K1. It was demonstrated that the S6K1-splicing protein SF2/ASF is a proto-oncogene and that, although p70 S6K1 has little oncogenic Introduction potential, the alternatively spliced p31 S6K1 is sufficient to cause cell transformation and has a major role for the The mammalian target of rapamycin (mTOR) is a SF2/ASF-mediated cellular transformation phenotype serine/threonine kinase having a central role within (Karni et al., 2007; Fenton and Gout, 2011). the insulin-signaling cascade regulating a wide variety S6Ks and RSKs (p90 ribosomal protein S6 kinases) of different cellular functions. Upstream of mTOR, are the two classes of kinases phosphorylating the the PI3K (phosphatidylinositol-3-kinase) regulates the ribosomal protein S6. S6 protein is dispersed throughout the cytoplasm and concentrated to the nucleoli within the nucleus, what is a consequence of the fact that Correspondence: Professor M Hengstschla¨ger, Institute of Medical eukaryotic ribosomes are assembled in the nucleolus Genetics, Medical University of Vienna, Wa¨hringer Strasse 10, 1090 before export to the cytoplasm. Besides S6, many other Vienna, Austria. E-mail: [email protected] S6K substrates have been reported, including, for Received 25 November 2010; revised 19 March 2011; accepted 4 April example, the cytoplasmic proteins eukaryotic elongation 2011; published online 23 May 2011 factor 2 kinase, eukaryotic translation initiation factor Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4510

Figure 1 Distinct expression patterns of S6K1 isoforms p85, p70 and p31 in various cell types. (a) Schematic presentation of the S6K1 family of ribosomal protein S6 kinases. Alternative splicing of the primary transcript of RPS6KB1 results in the generation of two different isoforms, a longer one, spanning 525 amino acids (p85 S6K1) and a shorter one spanning 316 amino acids (p31 S6K1). An alternative translation start site in the N-terminus of splicing variant 1 (isoform 1) gives rise to an additional protein, the p70 S6K1, which is identical to p85 S6K1 but lacks its first 23 amino acids and therefore comprises 502 amino acids. Similar diversity including different isoforms is known for S6K2 as well as for the p90 ribosomal protein S6 kinases (RSKs), but is not outlined and discussed in detail here. (b) Schematic alignment of isoforms 1 and 2 of S6K1. p85 S6K1 and p70 S6K1 are almost identical except that the shorter form lacks the N-terminal 23 amino-acids extension of p85 S6K1 containing a suggested nuclear-localization-signal (NLS). Isoform 2 of S6K1 (p31 S6K1) is identical to p85 S6K1 for amino acids 1–195 but differs in the adjacent 121 amino acids (dashed box). The kinase domain of p70 and p85 S6K1 is only partially existent in p31 S6K1. The major phosphorylation sites known to drive full activation of p70 S6K1 (and the corresponding sites in p85 S6K1) are highlighted. (c) Four non-tumor mammary gland-derived cell lines and four established breast cancer cell lines were examined for the expression level of SF2/ASF (upper panel) and S6K1 isoforms p85, p70 and p31 (lower panels) via immunoblotting using indicated antibodies. The N-terminal anti-p70S6K1 antibody used here detects both transcript variants, p85/p70 and p31. Although detected in a single step on the same membrane, different exposures of p85/p70 and p31 are separately presented to overcome apparent differences in their expression ratio and to allow direct comparison between samples. (d) Experiments presented in (c) were repeated using four distinct bladder cancer cell lines and a corresponding non- tumor derived cell line (nt). In addition, cells of different origin (different), including human non-transformed, non-immortalized IMR-90 fibroblasts and various immortalized and/or transformed cell lines were co-analyzed. (e) Immortalized NIH3T3 murine fibroblasts, primary IMR-90 fibroblasts and BT474 breast cancer cells, both of human origin, were analyzed for the expression pattern of S6K1 isoforms p85, p70 and p31 via immunoblotting using the aforementioned anti-p70S6K1 antibody directed against the N- terminus of S6K1 (left panel). In addition, same lysates were analyzed with a C-terminal p70 S6K1 antibody. A direct comparison of both immunodetections is separately presented (right panel). (f) BT474 and IMR-90 cells were treated with siRNAs specifically targeting human S6K1 and were examined for knockdown efficiency via immunoblotting using indicated antibodies.

4B, BAD, insulin receptor substrate, and mTOR (which not affect the nucleocytoplasmic localization of p31 and is also located to the nucleus) and the nuclear proteins p85, but triggers loss of nuclear p70 S6K1. Analyses of cAMP-responsive element modulator t, the 80-kDa p70 S6K1 mutants, either phosphorylation defective or subunit of the nuclear Cap-binding complex and S6K1 mimicking, demonstrated that phosphorylation of p70 Aly/REF-like target (Ruvinsky and Meyuhas, 2006; S6K1 at T389 is essential for its nuclear localization. In Meyuhas and Dreazen, 2010). primary cells only hyperphosphorylated p70 S6K1 can Although both, nuclear and cytoplasmic, S6K sub- be detected in the nucleus. In agreement with this role of strates have been reported, the nucleocytoplasmic the mTOR pathway for the p70 S6K1 localization, localization of the three S6K1 proteins, p31, p70 and activation of the mTOR cascade in TSC2À/À fibro- p85, has not been comparatively elucidated so far. blasts or via TSC2-specific small interfering (si)RNAs Using non-transformed, non-immortalized primary selectively induces nuclear localization of the p70 human fibroblasts we show here that p85 S6K1 is protein. Although cytoplasmic p70 S6K1 protein can cytoplasmic, the p70 protein is localized to both, be detected in all phases of the mammalian cell cycle, cytoplasm and nucleus, whereas p31 is nuclear. The this protein becomes nuclear only at mid G1 phase. p70 and p85 isoforms are known to be phosphorylated TSC2 siRNA experiments revealed that this cell cycle- by mTOR. Our presented findings here suggest that p31 dependent nucleocytoplasmic localization of p70 S6K1 is also a target of mTOR-mediated phosphorylation. is controlled by the TSC2/mTOR pathway. Our data However, blocking mTOR activity via rapamycin does show for the first time that the S6K1 proteins are

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4511 differently localized and that alternative splicing can be Furthermore, these data show that IMR-90 cells can a mechanism resulting in different cytoplasmic/nuclear be used to study the endogenous regulation of the three localization of proteins encoded by one gene. In S6K1 proteins. This is of importance, as we wanted to addition, we demonstrate that the TSC2/mTOR cascade study the localization and regulation of the endogenous not only regulates the activity of p70 S6K1 via phos- S6K1 proteins in a non-transformed, non-immortalized phorylation, but also its growth factor- and cell cycle- diploid primary human cell system showing no evidence dependent nucleocytoplasmic localization. Our findings for deregulated S6K1 expression (Figures 1d–f). IMR- allow a better understanding of the temporal and spatial 90 cells have the same DNA content as other normal regulation of TSC2/mTOR and S6K1 activities. primary human cells (see the comparison with human amniotic fluid cells in Figure 2a), are commercially available to all colleagues in the field and can easily be cultivated with reasonable proliferation rates. Addition- Results ally, for the planned experiments it was necessary to prove that the here chosen IMR-90 cells can be cell cycle Nucleocytoplasmic localization of the S6K1 proteins p85, synchronized via serum deprivation and restimulation p70 and p31 and can be used to perform nucleocytoplasmic fractio- The alternative splicing protein SF2/ASF is an oncogene nations of high purity (Figures 2a and b). responsible for the generation of p31 via splicing of Using IMR-90 fibroblasts we separated cytoplasmic S6K1 (Figures 1a and b; Karni et al., 2007). Western and nuclear fractions, the latter was proven not to be blot analyses revealed that compared with four non- significantly contaminated with cytoplasmic, mitochon- tumor mammary gland-derived cell lines (CRL7347, drial or endoplasmatic reticulum proteins. Although CRL7379, CRL7847 and MCF10a) the four analyzed p70 S6K1 can be detected in the cytoplasm and in the breast cancer cell lines (MCF7, BT474, MDA-MB468 nucleus (predominantly cytoplasmic), p85 S6K1 was and ZR-75-1) express significantly higher levels of found to be cytoplasmic. The alternative splicing SF2/ASF correlated with higher levels of p31 S6K1 isoform p31 S6K1 was observed to be a nuclear protein (Figure 1c). A similar tumor-associated pattern was (Figures 2b–e). Since p85 is less abundant than p70, we observed comparing four bladder cancer cell lines (5637, next wanted to exclude that the reason why p85 does not RT4, RT112 and T24) with HCV29 cells derived from appear in the nuclear fraction is just due to detection normal, non-malignant ureteric epithelium (Figure 1d). limits. (1) In Figure 2d we show a western blot In addition, comparing transformed HeLa or HEK293 localization experiment, in which the cytoplasmic/ cells with normal, non-tumor-derived IMR-90, provided nuclear protein loading ratio is 1:5 and p85 was not further support for the connection between transforma- detectable in the nucleus, neither the total protein nor tion and high levels of SF2/ASF and p31 S6K1 the phosphorylated form. (2) We performed an im- (Figure 1d). The following results prove the specificity munoprecipiation experiment (less unspecific protein of the analyzed band here for the p31 isoform: (1) in a background) analyzing fractionated IMR-90 with a wide variety of different cell types we confirm the cytoplasmic/nuclear protein loading ratio of 1:8. Again, correlation of the expression of SF2/ASF and p31 we could not detect p85 in the nucleus under the used (Figures 1c and d). (2) BT474 is a tumor-derived cell line experimental conditions here (Figure 3a). (3) This result with an S6K1 gene amplification (Karni et al., 2007). was confirmed in mouse embryonic fibroblasts (MEFs) We found all three protein bands (p85, p70 and p31) (Figure 3b). to be upregulated in this cell line (Figures 1c, e and f). It was interesting to see that the S6K1-phosphory- (3) Although using an N-terminal anti-S6K1 antibody lated forms of mTOR and S6 could be detected in higher allows the detection of p31, a C-terminal anti-S6K1 levels in the nucleus than in the cytoplasm (Figures 2e, antibody only detects p85 and p70, but not p31, which is 3a and 7f). In the future these localization patterns known to have a different C-terminal sequence (Figure 1e). could be of interest for further investigations of both, (4) We have performed S6K1-specific siRNA experi- the upstream regulators of these two proteins and their ments in two different cell lines demonstrating that the downstream targets. bands representing the p85, p70 and p31 isoforms were This is the first demonstration that in primary normal affected (Figure 1f). cells the three S6K1 proteins are differently localized It has already been shown in the past, that SF2/ASF and that alternative splicing of S6K1 can be a and p31 are upregulated in breast cancer cell lines mechanism resulting in different nucleocytoplasmic (Karni et al., 2007). Using a different set of breast cancer localization of the proteins encoded by the one gene cell lines and normal counterparts our presented data RPS6KB1. here confirm this correlation. In addition, for the first time we provide evidence that this deregulation could also be involved in bladder cancer development. Finally, Blocking mTOR activity causes delocalization of p70 these results prove that upregulation of SF2/ASF is S6K1, but is without effects on p85 and p31 localization always accompanied with high levels of p31 and that we p31 S6K1 does not contain the mTOR-mediated have established conditions allowing the analysis of all phosphorylation site T389 found in p70 S6K1, corre- three endogenous S6K1 proteins in human and murine sponding to T412 in p85 S6K1 (Figure 1b), and cells. accordingly cannot be detected via an anti-phospho

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4512

Figure 2 Rapamycin-sensitive S6K1 isoforms are differently distributed between the cytoplasm and the nucleus of primary IMR-90 fibroblasts. (a) Non-transformed, non-immortalized human IMR-90 fibroblasts were examined for chromosomal abnormalities via standard karyotyping procedures (upper panel) and cytofluorometrically analyzed for DNA content on a fluorescence-activated cell sorter (FACS). Freshly isolated and cultured human amniotic fluid-derived cells (hAFCs) served as a control for a normal, diploid DNA content and were analyzed in parallel (lower panel, left). Additionally, IMR-90 were serum deprived (0.2% serum for 48 h) and restimulated with 10% serum for 27 h. FACS analyses of so treated cells were performed to verify suitability of IMR-90 fibroblasts for cell synchronization and restimulation experiments (lower panel, right). (b) IMR-90 were separated into cytoplasmic and nuclear fractions and examined via immunoblotting. Purity of obtained fractions was proven by analyzing a panel of different subcellular marker proteins as indicated. Whole cell lysate from the same pool of cells was analyzed in parallel. In terms of purity, these data are representative for all fractionation experiments presented in this study. (c) Cytoplasmic/nuclear fractionation of IMR-90, treated with dimethyl sulfoxide (DMSO) or 100 nM rapamycin for 24 h, was performed. Equal amounts of fractions (cytoplasmic/nuclear protein loading ratio ¼ 1:1) were analyzed for the mTOR-phosphorylated forms of p85 and p70 S6K1 (upper panel) and for the total expression of p85, p70 and p31 S6K (lower panels) via iummunoblotting. Asterisks indicate non-related, cross-reactive bands. (d) Experiments were performed as described in (c) except that the amount of nuclear protein loaded was five times higher compared with the amount of cytoplasmic protein loaded (cytoplasmic/nuclear protein loading ratio ¼ 1:5). Asterisks indicate non-related, cross-reactive bands. (e) Equal amounts of cytoplasmic and nuclear fractions of rapamycin-treated IMR-90 cells (100 nM for 24 h) were analyzed for phosphorylated and total forms of p85 and p70 S6K1 and for phosphorylated and total forms of the S6K1 substrates ribosomal protein S6 and mTOR via immunoblotting. Fibrillarin (a marker protein specifically expressed in the nucleolus) and atubulin serve as fractionation and loading controls. The asterisk indicates non-related, cross-reactive bands.

T389 S6K1-specific antibody. Upon treatment with the is a cell cycle-regulated enzyme and the p31 mobility mTOR-specific inhibitor rapamycin phosphorylation of shift is cell cycle regulated (Figure 4c). (3) We have p70 T389 and p85 T412 dissapeared, always accom- performed calf intestine alkaline phosphatase experi- panied by a shift to the faster migrating forms of these ments showing that p31 is a phosphoprotein and that proteins (Figures 2c–e, 4a and b). Furthermore, our the rapamycin-sensitive mobility shift reflects phospho- data suggest but not necessarily prove that p31 is a rylation (Figure 4b). phosphorylation target of mTOR: (1) treatment with As described above, incubation with rapamycin the mTOR-specific inhibitor rapamycin triggers a p31 induced a shift to the faster migrating form of p85 mobility shift (Figures 2c, d, 4a and b). (2) mTOR S6K1 in the cytoplasm and of p31 S6K1 in the nucleus.

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Figure 3 Cytoplasmic localization of p85 S6K1 in human amd murine fibroblasts. (a) p85 and p70 S6K1 were immunoprecipitated from IMR-90 cytoplasmic and nuclear fractions at indicated ratios using an anti-p70 S6K1 antibody and were analyzed for their expression via immunoblotting (IP, upper panel; C(1X) corresponds to 60 mg of cytoplasmic protein lysate, N(1X) and N(8X) correspond to 60 and 480 mg of nuclear protein lysate, respectively, used for immunoprecipitation). Reactions in which the precipitating antibody was omitted were analyzed in parallel and serve as a negative control. In addition, supernatants of precipitates were examined for the depletion of p85 and p70 S6K1, for the expression of fractionation/loading controls atubulin and fibrillarin and for the S240/ 244 phosphorylated form of the S6K target S6. The asterisk indicates an immunoreactive band originating from the previous fibrillarin detection (IP-SN, lower panel). (b) TSC2 þ / þ MEFs were fractionated and cytoplasmic and nuclear fractions of indicated protein loading ratios were examined for p85 and p70 S6K1 expression via immunoblotting. atubulin and fibrillarin were co-analyzed and serve as a control for proper fractionation and loading.

Figure 4 Altered gel migration properties of the phosphoprotein p31 S6K1 under growth inhibitory and stimulated conditions. (a) IMR-90 ﬁbroblasts were treated with 100 nM rapamycin for indicated times. Whole cell lysates were prepared and analyzed for the phosphorylation status of p85 and p70 S6K1 at T412 and T389, respectively (upper panel) and the expression level of p85, p70 and p31 S6K1 (lower panel) via immunoblotting using indicated antibodies. Asterisks indicate non-related, cross-reactive bands. (b) IMR-90 cells were either left untreated or were incubated with 100 nM rapamycin or DMSO (vehicle control) for 1 h. Whole cell lysates of so treated cells were examined for the expression level of p85, p70 and p31 S6K1 and for p70 S6K1 phosphorylation at T389. In parallel, lysates of logarithmically growing IMR-90 derived from the same pool of cells were treated with or without CIAP (calf intestine alkaline phosphatase) for 1 h at 37 1C and were co-analyzed in the same immunoblotting experiment. The asterisk indicates non- related, cross-reactive bands. (c) IMR-90 were either grown under basal growth conditions (log for logarithmically growing) or serum deprived in medium containing 0.2% serum for 48 h and serum restimulated for additional 9 h. So treated cells were harvested, stained with propidiumiodide and cytoﬂuorometrically analyzed for DNA content. In addition, total protein was extracted and lysates were examined for the expression of cell cycle marker proteins cyclin A, cyclin D1 and p27 (upper panel). Cells grown under inhibitory or stimulated conditions as described were examined for the phosphorylation of p85 and p70 S6K1 at T412 and T389, respectively, and for p31 S6K1 expression (lower panel).

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Figure 5 Rapamycin-mediated inhibition of p70 S6K1 phosphorylation prevents its nuclear localization. (a) IMR-90 cells treated with 100 nM rapamycin for 24 h and separated into cytoplasmic and nuclear fractions were examined for the distribution of phosphorylated and total forms of p85 and p70 S6K1 via western blotting. For better visualization of the phosphorylation-mediated S6K1 gel shift duplicates of lysates were resolved by 7.5% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were cut, separately incubated with antibodies as indicated and remerged for chemiluminescence detection allowing direct comparison of cytoplasmic and nuclear p70 S6K1 phosphorylated at multiple sites (a, b, g and d forms of p70 S6K1, left panels) and p70 S6K1 phosphorylated at T389 (g and d forms of p70 S6K1, right panels). Different exposures have been merged together to facilitate interpretation. The asterisk indicates non-related, cross-reactive bands. (b) Cells were treated with low doses of rapamycin for 1 h and examined for phopshorylated p85 and p70 S6K1 in whole cell lysates (left panel) and for distribution of total p85 and p70 S6K1 in subcellular fractions (right panels). Co-analyses of atubulin and topoIIb serve as fractionation and loading controls.

However, the localization of these two proteins localization were a direct consequence of mTOR kinase remained unchanged (Figures 2c and d). Rapamycin activity or were associated with putative secondary treatment also caused a shift to the faster migrating effects due to cell cycle arrest. To answer this question form of p70 S6K1 in the cytoplasm, but triggered we performed 1 h short-term experiments using differ- complete loss of this protein in the nucleus (Figures 2c–e). ent concentrations of the mTOR-inhibitor rapamycin. Taken together, these data demonstrate that although We found the effects on p70 localization to be titratable mTOR is not involved in the localization of p85 and p31, depending on the remaining mTOR activity and to be p70 S6K1 nucleocytoplasmic localization is stringently independent of any mTOR-mediated cell cycle effects controlled via this kinase. (Figure 5b). The findings described above suggest that only phosphorylated p70 S6K1 is localized to the nucleus. To allow the detection of the different phosphorylated/ Loss of tuberin triggers increased nuclear p70 S6K1 hyperphosphorylated p70 forms a, b, g, d (Cheatham As described above, blocking mTOR activity triggers et al., 1995; Kanazawa et al., 2004) we next resolved delocalization of p70 S6K1, but is without effects on p85 IMR-90 protein fractions by 7.5% SDS gel conditions. and p31 localization. We next wanted to test whether These experiments revealed that indeed only the activation of mTOR can induce nuclear p70 localiza- hyperphosphorylated forms of p70 S6K1 (correspond- tion. To investigate this question we decided to ing to T389 phosphorylated p70 S6K1) localize to the modulate the activity of tuberin, which is the main nucleus. Rapamycin treatment confirmed again that negative regulator of mTOR (see introduction). hypophosphorylated p70 remains in the cytoplasm but Analyses using TSC2 þ / þ (MEFs) confirmed that p85 completely disappears from the nucleus (Figure 5a). is cytoplasmic whereas p70 can be detected in both, All rapamycin experiments described so far were long- the cytoplasm and the nucleus. Increased mTOR- term treatments for 24 h known to also trigger a mediated phosphorylation of p70 and p85 (studied pronounced cell cycle arrest of IMR-90 cells. The 1 h via the phospho-T389/T412-specific antibody and the short term incubation with rapamycin does not affect appearance of the slower migrating protein forms) the cell cycle of these cells (Rosner and Hengstschla¨ger, upon loss of TSC2 in TSC2À/À cells induces nuclear 2008; Rosner et al., 2009). Accordingly, it was of interest localization of p70 S6K1 but is without effects on to investigate whether the obtained effects on p70 S6K1 p85 localization (Figures 6a and b). Short-term serum

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Figure 6 Loss of TSC2 induces p70 S6K1 nuclear localization. (a) TSC2 knockout MEFs were grown in medium containing 10% serum (basal) or were starved in medium without serum (0% serum) for 12–16 h (starvation). Whole cell lysates were examined for the expression of tuberin, phosphorylated p85 and p70 S6K1 and phosphorylated S6 (upper panel, left). Additionally, DNA content was determined on a flowcytometer (lower panel, left). Cytoplasmic/nuclear fractions of the same pool of cells were analyzed for distribution of phosphorylated and total forms of p85 and p70 S6K1 via immunoprecipitation and immunoblotting (IP:a-p70 S6K1, IB:a-p70 S6K1 T389 and a-p70 S6K1) (right panels). (b) For short-term serum stimulation experiments TSC2 MEFs were starved overnight as described above and restimulated with 10% serum for 30 min. Where indicated, rapamycin or wortmannin was added 30 min before restimulation at a concentration of 100 nM. Whole cell lysates were examined for tuberin and phosphorylated and total forms of p70 S6K1 and S6 (left panel). Subcellular distribution of p70 S6K1 was analyzed in cytoplasmic and nuclear fractions (cytoplasmic:nuclear protein loading ratio ¼ 1:5) via immunoprecipitation and immunoblotting (IP:a-p70 S6K1, IB:a-p70 S6K1 T389 and a-p70 S6K1). Equal loading and purity of fractions was proven by co-analysing supernatants of precipitates for atubulin and topoIIb (right panel). (c) Experiments described in (a) were repeated with IMR-90 cells transfected with TSC2-specific or non-targeting siRNAs (si control). Whole cell lysates and subcellular fractions were investigated for the expression of indicated proteins via immunoblotting (left panel) or via immunoprecipitation and subsequent immunoblotting (IP:a-p70 S6K1, IB:a-p70 S6K1 T389 and a-p70 S6K1) (right panel). starvation triggers downregulation of mTOR activity and p70 is the same in murine embryonic fibroblasts and accompanied by loss of nuclear p70 S6K1 localization in in primary human fibroblasts (IMR-90), (2) that in both TSC2 þ / þ cells (again without effects on p85 localiza- cells induction of mTOR activity via downregulation of tion). The observation that p70 S6K1 localization is tuberin induces nuclear localization of p70, but is insensitive to serum starvation in TSC2À/À cells proves without effects on p85 localization, (3) that serum this serum-mediated regulation to be tuberin-dependent. regulates p70 localization, but not the localization of (Figures 6a and b). These results in murine cells were p85, in a PI3K/TSC2/mTOR-dependent manner. confirmed in human cells by downregulation of endogenous tuberin via TSC2-specific siRNA treatment of IMR-90 cells (Figure 6c). mTOR-mediated phosphorylation of p70 S6K1 at T389 is Furthermore, short-term restimulation of starved essential for its nuclear localization MEFs revealed that serum can induce nuclear p70 To determine whether phosphorylation at T389 or the S6K1 localization. Blocking mTOR activity, either by kinase activity of p70 S6K1 is required for nuclear the mTOR-inhibitor rapamycin or the PI3K-inhibitor translocation (or merely correlates with nuclear translo- wortmannin showed that this serum-induced nuclear cation), we performed experiments with different p70 localization of p70 depends on the PI3K/TSC2/mTOR S6K1 mutants (Figure 7a). First, we proved that wild- pathway (Figure 6b). In summary, these results demon- type p70 S6K1, ectopically expressed in primary IMR- strate (1) that the nucleocytoplasmic localization of p85 90 cells, is regulated and localized comparable to the

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4516

Figure 7 mTOR-dependent phosphorylation of p70 S6K1 at T389 is indispensable for its nuclear localization. (a) Schematic presentation of S6K1-constructs used in this study. (b) IMR-90 fibroblasts were transfected with plasmids harboring wild-type p70 S6K1 (S6K1-WT) or the empty vector control and were further treated with rapamycin under basal growth conditions or starvation/ restimulation conditions according to the experimental flow chart presented (TR ¼ transfection, þ R ¼ addition of rapamycin at a final concentration of 100 nM, S ¼ serum starvation, 0% serum, RS ¼ serum restimulation, 10% serum). Whole cell lysates were prepared and S6K1 expression and phosphorylation was examined via immunoblotting. (c) Transiently transfected and rapamycin-treated IMR- 90 cells were separated into cytoplasmic and nuclear fractions and analyzed for the distribution of ectopic p70 S6K1 via immunoblotting. The asterisk indicates non-related, cross-reactive bands. (d) Whole cell lysates and subcellular fractions of IMR-90 cells overexpressing either wild-type or a TOS motif-mutated form of p70 S6K1 (S6K1-DTOS) were analyzed for p70 S6K1 phosphorylation at T389 (left panel) and p70 S6K1 distribution (right panel), respectively. (e) Whole cell lysates of IMR-90 cells transiently transfected with S6K1-WT, S6K1-DTOS or a kinase-dead form of p70 S6K1 (S6K1-KD) were further treated as indicated (compare flow chart) and were subjected to immunoblot analyses of the T389 phosphorylated form of p70 S6K1 (left panel). Fractions derived from the same pool of cells were analyzed for the cytoplasmic/nuclear distribution of total p70 S6K1 (right panel). (f) IMR-90 fibroblasts transfected with either S6K1-WT or a rapamycin-resistant mutant of p70 S6K1 (S6K1-RR) were further treated with or without 100 nM rapamycin for 24 h and lysed to obtain extracts of cellular total protein and cytoplasmic and nuclear fractions. Whole cell lysates were examined for the phosphorylation status of ectopic p70 S6K1 at T389 via immunoprecipiation and immunoblotting of HA-tagged proteins (upper panel, left, IP-SN ¼ supernatant of precipitates) and for the phosphorylation status of the S6K1 substrate S6 at S240/244 (upper panel, right) via immunoblotting; subcellular fractions were analyzed for the distribution of transfected proteins (middle panel) and for the distribution of the phosphorylated form of the S6K1 target S6 (lower panel). (g) Immunoblot analyses of ectopic p70 S6K1 in cytoplasmic and nuclear fractions of IMR-90 cells transiently transfected with S6K1-WT or an N- and C-terminal S6K1 truncation mutant (S6K1-DNTDCT). Asterisks indicate non-related, immunoreactive background bands (in Figures 7b–g co-analyses of atubulin and fibrillarin have been included and serve as fractionation and loading controls). (h) Brief overview summarizing the main features of studied S6K mutants with respect to phosphorylation status at T389, in vitro kinase activity, eIF3-PIC (translation preinitiation complex) binding and nuclear localization (ND, not determined). Presented data originate from this study and studies previously published and referenced/discussed in the text.

endogenous protein (Figures 7b and c). The S6K1- pared to the wild-type protein (Figures 7d and e). The DTOS (S6K1-F5A) mutant, which cannot bind to raptor kinase-dead mutant S6K1-KD (S6K1-K100R, mutated and is therefore neither phosphorylated at T389 (with in the ATP binding site), which in addition to its loss of other S6K1-phosphorylation sites remaining largely kinase activity is also not T389 phosphorylated (Chea- unaffected by the mutation) nor exerts kinase activity tham et al., 1995; Holz et al., 2005; Figure 7e), also (Schalm and Blenis, 2002; Holz et al., 2005; Figures 7d exhibited diminished nuclear localization (Figure 7e). and e), exhibits diminished nuclear localization com- Vice versa, the constitutively active, rapamycin-resistant

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4517 mutant S6K1-RR (S6K1-F5A-E389-R3A; Schalm et al., processes, including, for example, translation, ribosome 2005), which remains to be phosphorylated at T389 biogenesis or transcription. Deregulation of mTOR is upon rapamycin treatment (Figure 7f), retains its involved in tumor development, differentiation pro- nuclear localization compared with the wild-type cesses, apoptosis and cell size control (Yang and Guan, protein (Figure 7f). In addition, we found a p70-S6K1- 2007; Wang and Proud, 2009). The specific roles of the truncated protein without the N-terminal TOS motif, different mTOR targets for these different effects are without the C-terminal autoinhibitory domain (its loss poorly understood. To allow a better understanding of partially reverts the negative effects of loss of functional these regulations it is required to examine the temporal TOS motif in respect to T389 phosphorylation and and spatial dynamics of endogenous mTOR targets. kinase activity) and without the PDZ-binding motif Compartmentalization could keep different upstream (Cheatham et al., 1995; Weng et al., 1995; Schalm et al., regulators and downstream effectors of mTOR in 2005) to exhibit proper localization (Figure 7g). different localizations allowing coordinated regulations Taken together, these findings demonstrate that the in response to different stimuli or throughout cellular mTOR-dependent phosphorylation of p70 S6K1 at T389, processes, such as differentiation, apoptosis or cell cycle respectively the p70 S6K1 kinase activity, determines p70 (Shaw, 2008; Sengupta et al., 2010). S6K1 nucleocytoplasmic localization (Figure 7h). In this study we compared the nucleocytoplasmic localization of the different isoforms of S6K1, a major The nucleocytoplasmic p70 S6K1 localization is regulated target of mTORC1. This is of special interest as a variety throughout the cell cycle via TSC2/mTOR of nuclear and cytoplasmic substrates of S6K1 proteins The results presented in Figure 6 demonstrate that short- have been described (Ruvinsky and Meyuhas, 2006; term (30 min) serum restimulation triggers p70 S6K1 Meyuhas and Dreazen, 2010; Fenton and Gout, 2011). nuclear localization in a PI3K/TSC2/mTOR-dependent Using primary non-immortalized, non-transformed hu- manner. As 30 min restimulation does not induce G0 man fibroblasts we found p85 to be cytoplasmic, p31 to cells to enter the cell cycle, these observations prove be nuclear and p70 S6K1 to be localized to both this regulation to be a direct consequence of mTOR compartments. This is the first comparative analysis of activity rather than of secondary cell cycle effects. the nucleocytoplasmic localization of all three S6K1 The results made it interesting to investigate the cell proteins. The S6K1 isoform p31 is generated via cycle regulation of the nuclear localization of p70 S6K1. alternative splicing mediated by SF2/ASF. It was IMR-90 cells were synchronized in G0/G1 via serum reported that p70 S6K1 has little oncogenic potential, deprivation. Flow cytometric analyses of the amount of but the alternatively spliced p31 S6K1 is sufficient to G0/G1, S and G2/M phase cells and western blot cause cellular transformation (Karni et al., 2007). These analyses of typically cell cycle regulated proteins, such as findings on this dramatic difference between the two jun, cyclin D1, cyclin A or p27, were performed to S6K1 isoforms made it interesting to investigate how the qualify the re-entering of these cells upon serum properties of S6K1 may be altered as a consequence of stimulation (Figure 8a). the splicing switch. We here report that this splicing Although cytoplasmic p70 S6K1 can be detected in all switch triggers different localization of the oncogenic phases of the cell cycle, this protein becomes nuclear and the non-oncogenic S6K1 isoforms. Taken together, only in mid G1 phase. Comparing the pattern of total alternative splicing via SF2/ASF results in an oncogenic p70 S6K1 protein with the cell cycle regulation of p70 smaller p31 S6K1 isoform 2, which in contrast to the S6K1 protein phosphorylated at T389 demonstrated non-oncogenic p70 S6K1 isoform 1 exclusively localizes that in G0/G1 hypophosphorylated protein exists in the to the nucleus. cytoplasm. However, in the nucleus total p70 protein SF2/ASF and p31 have been reported to be upregu- et al. cannot be detected before it becomes phosphorylated via lated in breast cancer cell lines (Karni , 2007). mTOR (Figure 8a). Furthermore, induction of mTOR Using a different set of breast cancer cell lines and activity via a combined approach of serum restimulation normal counterparts we confirmed this observation in and TSC2-specific siRNA treatment is able to induce the the presented study here. Analyzing a set of bladder nuclear localization of phosphorylated p70 S6K1 in G0/ cancer cell lines we here provide first evidence that G1 (Figure 8b). overexpression of SF2/ASF accompanied by induced These findings confirm that the mTOR-dependent levels of p31 S6K1 could also be involved in the phosphorylation induces nuclear localization of p70 molecular development of bladder cancer. S6K1. In addition, we report for the first time that the Although this is the first analysis of the nucleocyto- nucleocytoplasmic localization p70 is strictly cell cycle- plasmic localization of p31, the localization of p70 and dependent and that the TSC2/mTOR pathway is p85 has already been investigated in the past. In responsible for this regulation. agreement with our data, p70 S6K1 has always been localized to both, cytoplasm and nucleus, but the underlying regulating mechanism remained elusive (Coffer and Woodgett, 1994; Kim and Kahn, 1997; Discussion Kim and Chen, 2000). An earlier report described growth factor-dependent fluctuation of S6K1 localiza- The PI3K/TSC2/mTOR cascade is known to have tion. This report could not investigate the later important roles in many cytoplasmic and nuclear discovered p31 isoform and moreover did not experi-

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4518

Figure 8 Cell cycle-dependent nucleocytoplasmic distribution of phosphorylated p70 S6K1 is regulated by TSC2. Non-treated IMR- 90 or IMR-90 with modulated TSC2 expression were synchronized in G0/G1 via serum deprivation and serum restimulated to re-enter the cell cycle. (a) Schematic outline of the synchronization procedure. Briefly, cells growing under full serum conditions (10% serum) for 24–72 h were serum deprived in medium containing 0.2% serum for 48 h and serum restimulated (10% serum) for additional 36 h. At indicated time points of restimulation cells were harvested and cytofluorometrically analyzed for DNA content. Representative DNA profiles and the relative amount of cells in different cell cycle phases are presented. In addition, whole cell lysates were prepared and examined for the expression level of cell cycle regulated proteins jun, cyclin D1, cyclin A and p27 via immunoblotting. Subcellular fractions of the same pool of cells were analyzed for the distribution of phosphorylated and total p70 S6K1. Equal loading and purity of fractions was proven by co-analysing atubulin and topoIIb.(b) Experiments were performed as described in (a) except that cells were transfected with TSC2-specific or non-targeting siRNAs 48 h before restimulation. Also compare schematic outline of the procedure presented. At indicated time points, cells were harvested and investigated for DNA distribution and expression of tuberin, cyclin D1, atubulin and phosphorylated and total p70 S6K1. In addition, cytoplasmic and nuclear fractions were prepared and analyzed for T389 phosphorylated p70 S6K1 via immunoblotting.

mentically differentiate between the p70 and p85 specific siRNAs revealed that mTOR regulates the proteins (Edelmann et al., 1996). nucleocytoplasmic localization of p70, but not of p31 The S6K1 isoforms p70 and p85 are well known to be and p85 S6K1. Activation of mTOR induces nuclear targets of mTOR-mediated phosphorylation (see Intro- p70 S6K1 localization and inhibition of mTOR leads to duction). Data presented in this report suggest but not loss of nuclear p70. We found that only mTOR- necessarily prove that p31 is also a phosphorylation mediated hyperphosphorylated p70 S6K1 is located to target of mTOR. We found rapamycin treatment to the nucleus. This is the first demonstration that mTOR trigger a mobility shift of p31 in western blot analyses. not only regulates p70 S6K1 activity, but also its This p31 mobility shift was proven to be the conse- localization. As we and other colleagues have found quence of protein phosphorylation and is cell cycle- little to no mTORC1 kinase complex in the nucleus regulated correlating with mTOR activity. (Rosner and Hengstschla¨ger, 2008; Sancak et al., 2008), However, inhibiting endogenous mTOR activity via it is very likely that mTOR-mediated phosphorylation of rapamycin and activation of mTOR in TSC2À/À MEFs p70 S6K1 in the cytoplasm triggers its localization into and in IMR-90 human fibroblasts treated with TSC2- the nucleus. This notion is further supported by our

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4519 finding that during restimulation of G0 cells into the cell demonstrate this regulation of phosphorylation to be cycle mTOR-mediated phosphorylation of p70 S6K1 in responsible for p70 nuclear localization. On the other the cytoplasm occurs at earlier time points than it is hand, one could speculate that binding to eIF3-PIC detectable in the nucleus. Furthermore, performing might have an active role in p70 S6K1 cytoplasmic short-term serum starvation and restimultation experi- retention. In future, it will be of interest to clarify this ments we here demonstrate that the growth factor- issue. mediated localization of p70 S6K1 is regulated via It was earlier reported that phosphorylation of p70 PI3K/TSC2/mTOR. (1) Serum starvation triggers S6K1 via CK2 protein kinase promotes its export from downregulation of mTOR activity accompanied by loss the nucleus, but this regulation is not responsible for the of nuclear p70 S6K localization. (2) Our finding that above described growth factor-dependent fluctuation of p70 S6K1 localization is insensitive to serum starva- nucleocytoplasmic p70 localization (Panasyuk et al., tion in TSC2À/À cells demonstrates this regulation to be 2006). tuberin dependent. (3) All these results obtained in In the past, two arguments have often been cited to murine cells were confirmed in human cells by down- support the notion that p85 S6K1 is a nuclear protein: regulation of endogenous tuberin via TSC2-specific the first 23 amino acids contain a suggested nuclear- siRNA treatment. (4) Blocking mTOR activity, either localization-signal and an immunocytochemical ap- by the mTOR-inhibitor rapamycin or the PI3K-inhibi- proach using a specific antibody provided evidence for tor wortmannin again showed that the serum-induced nuclear localization (Reinhard et al., 1994). However, nuclear localization of p70 depends on the PI3K/TSC2/ we show here that p85 S6K1 is not a nuclear but a mTOR pathway. cytoplasmic protein in non-transformed, non-immorta- The analyses of a variety of different p70 S6K1 lized primary human cells and in MEFs. During the mutants provided more detailed insights into the course of our project, a report has been published relevance of the different p70 S6K1 protein motifs describing a similar observation on p85 localization for nucleocytoplasmic localization (summarized in (Kim et al., 2009). Here it is important to note that Figure 7h). Studying two mutants, which cannot be earlier immunocytochemical studies could have used phosphorylated at T389, and one T389 phosphoryla- antibodies, which must be clarified for their potential to tion-mimicking mutant demonstrated the requirement differentiate between the three different S6K1 isoforms. of this site-specific phosphorylation for p70 S6K1 In long-term restimulation experiments we further nuclear localization. These ectopic expression experi- found that re-entry of G0 cells into the cell cycle triggers ments of different mutants further revealed that both, nuclear localization of p70 S6K1 in an mTOR- the TOS motif of p70 S6K1 (essential for binding to the dependent manner. Furthermore we found that cyto- mTORC1 component raptor) and the ATP binding site plasmic p70 S6K1 protein can be detected throughout are indispensable for proper p70 localization. In the entire cell cycle but p70 does not enter the nucleus addition, we found a p70-S6K1-truncated protein with- before mid G1 phase. We demonstrated that induction out the N-terminal TOS motif, without the C-terminal of mTOR activity via TSC2-specific siRNA treatment is autoinhibitory domain (its loss partially reverts the able to induce the nuclear localization of phosphory- negative effects of loss of functional TOS motif in lated p70 S6K1 in G0/G1. These data prove that the cell respect to T389 phosphorylation and kinase activity) cycle-dependent nuclear localization of p70 S6K1 is and without the PDZ binding motif to exhibit proper mediated by its phosphorylation at T389 via mTOR. localization. From the here obtained results it appeared For a better understanding and for further investiga- that the latter mutant missing the PDZ binding motif, tions of the regulation of the different cytoplasmic and earlier shown to be involved in the binding of p70 S6K1 nuclear targets of p70 S6K1, the discovery of this cell to neurabin (reviewed in Fenton and Gout, 2011), even cycle control is of highest relevance. exhibited an increased nuclear localization (Figure 7g). Deregulation of mTOR is a hallmark of many human Further experiments are warranted to investigate a cancers and genetic diseases. Owing to their antiproli- putative role of this motif in cytoplasmic retention of ferative and immunosuppressive potential, rapamycin p70 S6K1. and related drugs (analogs) are being currently eval- It was earlier reported that unphosphorylated p70 uated in many clinical trials for a variety of different S6K1 binds to a protein complex named eIF3-PIC cancers, for diabetes and as parts of specific transplant (translation preinitiation complex). This complex has immunosuppresion regimens (Chiang and Abraham, been discussed to function as a scaffold on which mTOR 2007; Plas and Thomas, 2009). Especially as it already phosphorylates p70 S6K1 at T389, what triggers became evident that different tumors (as well as dissociation of p70 S6K1 from eIF3-PIC (Holz et al., different patients) respond differently to rapamycin, 2005). We found that two p70 S6K1 mutants, earlier a more detailed understanding of the spatial and shown to bind this complex, exhibit diminished nuclear temporal intracellular effects of rapamycin-induced localization, whereas one p70 mutant defective in mTOR inhibition is critical for this drug to most binding this protein complex (Holz et al., 2005), shows effectively be used in the clinic. Our observation that induced nuclear localization (Figure 7). On the one rapamycin triggers different effects on the three S6K1 hand, these data could be interpreted to only be a forms in the cytoplasm and in the nucleus might be of consequence of the correlation of eIF3-PIC binding and relevance for the future usage of this mTOR-inhibitor S6K1 phosphorylation taking into account that we in clinical trials for different cancer syndromes and

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4520 human genetic diseases involving deregulation of differ- nized cells were fixed by rapid submersion in ice-cold 85% ent S6K1 proteins. ethanol. After overnight fixation at À20 1C, DNA was stained with 0.25 mg/ml propidium iodide, 0.05 mg/ml RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8. DNA distribution of a total of 2 Â 104–4 Â 104 cells per sample was analyzed on a Materials and methods Beckton Dickinson FACScan (Becton Dickinson, San Jose, CA, USA). Karyotyping of primary IMR-90 fibroblasts was Cells performed according to standard procedures. Chromosome IMR-90 (#CCL-186, American Type Culture Collection (ATCC), banding was produced by means of a conventional 550-band Wesel, Germany) are fetal lung-derived, non-transformed, trypsin-Giemsa analysis (GTG) and a significant number of non-immortalized human diploid fibroblasts with finite life- metaphases were analyzed for numerical and structural time and were obtained at passage number 10 (population chromosome aberrations (Hengstschla¨ger et al., 2001). doubling 25). To avoid potential effects due to cellular senescence, cells were grown for no more than eight addi- RNA interference tional passages (p47 total population doublings) and were regularly analyzed by standard karyotyping to confirm a The siRNA transfection experiments were performed using normal diploid karyotype. Mammary gland-derived cell lines Lipofectamine 2000 transfection reagent (TSC2-specific siR- analyzed in this study include CRL7347, CRL7379, CRL7847, NAs) or Lipofectamine RNAiMAX (S6K1-specific siRNAs) all obtained from apparently normal tissue of patients with (both Invitrogen) following the guidelines provided by the manu- a carcinoma of the breast, MCF10a, an immortalized breast facturer. Pooled siRNAs specifically targeting human TSC2 or cell line from a patient with fibrocystic disease, and MCF7, human S6K1 (Dharmacon, ON-TARGETplus SMART pool BT474, MDA-MB 468 and ZR-75-1, four established breast reagents, Dharmacon, Lafayette, CO, USA) were delivered to the cancer cell lines. Urothelial cell lines used are HCV29, derived cells at a final concentration of 100 nM and 50 nM, respectively. from normal, non-malignant ureteric epithelium of a patient A pool of four non-targeting siRNAs was used as a control for with transitional cell carcinoma and bladder tumor-derived non-sequence-specific effects. After 96 h of incubation, cells were cell lines 5637, RT4, RT112 and T24. In addition, HeLa harvested for analyses (Rosner et al., 2009, 2010). Combined cervical adenocarcinoma cells, HEK293 human embryonic approaches of cell synchronization and siRNA transfection are kidney cells, NIH3T3 mouse fibroblasts, immortalized MEFs outlined in the corresponding Figure and Figure legend. (TSC2 þ / þ and TSC2À/À) and primary human amniotic fluid cells (hAFCs) were analyzed. All cells are available from Plasmid constructs and transfection ATCC, except two lines of the collection of urothelial cell HA-S6K1 constructs used in this study were generated in the lines (Knowles et al., 2003) and the TSC knockout MEFs used laboratories of Drs J Blenis and J Avruch and were purchased here, kindly provided by Drs M Knowles (Leeds, UK) and via Addgene (Cambridge, MA, USA): HA-S6K1 wild type, D Kwiatkowski (Boston, MA, USA), respectively. Primary HA-S6K1-DTOS (S6K1-F5A), HA-S6K1-KD (HA-S6K1- hAFCs were obtained from amniocentesis performed for K100R, kinase-dead), HA-S6K1-RR (HA-S6K1-F5A-E389- routine prenatal diagnosis. Usage of hAFCs was approved R3A, rapamycin-resistant/constitutively active) and HA- by the ethics committee of the University of Vienna (project S6K1-DNTDCT (deletion of the N- and C-termini retaining number: 036/2002). All cells were grown at 37 1C and 5% CO2. the kinase domain and the hydrophobic motif at T389). Media used for cell cultivation were the following: Dulbecco’s Plasmids were transfected into IMR-90 fibroblasts using modified Eagle’s medium (PAA, Pasching, Austria) at 4.5 g/l Lipofectamine 2000 transfection reagent. glucose for IMR-90, HeLa, HEK293, NIH3T3, TSC MEFs, CRL7347, CRL7379, CRL7847 and MCF7. Dulbecco’s Preparation of whole cell lysates and cytoplasmic/nuclear modified Eagle’s medium/F12 (Invitrogen, Lofer, Austria) fractions (supplemented with EGF and insulin, both form Sigma, Whole cell lysates containing both, cytoplasmic and nuclear Vienna, Austria) for MCF10a. RPMI/NCTC135 (Invitrogen) proteins, were prepared by physical disruption of cell (supplemented with insulin) for BT474, McCoy’s Medium membranes by repeated freeze and thaw cycles. Briefly, cells (PAA) for MDA-MB 468, RT4 and T24, RPMI (PAA) for were washed with PBS and harvested by trypsinization. Pellets ZR-75-1 and urothelial cell lines HCV29, 5637 and RT112 were washed twice with ice-cold PBS and lysed in buffer A and Ham’s F10 (Invitrogen) (supplemented with Ultroser G, containing 20 mM Hepes, pH 7.9, 0.4M NaCl, 25% glycerol, PALL, Vienna, Austria) for hAFCs. All media were supple- 1mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfo- mented with 10% calf serum, 2 mML-Glutamine, antibiotics nyl fluoride, 0.5 mM NaF, 0.5 mM Na3VO4 supplemented with (60 mg/l penicillin, 100 mg/l streptomycin sulfate) and addi- 2 mg/ml aprotinin, 2 mg/ml leupeptin, 0,3 mg/ml benzamidin- tives as indicated. chlorid, 10 mg/ml trypsininhibitor by freezing and thawing. Supernatants were collected by centrifugation at 20 000 g for Cell culture and reagents 20 min at 4 1C. For cytoplasmic and nuclear fractionation cell For short-term serum stimulation experiments cells were pellets were lysed in five packed cell volume buffer F1 con- starved in growth medium without serum (0% serum) for taining 20 mM Tris–HCl, pH 7.6, 50 mM 2-mercaptoethanol, 12–16 h and then treated and stimulated as indicated. For 0.1 mM EDTA, 2 mM MgCl2,1mM phenylmethylsulfonyl G0/G1 cell synchronization experiments, IMR-90 fibroblasts fluoride supplemented with protease inhibitors (2 mg/ml at passage number 15 were deprived of serum in growth aprotinin, 2 mg/ml leupeptin, 0,3 mg/ml benzamidinchlorid, medium containing 0.2% serum for 48 h. Cells were stimulated 10 mg/ml trypsininhibitor) for 2 min at room temperature and to re-enter the cell cycle by the addition of 10% serum for subsequent incubation on ice for 10 min. Thereafter NP-40 was another 36 h. Experiments with the mTOR inhibitor rapa- added at a final concentration of 1% (v/v) and lysates were mycin or the PI3K inhibitor wortmannin (both Calbiochem, homogenized by passing through a 20-gauge needle or La Jolla, CA, USA) were performed in the absence (DMSO equivalent through a 200 ml tip for three times. Nuclei were vehicle control) or presence of the drug at indicated pelleted by centrifugation at 600 g for 5 min at 4 1C and concentrations. For cytofluorometric DNA analyses trypsi- supernatant containing cytoplasmic proteins was collected and

Oncogene Nucleocytoplasmic localization of S6K1 isoforms M Rosner and M Hengstschla¨ger 4521 stored at À80 1C. Remaining nuclei were washed three times in specific for the following proteins were used: p70S6K (clone buffer F1 containing 1% NP-40. Pellets of pure nuclei were 49D7), N-term (#2708, Cell Signaling), p70S6K T389 (clone lysed in buffer containing 20 mM Hepes, pH 7.9, 0.4M NaCl, 108D2) (#9234, Cell Signaling), SF2/ASF (clone 96) (#32– 25% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl 4500, Invitrogen), tuberin C-20 (#sc-893, Santa Cruz), cyclin A fluoride, 0.5 mM NaF, 0.5 mM Na3VO4, 0.5 mM dithiothreitol, H-432 (#sc-751, Santa Cruz), cyclin D1 M-20 (#sc-718, Santa supplemented with protease inhibitors by repeated freezing Cruz), p27Kip1 (clone 57) (#K25020, Transduction Labora- and thawing. Supernatants containing soluble nucleic proteins tories, Becton Dickinson), atubulin (clone DM1A) (#CP06, were collected by centrifugation at 20 000 g for 20 min (Rosner Calbiochem), SOD-2 (#SOD-110, Stressgen Biotechnologies, et al., 2007; Rosner and Hengstschla¨ger, 2008). San Diego, CA, USA), calnexin (clone 37) (#610524, Transduction Laboratories), jun (clone 3) (#610326, Transduc- Phosphatase treatment tion Laboratories), topoisomerase IIb (clone 40) (#611492, For dephosphorylation of protein extracts cells were lysed in Transduction Laboratories), lamin A/C (clone 14) (#612162, calf intestine alkaline phosphatase buffer containing 20 mM T, Transduction Laboratories), HSP90 (#4874, Cell Signaling), importin (or karyopherin a, 2, #610486, Transduction pH 8.0, 150 mM NaCl, 1 mM MgCl2,1mM dithiothreitol, 0.5% Triton X-100 supplemented with protease inhibitors (Shin et al., Laboratories), S6 ribosomal protein (clone 54D2) (#2317, Cell 2005). Lysates were treated with or without 0.4 units of calf Signaling), S6 ribosomal protein S240/244 (#2215, Cell intestine alkaline phosphatase (Promega, Mannheim, Germany) Signaling), mTOR (#2972, Cell Signaling), mTOR S2448 per mg protein for 1 h at 37 1C. Reactions were stopped by (#2971, Cell Signaling), fibrillarin (clone C13C3) (#2639, Cell Signaling) and HA (clone 3F10) (#11867423001, Roche). adding phosphatase inhibitors (1 mM Na3VO4,50mM NaF) and boiling in SDS sample buffer at 95 1C for 5 min. Rabbit polyclonal and monoclonal antibodies were detected using anti-rabbit immunoglobulin G, a horseradish peroxidase-linked heavy and light chain antibody from goat (#A120- Immunoprecipitation and immunoblotting 101P, Bethyl Laboratories, Montgomery, TX, USA); mouse In some cases where indicated, S6K1 was immunoprecipitated monoclonal antibodies and rat anti-HA antibody were from total lysates or subcellular fractions before immunoblot- detected using anti-mouse immunoglobulin G, a horseradish ting. For immunoprecipitation crude cell extracts (150 mgof peroxidase-linked heavy and light chain antibody from goat total lysates or 350 mg of cytoplasmic and nuclear fractions) (#A90-116P, Bethyl Laboratories) and anti-rat IgG1, a horse- were diluted in NP-40-containing total cell lysis buffer and radish peroxidase-linked heavy and light chain antibody from precleared with 30 ml protein A/G-Sepharose beads (Pierce, goat (#A110-106P, Bethyl Laboratories), respectively; Signals Thermo Fisher Scientific, Vienna, Austria) for 30–60 min at were detected with the enhanced chemiluminescence method 1 4 C. Indicated primary antibodies were added to the cleared (Pierce) (Rosner et al., 2007). To avoid any interference in the lysates and incubated with constant rotation for 12–16 h sequential detection of S6K1 and phosphorylated S6K1 (both 1 (overnight) at 4 C. A volume of 30 ml of a 50% slurry of detected by rabbit polyclonal antibodies) on the same protein A/G sepharose were then added and the incubation membrane, same lysates or precipitates were divided into 1 continued for another 90 min at 4 C. So captured immuno- equal amounts (50:50 ratio), loaded and detected separately precipitations were washed four times with lysis buffer with antibodies against S6K1 and phosphorylated S6K1. containing 50 mM Tris–HCl, pH 8.0, 1% NP-40, 150 mM NaCl, 10 mM b-glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride supplemented with protease inhibitors aprotinin, leupeptin, benzamidinchlorid and trypsinin- Abbreviations hibitor as described above. The final washing step was carried out in wash buffer containing 10 mM Tris pH 7.5 and 0.1% NP-40. mTOR, mammalian target of rapamycin; PI3K, phosphatidy- Immunoprecipitated proteins were then denatured and sepa- linositol-3-kinase; siRNA, small interfering RNA; S6K, ribosomal rated from the sepharose beads by adding SDS-sample buffer protein S6 kinase; TSC2, tuberous sclerosis complex gene 2. and boiling for 5 min (Rosner et al., 2007). For immunoprecipitation antibodies specific for the following proteins were used: rabbit anti-p70S6K (C-18), C-term (#sc-230, Santa Cruz Conflict of interest Biotechnology, Santa Cruz, CA, USA), rabbit anti-p70S6K (clone 49D7), N-term (#2708, Cell Signaling, Danvers, MA, USA) The authors declare no conflict of interest. and HA (clone 3F10) (#11867423001, Roche, Basel, Switzerland). Equal amounts of denatured samples with or without previous immunprecipitation (10–25 mg, cytoplasmic/nuclear ratio of used/loaded protein amount ¼ 1:1, unless otherwise Acknowledgements indicated) were resolved by 7.5 or 8.5% (S6K mobility shift), 10% or 12.5% SDS–polyacrylamide gel electrophoresis accord- Research in our laboratory is supported via grants from ing to the molecular weight of the analyzed proteins and the Herzfelder’sche Familienstiftung and the O¨sterreichische transferred to nitrocellulose. For immunodetection antibodies Nationalbank.

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