Receptor association and tyrosine phosphorylation of S6 kinases Heike Rebholz 1,2 , Ganna Panasyuk 3 , Timothy Fenton 1,2 , Ivan Nemazanyy 3 , Taras Valovka 4 , Marc Flajolet 5 , Lars Ronnstrand 6 , Len Stephens 7 , Andrew West 7 and Ivan T. Gout 2,3 1 Ludwig Institute for Cancer Research, London, UK 2 Department of Biochemistry and Molecular Biology, University College London, UK 3 The Institute of Molecular Biology and Genetics, Kyiv, Ukraine 4 Institute of Veterinary Biochemistry and Molecular Biology, University Zurich, Switzerland 5 Rockefeller University, New York, NY, USA 6 Lund University, Department of Experimental Clinical Chemistry, Malmo, Sweden 7 Babraham Institute, Cambridge, UK 8 GlaxoSmithKline, Harlow, UK Keywords AGC kinases; platelet-derived growth factor receptor; receptor tyrosine kinases; ribosomal protein S6 kinase; src Correspondence H. Rebholz, Box 296, Rockefeller University, 1230 York Ave, New York, NY 10021, USA Fax: +1 212 327 7888 Tel: +1 212 327 8486 E-mail: hrebholz@rockefeller.edu (Received 17 August 2005, revised 6 February 2006, accepted 8 March 2006) doi:10.1111/j.1742-4658.2006.05219.x Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser ⁄ Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB ⁄ Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein–protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme com- plex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Fur- thermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localiza- tion. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine ⁄ s in response to PDGF or serum. Abbreviations btk, Bruton’s tyrosine kinase; CSFR, colony stimulating factor receptor; DBS, donor bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FITC, fluoroscein isothiocyanate; HGFR, hepatocyte-growth factor receptor; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PDK1, 3-phosphoinositide-dependent protein kinase-1; PH, pleckstrin homology; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKB ⁄ Akt, protein kinase B; PKC, protein kinase C; PTB, phosphotyrosine binding domain; RTK, receptor tyrosine kinase; S6K, ribosomal protein S6 kinase; SH2, Src homology 2. FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2023 Ribosomal protein S6 kinase (S6K) is a serine ⁄ threon- ine kinase belonging to the family of AGC kinases, which includes protein kinase A (PKA), protein kinase B (PKB ⁄ Akt), protein kinase C (PKCs), p90 ribosomal S6 kinase and 3-phosphoinositide-dependent protein kinase-1 (PDK1). AGC kinases share a high homology in their kinase domains and have a similar mode of activation [1]. There are two isoforms of S6 kinase, S6K1 and 2. Both have highly homologous kinase and kinase extension domains flanked by the less conserved N- and C-terminal regulatory regions which are responsible for their differential regulation [2,3]. S6K1 and S6K2 have cytoplasmic and nuclear isoforms, which originate from different translational start sites. Nucleocytoplasmic shuttling has been shown for both cytoplasmic forms of S6Ks. All four isoforms lack canonical protein–protein interaction domains, such as Src homology 2 (SH2), phosphotyrosine binding domain (PTB), Src homology 3 and WW, and have no pleckstrin homology (PH) domain, which would enable membrane association via lipid-binding. Instead, in their C-terminal regions, S6K1 and S6K2 possess either a PDZ domain-binding motif or a proline-rich region, respectively, through which S6Ks could bind other signaling molecules. S6 kinases are activated through mitogen- and nutri- ent-mediated pathways. Growth factor-activated recep- tor tyrosine kinases (RTKs) recruit PI3K which, via its effectors PKB ⁄ Akt and PDK1, mediates S6K activa- tion [4]. Another major player in the activation of S6K is the mammalian target of rapamycin, mTor (FRAP) which senses the level of amino acids and possibly other nutrients within a cell [5]. The activation of S6K is a multistep phosphorylation event, involving several ser ⁄ thr kinases. Initially, a series of serines and threo- nines in the C-terminal autoinhibitory domain become phosphorylated, followed by two sites within the hydrophobic linker domain (S371 and T389) [6,7]. Phosphorylation at T389 by mTor or an mTor- dependent kinase enables PDK1 to bind to S6K via its PIF binding pocket [8]. Finally, PDK1 phosphorylates T229 in the activation loop and hereby fully activates S6K [8]. Protein phosphatases PP2A and PP1 have been found in a complex with S6Ks [9,10]. PP2A has further been shown to be the major phosphatase responsible for the dephosphorylation and inactivation of S6K [11] and its activity is stimulated upon inhibi- tion of mTor [12]. The main known physiological substrate of S6 kin- ases is the 40S ribosomal protein S6. Several other in vitro and in vivo substrates have been recently identi- fied, including pro-apoptotic protein Bad1 [13], cyto- skeletal protein neurabin [14] and transcriptional activator CREM [15]. Knockout studies in mice and Drosophila provided evidence that S6K is an important regulator of cell size and growth [16,17]. In S6K2(– ⁄ –) cells S6 phosphoryla- tion is strongly reduced whereas in S6K1(– ⁄ –) almost no reduction can be observed. This finding indicates that S6 protein is not the major substrate for S6K1 in vivo as it cannot compensate for the lack of S6K2. Hence, it is possible to imagine that S6K1 exerts some effects via other substrates. It is also plausible that changes in subcellular localization bring S6K in contact with different substrates. Indeed, we have shown that PKC-mediated phosphorylation of S6K2 at S486 leads to a retention of the kinase in the cyto- plasm [2]. Here we report, for the first time, that both isoforms of S6 kinase, S6K1 and S6K2, are associated with RTKs and recruited to membrane ruffles upon growth factor stimulation. Furthermore, we have shown that S6Ks become phosphorylated on tyrosine in response to mitogenic stimuli and that this phosphorylation coincides with receptor recruitment. The use of platelet derived growth factor (PDGF) receptor mutants defici- ent in signaling via specific pathways and SU6656, a src-specific inhibitor, indicated that both, RTK and src activities are needed for tyrosine phosphorylation of S6Ks. We have mapped the major src-dependent tyro- sine phosphorylation site to a tyrosine in the N-termi- nus of S6K1 and 2. Tyrosine phosphorylation does not affect the activity or subcellular localization of S6Ks. Results S6 kinases are tyrosine phosphorylated by various receptor and nonreceptor tyrosine kinases In the present study, we addressed whether S6K acti- vation involves tyrosine phosphorylation and trans- location to the plasma membrane. In recent years, a number of AGC kinases such as PKB ⁄ Akt, PDK1, various PKCs and PKD but not S6Ks have been shown to be tyrosine phosphorylated [18–23]. Initially, we used a baculoviral expression system in Sf9 insect cells. We infected Sf9 cells with viruses expressing either cytoplasmic EE[Glu-Glu]-tagged S6K1 or S6K2 together with a panel of RTKs or the cytosolic tyro- sine kinase fyn. When we immunoprecipitated S6Ks with an anti-EE-tag IgG and probed the membrane with phosphotyrosine antibody (4G10), tyrosine phos- phorylation of S6K1 and 2 was reproducibly observed when HGFR (hepatocyte-growth factor receptor), PDGFR (platelet-derived growth factor receptor) and S6K tyrosine phosphorylation H. Rebholz et al. 2024 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS CSFR (colony stimulating factor receptor) were coex- pressed. The cytoplasmic tyrosine kinase fyn induced tyrosine phosphorylation of S6K2 but not S6K1 (Fig. 1A). Next, we investigated tyrosine phosphorylation of S6Ks in an in vitro kinase assay with a panel of recom- binant tyrosine kinases. As PDGFRb induced a strong phosphotyrosine signal for S6K in insect cells, we tes- ted this receptor for the ability of its kinase domain to phosphorylate S6Ks in vitro. As shown in Fig. 1B, recombinant PDGFRb kinase domain phosphorylated both S6Ks. We further tested a panel of nonreceptor tyrosine kinases, including src and fyn, Bruton’s tyro- sine kinase (btk) and syk in an in vitro kinase assay using S6K1 and 2 as substrates. As shown in Fig. 1C, all tested tyrosine kinases, in particular src, phosphor- ylated both isoforms of S6K in vitro. When tyrosine kinases were not present in the assay, autophosphory- lation of S6Ks was hardly detectable. Src kinase and S6K2 both migrate at 60 kDa in a SDS ⁄ PAGE gel. Therefore, both autoradiography signals are merged in the S6K2 sample treated with src. However, when S6K1 is treated with src, the src autophosphorylation signal is low in our experiment. For this reason, the autoradiography signal from the S6K2 plus src sample should stem mainly from S6K2 phosphorylation. S6Ks are tyrosine phosphorylated and associated with receptor tyrosine kinases upon growth factor stimulation To test whether tyrosine phosphorylation would also occur in mammalian cells, we transiently transfected Cos7 cells with S6Ks and PDGFR. Cells were starved for 24 h and stimulated for 30, 60 or 180 min with PDGF-BB. When S6Ks were immunoprecipitated via their EE-tag, we found PDGF-dependent tyrosine phosphorylation of S6Ks. Tyrosine phosphorylation reached its maximum at 30 min of stimulation and decreased after 1 h (Fig. 2A). Time course experiments using 5 and 10 min of stimulation were also performed and indicated that S6Ks are already tyrosine phos- phorylated within 5 min (data not shown). Further- more, PDGFR was found to coimmunoprecipitate with S6Ks. In this system, the association appears to be constitutive. However, one has to take into account that the receptor is strongly overexpressed and there- fore may be partially active even in starved cells. The fact that the top band of the coimmunoprecipitated PDGFR (representing the mature receptor) exhibits a slightly weaker pY signal in the starved sample than in the PDGF-treated sample further indicates that the receptor is partially but not fully active when cells are A C B Fig. 1. Tyrosine phosphorylation of S6K1 and S6K2 in Sf9 cells and in vitro. (A) Sf9 cells were infected with baculoviruses encoding either EE-tagged S6K1 or S6K2 and a receptor tyrosine kinase (EGFR, HGFR, PDGFR) or the cytosolic tyrosine kinase fyn. Cells were lyzed 2 days postinfection and S6Ks were immunoprecipitated with anti-EE IgG. Samples were resolved by SDS ⁄ PAGE, transferred onto nitrocellulose membrane and analyzed by immunoblotting with monoclonal antibodies against phosphotyrosine (4G10). (B) In vitro tyrosine phosphorylation of S6K by PDGFR. S6Ks were immunoprecipitated from Sf9 cells (using anti-EE IgG) and then subjected to an in vitro tyrosine kinase assay with PDGFR as kinase for 30 min at 30 °C. 100 ng of PDGFR were used per sample. An autoradiograph and the Coomassie-stained gel are shown. (C) In vitro tyrosine phosphorylation of S6K by cytosolic tyrosine kinases. P70S6Ks were immunoprecipitated from Sf9 cells (using anti-EE IgG), then subjected to an in vitro tyrosine kinase assay for 30min at 30 °C. Per sample, 7 pmol of the different tyrosine kinases src, lyn, syk and btk were used. Autoradiograph and the Coomassie-stained gel are shown. H. Rebholz et al. S6K tyrosine phosphorylation FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2025 starved. This activation may be sufficient for S6K recruitment but not for maximal S6K activation and tyrosine phosphorylation. The anti-S6K western blot confirms this hypothesis as in this system S6K is parti- ally active in starved cells as indicated by the partial bandshift, with most S6K being the bottom inactive S6K. With PDGF there is a stronger band shift which is decreased again after 180 min of stimulation. In a control experiment it was established that PDGFR, when expressed alone, does not precipitate with Pro- tein A Sepharose beads coupled with anti-EE IgG (data not shown, or also see Fig. S1). To strengthen our observation, we tested whether endogenous S6K would also become tyrosine phos- phorylated. Since NIH3T3 cells express high levels of endogenous S6K we used them in this study. To achieve maximal stimulation of multiple RTKs, we stimulated the cells with serum rather than PDGF. Endogenous S6K1 was immunoprecipitated from cells after 30, 60 and 180 min of serum-stimulation. As shown in Fig. 2B, both variants of S6K1 (p70 and p85) are phosphorylated on tyrosine in an inducible manner. Interestingly, the phosphorylation of the nuc- lear isoform, p85 S6K1 appears delayed compared to p70 S6K1. Activated S6K usually migrates as four dis- tinct bands on a SDS ⁄ PAGE gel due to multiple phos- phorylation. The tyrosine phosphorylated bands of S6K overlap with two of the activated and slower migrating bands. We have shown that S6Ks can be detected in a complex with PDGFR when they are transiently expressed. We further tested the interaction between endogenous S6Ks and PDGFR (Fig. 2C). We found that PDGFR is specifically associated with S6K1 in a serum-inducible manner indicating that, under physio- logical circumstances, S6K is only recruited to acti- vated RTKs. S6K translocates to PDGF-induced membrane ruffles Immunofluorescence studies in fibroblasts showed that PKB ⁄ Akt is recruited to membrane ruffles upon mito- gen treatment [24]. We therefore decided to investigate if S6K is also recruited to the plasma membrane upon mitogenic stimulation. We used PDGF as a stimulus as it is well known to generate ruffling in NIH3T3 cells. Serum-starved NIH3T3 cells were stimulated for various times, fixed and stained with an antibody against the C-terminus of S6K1. Using an fluoroscein isothiocyanate (FITC)-labeled secondary anti-rabbit IgG and phalloidin to stain actin, S6K was shown to be evenly distributed in the cytoplasm of starved cells. We could also detect colocalization with stress fibers which has been described previously [25] (data not shown). Platelet-derived growth factor (PDGF) Fig. 2. S6Ks are tyrosine phosphorylated and associated with RTKs. (A) PDGFR and S6K1 or 2 were expressed in Cos7 cells. Twenty- four hour post-transfection cells were starved for 20 h and stimul- ated with 40 ngÆmL )1 PDGF as indicated. Immunoprecipitated EE-S6Ks and complexed were separated by SDS ⁄ PAGE, trans- ferred onto nitrocellulose and blotted with phosphotyrosine (4G10) antibodies. The upper half of the membrane was reprobed with anti-PDGFR IgG and the lower part with anti-EE IgG. (B) Tyrosine phosphorylation of endogenous S6K1. NIH 3T3 cells were starved in 0.3% DBS for 24 h and stimulated with 10% DBS as indicated. Endogenous S6K1 was immunoprecipitated using an antibody against its C-terminus. The immunoprecipitates were treated as in (A) and the membrane reprobed with the C-terminal antibody. In this experiment we focused on S6K1 as NIH3T3 cells do not express S6K2. The results of three individual experiments for p70 S6K1 were quantified and are shown as histogram. (C) Endo- genous PDGF receptor coimmunoprecipitates with S6K1 in a stim- ulation-dependent manner. NIH3T3 cells were starved and stimulated with 10% DBS for the indicated times. Endogenous S6K1 was immunoprecipitated with antibody against the C-termin- us of S6K and immunocomplexes were analyzed by immunoblot- ting using anti-S6K or anti-PDGFR IgG. S6K tyrosine phosphorylation H. Rebholz et al. 2026 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS treatment leads to a redistribution of the bulk of S6K towards the nucleus or the perinuclear region. In addition, we reproducibly observed a small frac- tion of S6K1 in membrane ruffles for various time points tested. Fig. 3A shows a 5-min treatment with PDGF. In addition, we used v-src transformed Swiss 3T3 cells in this study as they show very strong PDGF- inducible ruffling. Serum-starved Swiss 3T3 cells were stimulated with PDGF for 5 min, fixed and stained with antibody against the C-terminus of S6K1. Simi- larly to NIH3T3 cells, PDGF treatment lead to a redistribution of the bulk of S6K towards the nucleus or the perinuclear region and of a small fraction of S6K1 to membrane ruffles (Fig. 3B). In a western blot on total cell lysate from NIH3T3, this S6K-antibody is very specific and solely recognizes S6K1 (p70 and p85). We used the same antibody for immunofluorescence studies. These data suggest that S6K may translocate towards the membrane where it could participate in multienzyme complexes consisting of RTKs and other signaling molecules. Tyrosine phosphorylation is dependent on PDGFR-src signaling Upon stimulation, the PDGF receptor dimerizes and autophosphorylates. The generated phosphotyrosine sites constitute binding sites for a variety of down- stream proteins with SH2 domains. In order to deter- mine the signaling pathways resulting in S6K tyrosine phosphorylation, we utilized a panel of PDGFR mutants where specific tyrosine sites were mutated to phenylalanines. The PDGFRb Y763 ⁄ 1009F mutant is deficient in signaling via Shp2 phosphatase, while PDGFRb Y579 ⁄ 581F is unable to bind and activate src [26,27]. PDGFRb K634A is kinase dead. We transfected Cos7 cells with S6K1 ⁄ 2 and various PDGFRb mutants. After serum starvation, cells were stimulated with PDGF and both S6Ks were immuno- precipitated and analyzed by western blotting. In the control experiment with kinase dead receptor (PDGFRb K634A), there was no detectable S6K tyro- sine phosphorylation (Fig. 4A). Notably, S6K expres- sion was always reduced when expressed together with Fig. 3. S6K1 is localized in membrane ruf- fles upon PDGF stimulation in NIH3T3 cells. NIH3T3 cells were starved for 24 h, followed by stimulation with PDGF (10 ngÆmL )1 ) for 5 min. Cells were fixed, permeabilized, blocked and probed with anti-C-terminal S6K1 IgG and secondary FITC-anti-rabbit IgG. Actin was visualized by phalloidin staining which was added during the last 10 min of incubation with FITC-anti- rabbit IgG. Arrows indicate membrane ruf- fles in which S6K is present. We also used v-src transformed Swiss3T3 cells as they generate very strong PDGF-induced ruffles. Cells were grown at 35 °C and treated simi- larly to NIH3T3 cells. H. Rebholz et al. S6K tyrosine phosphorylation FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2027 KD PDGFR. However, in the S6K2 ⁄ KD PDFGR sample the expression level is comparable to S6K ⁄ wtPDGFR of the starved sample. Expression of the Y763 ⁄ 1009F mutant when compared to wtPDGFR did not alter tyrosine phosphorylation of S6K. Interestingly, the phosphotyrosine signal of S6Ks from cells expressing the Y579 ⁄ 581F receptor is strongly reduced. This result suggests that src kinase may be involved in tyrosine phosphorylation of S6 kinase. When the membrane was re-probed with anti-S6K1 IgG, the migration of multiple bands representing S6K1 was similar in wtPDGFR and the Y579 ⁄ 581F mutant hinting that the activation process was probably not altered by the lack of tyrosine phos- phorylation. To investigate the involvement of src and PDGFR in tyrosine phosphorylation of S6K further, we studied the effect of inhibitors on tyrosine phosphorylation of S6Ks. As expected, genistein, a broad-range tyrosine kinase inhibitor, reduced the PDGF-induced phospho- tyrosine signal of both S6Ks. Similarly, PP1, an inhib- itor acting on src, but also PDGFR, c-kit and abl [28] reduced tyrosine phosphorylation of S6K very strongly. Finally, the src-specific SU6656 also showed an inhibi- tory effect on the phosphotyrosine signal in S6K (Fig. 4B). Interestingly, LY294002 and rapamycin, inhibitors of PI3Kand mTor, respectively, while being effective in inhibiting S6K activity (as shown by phos- pho-S6 blot), did not reduce but rather slightly enhanced tyrosine phosphorylation of S6K (supple- mentary Fig. S2). To further investigate if tyrosine phosphorylation was mediated by the action of src in vivo, we transi- ently expressed various mutants of src together with S6K. Expression of wild-type src leads to weak basal tyrosine phosphorylation which could be enhanced by serum ⁄ vanadate stimulation. A constitutively active src (Y527F) induced a much stronger tyrosine phosphory- lation of S6K1 (Fig. 5A). Dominant-negative src lead to a complete loss of the phosphotyrosine signal in immunoprecipitated S6Ks. Interestingly, in starved cells we could observe that overexpression of a consti- tutively active version of src (527F) led to a band shift of S6K1 that was similar to the shift in stimulated cells. Furthermore the pT389 signal, a marker of S6K activity, in these starved cells was equal to the signal from the stimulated cells. This activation was not reflected by the state of tyrosine phosphorylation, which was significantly lower in starved than in stimu- lated cells. In serum-stimulated cells we did not see a significant effect of src 527F on the activity of S6K even though src 527F led to its strong tyrosine phos- phorylation. Phospho-T389 levels and the band shift of S6K were similar and independent of the src variant (DN, WT, 527F) in stimulated cells. The most highly tyrosine phosphorylated S6K from src 527F expres- sing, stimulated cells was no more active than the non-tyrosine phosphorylated S6K derived from cells overexpressing DN src. Interestingly, this constitutively active src variant still needed stimulation in order to generate a maximal phosphotyrosine signal on S6K1. The reason therefore may be that stimulation leads to S6K translocation towards the plasma membrane where it may interact with src. Furthermore, we could B A Fig. 4. Tyrosine phosphorylation of S6K is mediated via a PDGFR- src pathway. (A) Cos7 cells transfected with wt or mutant forms of PDGFRb (KD PDGFRb K634A, PDGFRb579 ⁄ 581F, PDGFRbY763 ⁄ 1009F) and EE-tagged S6K1 or 2, starved and stimul- ated with PDGF (40 ngÆmL )1 ) for 15 min. Lysates were incubated with anti-EE IgG bound to protein A-sepharose followed by western blot analysis using anti-pY IgG. The membrane was stripped and reprobed with anti-S6K IgG. The total lysate (30 lg) was also tested for PDGFR expression. (B) Effect of inhibitors on tyrosine phos- phorylation of S6Ks. Hek293 cells transiently expressing PDGFR and either S6K1 or S6K2 were starved for 24 h. Sixty minutes before stimulation, cells were incubated with a panel of inhibitors (genistein 100 l M, PP1 50 lM and SU6656 4 lM), then stimulated with PDGF (40 ngÆmL )1 ). EE-S6Ks were immunoprecipitated with anti–EE IgG, transferred to nitrocellullose membrane and probed with antiphosphotyrosine (4G10) followed by anti–S6K IgG. Total lysate (30 lg) was tested for PDGFR expression. S6K tyrosine phosphorylation H. Rebholz et al. 2028 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS detect endogenous S6K1 and src in a complex in expo- nentially growing Hek293 cells (Fig. 5B), strengthening the hypothesis that src kinase, which localizes to an activated receptor tyrosine kinase, is a major kinase responsible for tyrosine phosphorylation of S6K in vivo. We also found endogenous S6K1 to be tyrosine phosphorylated in v-src transformed Swiss3T3 cells but not in the parental cell line. The src-specific inhib- itor SU6656 could inhibit this phosphorylation (Fig. 5C). This is another indication that the phos- phorylation of native S6K occurs in cells in a src- dependent manner. It is possible to imagine that S6K tyrosine phosphorylation occurs during the process of oncogenic transformation. In these Swiss3T3 cells we could also observe higher levels of phospho-S6 than in parental cells confirming earlier reports of elevated S6K activity [29] (data not shown). Src kinase phosphorylates S6K in the N-terminus In order to determine the sites of tyrosine phosphory- lation, we used N- and C-terminally truncated S6K1. When these mutants were immunoprecipitated from Hek293 cells that also transiently expressed activated src (Y527F), S6K1DC was tyrosine phosphorylated but not the S6K1DN mutant (Fig. 6A). This indicated that the major tyrosine phosphorylation site ⁄ s may be located at the N-terminus of S6K1. To verify our hypothesis and to exclude that the lack of tyrosine phosphorylation in the S6KDN mutant might be due to a conformational change that hinders the access of tyrosine kinases to their substrate residues, we gener- ated and purified recombinant S6K1 N-terminal domain and subjected it to an in vitro kinase assay with several cytoplasmic tyrosine kinases such as src, lyn, syk and btk. As a result, all kinases were able to phosphorylate the S6K1 N-terminal domain (Fig. 6B). An almost complete mobility shift of the domain could be seen in the presence of src. Even though N-terminal sequences of S6K1 and S6K2 are only conserved to 38%, both contain a tyrosine residue, S6K1Y39 and S6K2Y45, equally followed by a glutamate at +1 indicative for a src phosphorylation site. Using mass spectrometry, we could confirm the S6K1Y39 site as being tyrosine phosphorylated in vitro (supplementary Fig. S3). In order to determine if these residues consti- tute major phosphorylation sites in full length S6K we generated EE-tagged phenylalanine mutants. When these mutants were subjected to an in vitro tyrosine kinase assay, they were much less tyrosine phosphoryl- ated by src than wt S6K (Fig. 6C). The level of S6K autophosphorylation was also assessed and was hardly detectable under the experimental conditions. More importantly, overexpression of the mutants together with src (527F) in Hek293 cells led to a strongly Fig. 5. Tyrosine phosphorylation of S6K is dependent on Src activity. (A) Hek293 cells were transfected with S6K1 and either pcDNA3.1 or wild-type src, dominant negative src (DN) or constitutively active src (Y527F). Starved cells were stimulated with 10% FBS (15 min) followed by a brief treatment with pervanadate (2 min). Phosphotyrosine levels of S6K were assessed by western blot using 4G10 antibody. The membrane was stripped and reprobed twice with antibodies against pT389 and S6K1. Total lysates (30 lg) were probed with anti-src IgG. (B) Exponentially growing Hek293 cells were lyzed. Endogenous S6K1 was immunoprecipitated using an anti-S6K1 IgG, immunocomplexes were separated by SDS ⁄ PAGE and membrane was probed with anti-src IgG. As a control, we used ProteinA-sepharose beads to test for the specificity of the coimmunoprecipitation. (C) S6K is tyrosine phosphorylated in v-src transformed cells. Exponentially growing v-src trans- formed Swiss 3T3 and parental cells were treated with 4 l M SU6656 for 16 h before lysis. S6K1 was immunoprecipitated and blotted with 4G10 antibody. The membrane was reprobed with anti-S6K1 IgG. H. Rebholz et al. S6K tyrosine phosphorylation FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2029 reduced phosphotyrosine signal (by 88 and 95% for S6K1 and S6K2, respectively) (Fig. 6D) indicating that the N-terminal site is the major phosphorylation site in vivo. However, the possibility that another minor site exists cannot be excluded. As tyrosine phosphorylation was detectable upon growth factor stimulation and therefore paralleled the activation by S ⁄ T phosphorylation, it was logical to hypothesize that tyrosine phosphorylation may be involved in the regulation of S6K activity. As previ- ously shown, tyrosine phosphorylation is strongly reduced when the src signaling-deficient PDGFRY579 ⁄ 581F mutant is expressed (Fig. 4A). We assayed the in vitro activity of S6K coexpressed with wild-type PDGFR or Y579 ⁄ 581F in starved or PDGF-stimula- ted cells. S6K activity was not altered in the presence of the src signaling deficient mutant when compared with wild type (supplementary Fig. S4). Next, we tes- ted if mutation of Y39 ⁄ Y45 to phenylalanine would affect the activity of S6Ks. No difference between wild-type and mutant activities could be observed in stimulated or starved cells in an in vitro kinase assay, indicating that tyrosine phosphorylation of this site does not modulate kinase activity (Fig. 6E). The S6K1Y39D mutant was also tested and had similar activity to the wild type (data not shown). Src-induced tyrosine phosphorylation of atypical PKC has been shown to alter its subcellular localization. Therefore, we tested the subcellular localization of wild-type S6K and mutants (S6K1Y39F, S6K2Y45F) by confocal microscopy in NIH3T3 cells but did not observe significant differences. In addition, the subcellu- lar localization of S6K1 was similar in src-deficient (syf) or syf + src fibroblasts (data not shown). This data A D B C E Fig. 6. Determination of a N-terminal tyrosine as src-dependent phosphorylation site. (A) Deletion of the N-terminus leads to a loss of phos- photyrosine in S6K. Hek293 cells were transiently transfected with WT and truncated mutants of S6K (S6K1, S6K1 DN and S6K1DC) and src 527F. Cells were starved for 24 h and stimulated with FBS (15 min) followed by a 2-min treatment with Na 3 VO 4 . S6Ks were precipitated, immunocomplexes separated via SDS ⁄ PAGE and blotted with pY antibody. Membrane was stripped and reprobed with anti-EE IgG. Total lysate (30 lg) was also analyzed for src expression. Arrows indicate the truncated S6Ks. (B) The N-terminal domain of S6K1 is a substrate for tyrosine kinases. One microgram of the purified recombinant N-terminal fragment was used for an in vitro kinase assay using 7 pmol of cytosolic tyrosine kinases src, btk, lyn and syk. (C) Tyrosine Y39 ⁄ 45 in S6K1 ⁄ 2 is a substrate for src kinase in vitro. S6K1 ⁄ S6K2 and S6K1Y39F ⁄ S6K2Y45F mutants were immunopurified from Hek293 cells and subjected to an in vitro kinase assay using recombinant src kinase. Reaction products were analyzed by autoradiography and Coomassie staining as indicated. (D) Tyrosine Y39 ⁄ 45 in S6K1 ⁄ 2 is a sub- strate for src kinase in vivo. S6K WT and mutants and src kinase were overexpressed in Hek293 cells, which were starved and stimula- ted with 10% FBS for 15 min and for 2 min with Na 3 VO 4 . Immunoprecipitated S6Ks were tested with anti-pY IgG and membrane was reprobed with S6K antibody. Total lysate was also analyzed for src expression. (E) The activity of S6K1 ⁄ 2 Y39F ⁄ Y45F mutants is not altered. Hek293 cells were transfected with S6K1 ⁄ 2 or Y39F ⁄ 45F. Cells were starved and stimulated as indicated (15 min FBS). S6K was immuno- precipitated from these cells, subjected to an in vitro kinase assay using S6 as a substrate. The expression of S6Ks was assessed by western blotting. S6K tyrosine phosphorylation H. Rebholz et al. 2030 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS indicate that src-mediated tyrosine phosphorylation of S6K does not affect its subcellular localization. Taken together, we found that S6K becomes tyro- sine phosphorylated in a PDGFR-src mediated path- way which involves membrane recruitment of S6K. We have shown that a subpopulation of S6K is present at the membrane upon PDGF stimulation and thus in the vicinity of PKB and PDK1 which are the major activators of S6K. Discussion In this study, we have shown for the first time that S6Ks become tyrosine phosphorylated and associated with PDGFR in a ligand-induced manner. In mamma- lian cells, both events, receptor association and tyro- sine phosphorylation occur simultaneously and peak within the first 30 min after stimulation. Membrane translocation in response to mitogenic stimuli has been shown for a variety of AGC kinases, including PKB ⁄ Akt, PDK1, PKD and various iso- zymes of the PKC family. This is mainly thought to occur via binding to second messengers such as phos- pholipids or via binding to phosphotyrosine residues on activated RTKs. Translocation of PKB ⁄ Akt or PDK1 is mediated through PH domains which specif- ically recognize the second messenger PIP3 [30,31]. A variety of signaling molecules such as PI3K, IRS1, Src or GRB2 translocate to the membrane and associate with activated receptors via their SH2 or PTB domains. PKC translocation is mediated by a variety of isoform-specific RACKs (receptors for activated C-kinase) [32]. In addition, many AGC kinases have been shown to be substrates for src kinase which itself associates with activated RTKs. Even though the phosphorylation events leading to full activation of S6K have been thoroughly studied, it is not clear if they involve translocation of S6K to the membrane. However, S6K, in order to be phosphorylated by PDK1, may be in the vicinity of the membrane. Fur- thermore, Rho family G proteins Rac and Cdc42, which control cytoskeletal organization, were shown to associate with and activate S6K [33]. As these small GTPases are most active when they are membrane- bound, it would be logical for S6K to be colocalized with its upstream effectors. Finally, it was reported that S6K is complexed with the receptor-associated p85 subunit of PI3K and that this complex formation is needed for mTor and PI3K-mediated activation of S6K [34]. We showed that PDGFR can specifically im- munoprecipitate with S6K which, to our knowledge, is the first report of coimmunoprecipitation of an RTK with an AGC kinase. We further used immunofluores- cence microscopy to show that S6K1 can be localized at the plasma membrane. In starved cells, S6K1 is evenly distributed within the cytoplasm and can also be detected along stress fibers. Upon stimulation, the majority of S6K1 molecules translocate to the nucleus, whereas a subpopulation is reproducibly found in membrane ruffles. The association between receptor and nonreceptor tyrosine kinases and S6K leads to its tyrosine phos- phorylation in vitro and in vivo. The recombinant kinase domain of PDGFR, as well as cytoplasmic tyrosine kinases such as src, is able to phosphorylate S6Ks on tyrosine. In vivo, using PDGFR mutants that are deficient in signaling via src kinase, we found that both PDGFR and src kinase activities are needed for maximal tyrosine phosphorylation of S6Ks. Studies employing tyrosine kinase inhibitors such as PP1 and SU6656 validated this finding. PI3K and mTor do not influence tyrosine phosphorylation of S6K as demon- strated by the use of the inhibitors LY294002 or rapa- mycin. This finding is in congruence with the finding that PDK1 tyrosine phosphorylation is independent of PI3K activity [20]. The major src-dependent phos- phorylation sites, S6K1 Y39 and S6K2 Y45 are located at the N-terminus of S6K. We observed a difference in phosphorylation kinetics of the p70 and p85 isoforms of endogenous S6K1 in NIH3T3 cells: Whereas P70 was already phosphoryl- ated after 30 min, we could only detect p85 phosphory- lation after 60 min of stimulation. In contrast to p70 S6K, the p85 isoform is thought to be exclusively localized in the nucleus, and thus, the delayed tyrosine phosphorylation may result from activation and ⁄ or translocation of the respective kinase. For example, as c-src was shown to be in part localized in the nucleus [35], one possibility could be that src translocates to the nucleus where it can phosphorylate p85 S6K. Very poss- ibly both isoforms are part of distinct feedback mecha- nisms via tyrosine phosphatases. For several AGC kinases such as PKB ⁄ Akt, PDK1, PKCs and PKD it was shown that tyrosine phosphory- lation results in increased kinase activity [19] [20,23,36]. It is known that PI3K activity is involved in v-src transformation and the level of PIP3 is elevated in v-src transformed cells [37]. In v-src transformed cells PKB ⁄ Akt activity is enhanced, due to elevated PIP3 levels [38,39]. In the case of S6K, there is also evidence pointing towards src-induced S6K activation: Src inhibitor PP1 interferes with S6K activation after insulin, IGF1 and pervanadate stimulation [40]. Fur- thermore, S6K activity in v-src transformed cells is higher than in nontransformed cells [29]. We could confirm that the level of phospho-S6 is higher in v-src H. Rebholz et al. S6K tyrosine phosphorylation FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2031 transformed cells. We also found that S6K in v-src transformed cells but not the parental cells is tyrosine phosphorylated. However, our experimental data indi- cate that S6K tyrosine phosphorylation does not corre- late with its activity. We propose that in v-src transformed cells S6K could be activated indirectly via the enhanced action of upstream kinases PKB ⁄ Akt, PDK1 or PI3K or via the inhibition of ser ⁄ thr phos- phatases [41,42]. It was shown that some PKCs act in a negative feed- back loop which controls kit tyrosine kinase activity by directly phosphorylating two serine residues in the kin- ase insert of the receptor in a stem cell factor-dependent manner [43]. Similarly, it was recently published that S6K activity is required in a negative feedback loop which down-regulates insulin receptor signaling via phosphorylation of IRS1 [44,45]. In order to achieve this, S6K must be recruited to IRS1 and therefore be in membrane vicinity. It is plausible to speculate that S6K might not only receive signaling information from acti- vated PDGF receptors or associated second messengers, but could regulate their function by phosphorylation. Bioinformatic analysis of PDGFR kinase domain does not show the presence of S6K phosphorylation motifs. An in vitro kinase assay indicated no obvious phos- phorylation of recombinant PDGFR kinase domain by S6K. One could speculate that tyrosine phosphorylation may create an SH2 recognition site and thus may alter the binding affinities of S6K. In this study, and for the first time, we demonstrate receptor association and tyrosine phosphorylation of S6Ks. Both events occur simultaneously and can be induced by growth factor stimulation. Experimental procedures Materials Monoclonal antibody to the EE-tag was a gift from J. Downward, Cancer Research UK. The antiphosphotyrosine 4G10 antibody, polyclonal phosphospecific S6 protein (S235 ⁄ 236) and anti-src IgG were from Upstate (Lake Placid, NY, USA). Phosphospecific antibody against p70S6Kinase (pT389) was purchased from Cell Signaling (Danvers, MA, USA). Anti-flag (M2) IgG and anti- b -actin were from Sigma (St. Louis, MO, USA). Polyclonal anti- bodies against the C-terminus of S6K1 and 2 were des- cribed previously [2]. Recombinant human PDGF-BB was purchased from AutogenBioclear (Calne, UK). LY294002 and rapamycin were from Calbiochem (Nottingham, UK), genistein from Oxford Biomedical Research (Oxford, MI, USA), PP1 from Biomol (Exeter, UK), SU6656 and phal- loidin from Sigma. Construction of expression vectors Baculoviruses containing S6K1 and S6K2, fyn and RTKs have been made as described elsewhere [46]. The con- struction of mammalian expression vectors encoding wt S6Ks1 ⁄ 2, activated and kinase-dead forms of S6K (p70S6K1T389D, p70S6K2T388D and p70S6K1K100R) was previously reported [2]. The flag–tagged truncated S6Ks (S6K1DNDC and S6K2DNDC) were from K. Yone- zawa (Kobe University, Japan). The mammalian expres- sion constructs for wild-type PDGFRb and kinase dead PDGFR, PDGFR Y579 ⁄ 581F, PDGFR Y763 ⁄ 1009F were made as reported [26] [27]. Mouse ⁄ chicken activated Src (Y527F) and DN src (mouse K296R, Y528F) mam- malian expression constructs were purchased from Upstate. Expression of recombinant proteins in bacteria and Sf9 cells EE-tagged S6Ks were expressed in Sf9 cells, affinity purified using monoclonal EE-antibody and eluted with EE-peptide. PDGFRb cytoplasmic domain recombinant protein was purchased from Upstate. Tyrosine kinases src, fyn, btk and syk were purified as described [47]. The N-terminal domain of S6K1 was subcloned into pET42a (Novagen, Notting- ham, UK) in frame with a C-terminal His-tag, expressed in BLR21 DE3 cells, induced and purified with NiNTA agarose and eluted with 400 mm imidazole. Cell culture and transfection Sf9 cells were maintained at 27 °C in IPL41 insect medium (Invitrogen, Paisley, UK) with yeastolate ultrafiltrate (Gib- co ⁄ Invitrogen), lipid concentrate and gentamycin (Invitro- gen). NIH3T3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% donor bovine serum (DBS, Invitrogen), 50 lgÆmL )1 streptomycin, 50 UÆmL )1 penicillin and 2 mml-glutamine. Cos7 and Hek293 cells were cultured in the same conditions than NIH3T3, but 10% fetal bovine serum (FBS, Invitrogen) was added instead of DBS. Swiss 3T3 parental and tem- perature-sensitive v-src transformed cells (F29) were a gift from M. Frame (Beatson Institute, Glasgow, UK) and were grown at 35 °C. Cos7 cells were electroporated as described previously [31]. Hek293 cells were transiently transfected with LipofectAMINE (Qiagen, Crawley, UK). Immunoprecipitation Two days postinfection, Sf9 cells were lyzed in 50 mm Tris- HCl (pH 7.6), 150 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1% Triton X-100, 20 mm NaF, 50 lgÆmL )1 leupeptin, 0.5% aprotinin, 1 mm PMSF, 3 mm benzamidine and S6K tyrosine phosphorylation H. Rebholz et al. 2032 FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... domain of S6K interacts with the cytoplasmic domain of PDGFR, we transfected Hek293 cells with PDGFR and either full-length S6K1 ⁄ 2 (EE-S6K1 ⁄ 2 WT) or deletion mutants which lack both N- and C-terminal regulatory domains (flag-p70S6K1 ⁄ 2DNDC) S6Ks were immunoprecipitated via their tags and western blotting was performed with an EE ⁄ anti-flag antibody and anti-PDGFR antibody Both full-length S6Ks and. .. Srcfamily tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases Biochem J 366, 57–62 S6K tyrosine phosphorylation 41 Belandia B, Brautigan D & Martin-Perez J (1994) Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus Mol Cell Biol 14, 200–206 42 Villa-Moruzzi E & Puntoni F (1996) Phosphorylation of phosphatase-1alpha... LR, Anderson KE & Hawkins PT (2001) Src family kinases mediate receptor- stimulated, phosphoinositide 3-kinase-dependent, tyrosine phosphorylation of dual adaptor for phosphotyrosine and 3-phosphoinositides-1 in endothelial and B cell lines J Biol Chem 276, 42767–42773 Supplementary material The following supplementary material is available online: Fig S1 The kinase or kinase extension domain of S6K... oxidized methionine Fig S4 Tyrosine phosphorylation mediated by PDGFR does not affect S6K activity Hek293 cells were transfected with WT or mutant PDGFR and either isoform of S6K1 or 2, starved and stimulated with PDGF (40 ngÆmL)1) for 15 min (and Na3VO4 for 2 min) S6Ks were immunoprecipitated with the EE-antibody, then used for an in vitro kinase assay with ribosomal protein S6 as a substrate The total... the receptor and S6K mutants could be detected (data not shown) Fig S2 Tyrosine phosphorylation occurs independently of PI3K and mTor LY 294002 (20 lm) and rapamycin (30 nm) were added 60 min before starved Hek293 cells were PDGF stimulated Transiently expressed EE-S6Ks were immunoprecipitated with anti-EE IgG, transferred to nitrocellullose and probed with pY antibody followed by anti-S6K IgG Total... give a final volume of 40 lL which was added to immune complexes After 30 min at 30 °C, reactions were stopped by one wash with cold 20 mm Tris HCl pH 7.5 ⁄ 150 mm NaCl and the addition of SDS ⁄ PAGE sample buffer Samples were subjected to 10% SDS ⁄ PAGE, and S6K tyrosine phosphorylation the amount of 32P incorporated into S6 was assessed by autoradiography Immunofluorescent staining and microscopy NIH3T3... vitro S6 kinase assay and tyrosine kinase assay The in vitro kinase assay was performed with immunopurified S6Ks and 40S ribosomes as substrate, which we described previously [2] To test for tyrosine kinase activity towards S6 kinase, EE-tagged S6Ks were immunoprecipitated from Sf9 cells with anti-EE IgG immobilized on protein A-Sepharose Immunocomplexes bound to beads were washed twice in lysis buffer and. .. the S6K kinase and ⁄ or the kinase extension domain The positions of the truncated S6Ks are indicated with arrows Similarly, when mutants lacking only one regulatory domain, either Nor C-terminus, were coexpressed with PDGF receptor, FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2035 S6K tyrosine phosphorylation H Rebholz et al the interaction between the receptor. .. target of rapamycin phosphorylates and activates p70, S6 kinase alpha in vitro J Biol Chem 274, 34493–34498 8 Biondi RM, Kieloch A, Currie RA, Deak M & Alessi DR (2001) The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB EMBO J 20, 4380–4390 9 Bettoun DJ, Buck DW, 2nd Lu J, Khalifa B, Chin WW & Nagpal S (2002) A vitamin d receptor- Ser ⁄ Thr phosphatase-p70, S6 kinase... complex and modulation of its enzymatic activities by the ligand J Biol Chem 277, 24847–24850 Epub 2002 May 29 10 Peterson RT, Desai BN, Hardwick JS & Schreiber SL (1999) Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein Proc Natl Acad Sci USA 96, 4438–4442 11 Petritsch C, Beug H, Balmain A & Oft M (2000) TGFbeta inhibits p70, S6 . to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational. involvement of src and PDGFR in tyrosine phosphorylation of S6K further, we studied the effect of inhibitors on tyrosine phosphorylation of S6Ks. As expected,