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Molecular and Cellular Biology, February 2003, p. 852-863, Vol. 23, No. 3
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.3.852-863.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase ßII and Regulates Its Subcellular Localization

Taras Valovka,¹ Frederique Verdier,¹ Rainer Cramer,^1,2 Alexander Zhyvoloup,^1,3 Timothy Fenton,¹ Heike Rebholz,¹ Mong-Lien Wang,¹ Miechyslav Gzhegotsky,⁴ Alexander Lutsyk,⁴ Genadiy Matsuka,³ Valeriy Filonenko,³ Lijun Wang,⁵ Christopher G. Proud,⁵ Peter J. Parker,⁶ and Ivan T. Gout^1,2,3*

Ludwig Institute for Cancer Research, London W1W 7BS,¹ Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, London WC1E 6BT,² Division of Molecular Physiology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH,⁵ Cancer Research United Kingdom, London WC2A 3PX, United Kingdom,⁶ Department of Structure and Function of Nucleic Acid, The Institute of Molecular Biology and Genetics, Kyiv 143,³ L'viv State Medical University, L'viv 290010, Ukraine⁴

Received 5 July 2002/ Returned for modification 10 September 2002/ Accepted 29 October 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G₁/S transition of the cell cycle. There are two forms of S6K, termed S6K and S6Kß, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6K, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kß, but not S6K, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KßII by PKC-mediated phosphorylation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ribosomal protein S6 (rpS6) kinase (S6K) belongs to the AGC family of Ser/Thr protein kinases which includes the protein kinase C's (PKCs), protein kinase B's, SGKs, and 90-kDa ribosomal S6 kinases (p90 RSKs). There are two forms of S6K, S6K and S6Kß, which have cytoplasmic (S6KII and S6KßII) and nuclear (S6KI and S6KßI) variants derived from alternative splicings at the N terminus (2, 15). S6K and S6Kß have a very high level of overall sequence similarity, with the greatest homology in the kinase and kinase extension domains. However, both kinases differ significantly at their N- and C-terminal regulatory regions, sharing only 28 and 25% homology, respectively. The C terminus of S6Kß contains a specific proline-rich region, which is absent in S6K and might be involved in mediating protein-protein interactions with SH3 and WW domain-containing molecules. The presence of a PDZ domain-binding motif at the C terminus of S6K may direct the kinase into distinct signaling complexes (8).

The activity of S6K is regulated by phosphorylation and dephosphorylation events in cellular responses to various extracellular stimuli. The treatment of cells with growth factors, cytokines, and hormones leads to a rapid activation of S6K (10), while growth inhibitory agents, such as steroids and transforming growth factor ß, suppress kinase activity (45, 52). The mechanism of activation of S6K has been studied in detail by various laboratories and was shown to be a multistep phosphorylation process involving several Ser/Thr kinases (14, 64).

No direct, highly specific S6K inhibitor has yet been identified. Under these circumstances, the use of two indirect inhibitors, namely wortmannin (a phosphatidylinositol 3-kinase [PI3-K] inhibitor) and rapamycin (an mTOR inhibitor), has been instrumental in dissecting signaling events involved in the regulation of both forms of S6K. Studies from numerous laboratories demonstrated that signals from the PI3-K and mTOR pathways are crucial for full activation of S6K (4, 7, 11, 20, 21).

rpS6 is the most widely studied physiological substrate of S6K. The phosphorylation of S6 protein was shown to closely correlate with the initiation of protein synthesis induced by various extracellular stimuli (16, 55). The transcriptional activator CREM, elongation factor 2 kinase, and the regulator of apoptosis, Bad 1, have also been shown to be phosphorylated by S6K in vitro and in vivo (13, 22, 63). However, the physiological relevance of these phosphorylations requires further investigation, since other protein kinases can phosphorylate these molecules at identical sites.

S6K was identified more than a decade ago, and S6Kß was identified only recently, hence most functional studies have involved the p70/p85 isoforms of S6K. S6K was proposed to be involved in translational up-regulation of a subset of mRNAs that are characterized by the presence of an oligopyrimidine tract at their 5' termini and generally encode ribosomal proteins and elongation factors (25). This view has been recently challenged by studies which show S6K-independent translation of mRNAs with oligopyrimidine tracts at their 5' termini (58). Microinjection studies with neutralizing antibodies against S6K demonstrated its importance in mediating the G₁/S transition of the cell cycle (29). Knockout studies of the S6K gene in mice and Drosophila melanogaster indicate that the kinase is a key player in the regulation of cell size, growth, and glucose homeostasis (36, 44, 53).

Accumulating evidence from several laboratories demonstrates that S6Kß is activated similarly to S6K when cells are treated with fetal calf serum (FCS), insulin, or phorbol 12-myristate 13-acetate (PMA) (19, 28, 30, 32). Both kinases receive an input signal from common effectors of the PI3-K pathway, including PDK1, protein kinase B, PKC, Rac, and CDC42 (1, 6, 9, 32, 47, 50). However, some differences in the regulation of S6K and S6Kß have started to emerge. Comparative analysis of both forms of S6K indicated that the basal activity of S6Kß is more sensitive to activation by myristoylated PKC than is that of S6K (32). The same group has also reported that the C-terminal region of S6Kß exerts a stronger inhibitory effect on the kinase than does the S6K C terminus. Moreover, a novel regulatory connection between the MEK/extracellular signal-regulated kinase and S6Kß signaling pathways has recently been demonstrated (33, 42, 61).

As mentioned above, S6K and S6Kß are each represented by two splice variants with distinct subcellular distributions. The 23- and 13-amino-acid extensions at the N-termini of S6KI and S6KßI contain nuclear localization signals (NLSs) that target these isoforms constitutively to the nucleus (35, 49). The cytoplasmic form of S6K (S6KII) or p70 S6K is predominantly cytosolic, but it can also accumulate in the nucleus when cells are treated with leptomycin B (LMB) (27). The presence of a functional NLS at the C terminus of S6Kß, which is found in both splice variants, has recently been reported (28). The nuclear functions of S6Ks are not known.

The PKC family of lipid-dependent serine/threonine kinases has been implicated in a multitude of physiological processes, including protein synthesis, mitogenesis, cell survival, and transcriptional activation (34). Based on sequence homology, domain organization, and mode of activation, PKCs can be subdivided into 3 classes: classical PKCs (, ß, and ) are activated by diacylglycerol (DAG) and calcium, novel PKCs (, , , and ) require DAG but not calcium, and atypical PKCs ( and /) require neither DAG nor calcium for their activation (38).

As mentioned above, both S6K and S6Kß are rapidly activated when cells are treated with PMA, an activator of classical and novel PKCs. Furthermore, prolonged treatment of cells with PMA, which leads to down-regulation of DAG-activated PKCs, eliminates mitogenic activation of S6K (56, 57). Recently, the PI3-K- and PDK1-activated atypical PKC isoforms and have been implicated in the regulation of S6K (1, 31, 50). Atypical PKCs have been found in complexes with S6K, but it is not yet known whether they can directly phosphorylate and modulate the function of S6Ks. Taken together, these data indicate that PKCs transduce signal(s) in activated cells to both forms of S6K, but the regulatory mechanisms and functional importance remain unclear.

We report here that S6KßII, but not S6KII, is specifically phosphorylated by PKC at a site located in the middle of its C-terminal NLS. Using phosphospecific antibodies, we found that phosphorylation of S6KßII at S486 is strongly induced by PMA and to a lesser extent by epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), insulin, platelet-derived growth factor (PDGF), and FCS. Furthermore, we found that S486 phosphorylation does not effect S6K activity, but it eliminates the function of the C-terminal NLS. Mutational analysis of S6KßII provided evidence that S486 phosphorylation results in retention of an activated form of the kinase in the cytoplasm, possibly by blocking its nuclear import.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials. Restriction enzymes and DNA modifying enzymes were obtained from standard commercial sources and used according to the manufacturers' recommendations. Oligonucleotides were synthesized by Genosys, and phosphopeptides were synthesized by Eurogentec. Recombinant PKCs were purchased from Calbiochem. The anti-Myc 9E10 monoclonal antibody was from Santa Cruz Biotechnology. Monoclonal antibody to the EE tag was a gift from Julian Downward.

Construction of expression vectors. The full-length coding sequences corresponding to both splicing forms of S6K and S6Kß were amplified by PCR with rat S6K and human S6Kß cDNAs as templates, respectively. The products of PCR amplification were then cloned into the BamHI/EcoRI sites of the pcDNA3.1 expression vector (Invitrogen) in frame with the amino-terminal EE tag epitope (MEFMPME). The C-terminal regions of S6K (amino acids 453 to 525) and S6Kß (amino acids 442 to 495) were PCR amplified and cloned into the pET23d vector (Novagen) in frame with six-His tag sequences by using NcoI/EcoRI restriction sites. Mutated forms of S6K and S6Kß were generated by using the QuikChange site-directed mutagenesis kit (Stratagene) as recommended by the manufacturer. All constructs were verified by restriction enzyme digestion and DNA sequencing. The generation of mammalian expression constructs for PKCs used in this study was previously described (31).

Cell culture and transient transfection. Human embryonic kidney HEK 293 cells and human breast cancer MCF7 cells were maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Inc.), 2 mM L-glutamine, 50 U of penicillin/ml, and 50 µg of streptomycin/ml. NIH 3T3 cells were grown in DMEM supplemented with 10% donor calf serum (Life Technologies, Inc.), 2 mM L-glutamine, 50 U of penicillin/ml, and 50 µg of streptomycin/ml. Transient transfections of HEK 293 cells were performed by using Lipofectamine according to the manufacturer's recommendations (Life Technologies, Inc.). NIH 3T3 cells were transfected by using the PolyFect reagent (Qiagen) according to the manufacturer's recommendations. At 24 h posttransfection, cells were starved in serum-free DMEM for 24 h and then stimulated with either 10% FBS, 1 µM PMA, 50 ng of EGF/ml, 50 ng of IGF-1/ml, 100 nM insulin, or 50 ng of PDGF/ml for the indicated time.

Adult rat ventricular cardiomyocytes (ARVC) were isolated from the hearts of adult rats as described previously (62). Isolated cardiomyocytes were seeded onto laminin-coated dishes, cultured overnight in medium 199 containing 1 g of glucose/liter, 0.68 mmol of glutamine/liter, 5 mmol of creatine/liter, 2 mmol of carnitine/liter, and 5 mmol of taurine/liter. The next day, the cells were treated with phenylephrine (10 µM), insulin (20 nM), or vehicle only for 30 min.

Immunoprecipitation and S6K assay. HEK 293 cells were washed with ice-cold phosphate-buffered saline (PBS) and extracted with lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 2 mM EDTA, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 50 µg of leupeptin (Boehringer Mannheim)/ml, 0.5% aprotinin (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), and 3 mM benzamidine (Sigma). Whole-cell extracts were centrifuged at 10,000 x g for 15 min at 4°C, and recombinant EE-S6Ks were immunoprecipitated with the anti-EE monoclonal antibody immobilized on protein G-Sepharose beads (Amersham Pharmacia Biotech). Immune complexes were washed three times with lysis buffer followed by a single wash with kinase assay buffer (50 mM HEPES [pH 7.5], 10 mM MgCl₂, 1 mM dithiothreitol, 10 mM ß-glycerophosphate). The kinase reaction was initiated by resuspending the beads in 25 µl of kinase assay buffer supplemented with 1 µM protein kinase A inhibitor (Calbiochem), 50 µM ATP, 5 µCi of [-³²P]ATP (Amersham Pharmacia Biotech), and 20 µg of 40S ribosomes isolated from rat liver. The reaction was carried out at 30°C for 10 min and terminated by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer and boiling the mixture for 5 min. Samples were subjected to SDS-10% PAGE, and the amount of ³²P incorporated into the S6 protein was assessed by autoradiography and quantitated by phosphorimaging (Bio-Rad).

Expression of recombinant proteins in bacteria. Recombinant His-tagged C-terminal regions of S6K and S6Kß (His-S6KC and His-S6KßC) were expressed in BLR21 DE3 cells. Expression was carried out at 22°C for 4 h in the presence of 1 mM isopropyl-ß-D-galactosidase. Recombinant His-S6KC and His-S6KßC were affinity purified by using Talon beads according to the manufacturer's recommendations. Purified proteins were dialyzed overnight at 4°C against 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM dithiothreitol in 50% glycerol and stored at -20°C.

In vitro phosphorylation of S6K by PKCs. Recombinant EE-tagged S6KII and S6KßII were immunoprecipitated from serum-starved HEK 293 cells with anti-EE monoclonal antibody immobilized on protein G-Sepharose. Beads were washed twice with lysis buffer containing 0.5 M NaCl and three times in PKC kinase buffer (20 mM HEPES [pH 7.5], 10 mM MgCl₂, 100 µM CaCl₂). Immune complexes and 1 µg of recombinant His-S6KC, His-S6KßC, histone H1, or -peptide were incubated with 0.5 U of different PKC isoforms (Calbiochem)/ml at 30°C in buffer containing 100 µM ATP, 5 µCi of [-³²P]ATP, 0.03% Triton X-100, 100 µg of phosphatidylserine/ml, and 20 µg of DAG/ml. After incubation for 10 min, reactions were terminated by the addition of SDS-PAGE sample buffer and boiling the mixtures for 5 min. The incorporation of ³²P into recombinant EE-S6KII, EE-S6KßII, His-S6KC, His-S6KßC, histone H1, and -peptide was determined by phosphorimager analysis following SDS-5 to 17.5% PAGE.

Sample preparation and MS. Recombinant EE-S6KßII and His-S6KßC were phosphorylated with PKCßII as described above but without [-³²P]ATP. The products of the reaction were either directly analyzed by infrared and UV matrix-assisted laser desorption ionization (mass spectrometry) [MALDI (MS)] or first digested with modified trypsin (Promega, Southampton, United Kingdom) or endoproteinase Lys-C (Roche, Lewes, East Sussex, United Kingdom) in 25 mM ammonium bicarbonate buffer (pH 8) at 37°C and then analyzed. Prior to proteolysis some samples were separated by one-dimensional SDS-PAGE, and an in-gel digest (51) was performed on excised bands of interest.

MALDI samples were prepared by using the dried droplet method, which involves mixing 0.5 µl of the analyte solution with 0.5 to 1 µl of the matrix solution on the target and drying them by means of a warm stream of air. For all measurements, external calibration was performed by using calibration mixture 2 of the Sequazyme peptide mass standards kit (5 peptides/protein in the 1- to 6-kDa mass range) from Applied Biosystems, Warrington, Cheshire, United Kingdom.

All measurements were conducted on a Voyager Elite XL (Applied Biosystems, Framingham, Mass.) MALDI-time of flight mass spectrometer equipped with delayed extraction and a reflector analyzer for improved mass resolution and accuracy. The instrument has been modified to enable infrared MALDI measurements at 2.94 µm with a Q-switched Speser 15Q (Spektrum GmbH, Berlin, Germany) Er:YAG laser as well as UV MALDI measurements at 337 nm utilizing a VSL-337ND nitrogen laser (Laser Science, Inc., Franklin, Mass.) as supplied by the manufacturer of the mass spectrometer. The technical details regarding the experimental setup have been reported elsewhere (12).

Production of a phosphoserine-specific S6Kß antibody. Polyclonal antiserum that recognizes a specific phosphorylation site was raised against phosphopeptide corresponding to the C-terminal 11 amino acids (residues 481 to 491) of S6Kß (SGTKKS⁴⁸⁶KRGRG) with serine 486 as a phosphorylated residue. The peptide was coupled to keyhole limpet hemocyanin and injected into rabbits by using standard immunization techniques. Rabbit antibody specific for pS486-S6Kß was affinity purified by using antigenic peptide coupled to Actigel (Sterogene) and screened for antigen reactivity by immunoblot analysis.

Immunoblot analysis. Protein samples were subjected to SDS-PAGE and transferred onto 0.45-µm-pore-size nitrocellulose or Immobilon-P membranes. After blocking with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20, the membranes were probed overnight at 4°C with anti-EE (1:1,500), anti-Myc, anti-pS486-S6Kß (1:1,000), or anti-phospho rpS6 (Ser235) (Upstate Biotechnology) antibody. The immunoblots were washed four times for 15 min with Tris-buffered saline containing 0.1% Tween 20 and incubated with peroxidase-conjugated secondary antibodies for 40 min at room temperature. The antigen-antibody complexes were detected with the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Immunofluorescent staining and microscopy. HEK 293 cells were plated onto poly-L-lysine-coated coverslips in 24-well dishes at a density of 2.5 x 10⁴ cells per well and cultured overnight. The cells were then transfected with 0.5 µg of expression vectors containing various S6K and S6Kß constructs. At 24 h posttransfection, the cells were starved in serum-free DMEM for 24 h and then stimulated with 1 µM PMA for 30 min. LMB-treated cells were cultured in the presence of 10 ng of LMB/ml for 16 h before stimulation. After a brief wash at room temperature with PBS, cells were fixed with 4% formaldehyde for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Nonspecific binding was blocked by incubation with 0.5% bovine serum albumin in PBS for 30 min. The cells were then incubated with anti-EE (1:1,500) (mouse) or rabbit anti-pS486 (1:800) antibodies for 2 h at room temperature. After extensive washing with PBS, the samples were incubated for 45 min with goat fluorescein isothiocyanate-conjugated anti-mouse or anti-rabbit antibodies (1:200), respectively. Finally, the coverslips were extensively rinsed with PBS, air dried, and mounted onto microscope slides. Immunofluorescent staining was analyzed with a laser scanning microscope (LSM51D; Zeiss, Oberkochen, Germany).

Top Abstract Introduction Materials and Methods Results Discussion References

 
S6Kß but not S6K is phosphorylated in vitro at the C terminus by different isoforms of PKC. Inspection of the amino acid sequence of S6Kß revealed a potential PKC phosphorylation site located within the C-terminal regulatory region (Fig. 1A). S6K displays a low level of identity with S6Kß at the C terminus and does not contain consensus sequences for phosphorylation by PKC. To test whether PKC phosphorylates S6Kß, we initially employed an in vitro kinase assay. The C-terminal regions of S6K and S6Kß (His-S6KC and His-S6KßC), expressed in bacteria as His-tag fusion proteins, were used as substrates in a PKC phosphorylation assay. As shown in Fig. 1B, all PKC isoforms tested efficiently phosphorylated His-S6KßC, whereas no significant phosphorylation of His-S6KC was observed under similar conditions. The activities of the PKC isoforms were analyzed with histone H1 or -peptide as substrates (Fig. 2). It should be noted that the efficiency of His-S6KßC phosphorylation by PKCs correlated with their specific activities.

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FIG. 1. S6KßII, but not S6KII, is phosphorylated at the C terminus by different PKC isoforms in vitro. (A) Schematic representation of S6KßI and S6KßII and their deletion mutants, which lack amino- and carboxyl-terminal sequences. Major domain boundaries are indicated. Structural features are indicated as follows: grey boxes indicate unique proline-rich sequences of S6Kß; solid black boxes indicate NLSs (NLS1 and NLS2); striped boxes correspond to potential NESs. The N- and C-terminal amino acid sequences, containing NES and NLS, are shown above the diagrams. All recombinant constructs carry an N-terminal EE-tag sequence, and deleted amino acids are indicated. (B) In vitro phosphorylation of bacterially expressed His-S6KC and His-S6KßC by various PKCs. Affinity-purified His-tagged S6K and S6Kß C-terminal peptides were incubated in the presence of different recombinant PKC isoforms and [-32P]ATP. The reaction mixtures were separated by SDS-PAGE and stained with Coomassie. The dried gel was analyzed by autoradiography. (C) In vitro phosphorylation of recombinant full-length S6KII, S6KßII, and deleted S6KßII mutants by PKCs. HEK 293 cells transiently transfected with wild-type EE-S6KII, EE-S6KßII, EE-S6KßIIN, or EE-S6KßIIC were serum starved for 24 h, and recombinant proteins were immunoprecipitated with anti-EE-tag antibody. The immunoprecipitates were incubated with [-32P]ATP in the absence or presence of different recombinant PKC isoforms. The reaction mixtures were analyzed as described above.

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FIG. 2. Analysis of enzymatic activities of recombinant PKC isoforms. In vitro kinase assays were performed as described in Materials and Methods. HI, histone H1.

Next, we investigated whether full-length S6KII and S6KßII could serve as substrates for PKCs in an in vitro kinase assay. In this experiment, transiently expressed EE-tagged forms of S6KII and S6KßII were immunoprecipitated from serum-starved HEK 293 cells and subjected to in vitro phosphorylation by different isoforms of PKC. The results demonstrated that all isoforms of PKC readily phosphorylated full-length S6KßII but failed to use S6KII as a substrate (Fig. 1C). We have also observed a higher efficiency of S6KßII phosphorylation by PKCßI, PKCßII, and PKC (2-, 1.5-, and 4-fold increases, respectively) when compared with other isoforms.

To confirm that the PKC phosphorylation site is located within the C terminus of S6Kß, we created N- and C-terminal deletion mutants and tested whether they were phosphorylated by PKCs under the conditions described for the full-length kinases. As shown in Fig. 1C, deletion of the N-terminal region of S6Kß did not affect the efficiency and the pattern of phosphorylation by PKC isoforms. However, the removal of the C terminus completely abolished PKC-mediated phosphorylation of S6KßII. The data presented above clearly indicate that S6Kß can be phosphorylated by PKC in vitro and that the site(s) of phosphorylation is located at the C terminus.

Identification of PKC phosphorylation site(s) and characterization of phosphospecific antibodies. The precise identification of the PKC phosphorylation site(s) in S6KßII was carried out by MS. Affinity-purified His-S6KßC was used as a substrate for PKCßII in the presence of cold ATP. The products of the reaction were digested by trypsin or endoproteinase Lys-C, and the resulting peptides were analyzed by MS. Initial MALDI (MS) analysis of the intact or trypsin in-gel-digested His-S6KßC was inconclusive with regard to PKC phosphorylation. However, proteolysis with the endoproteinase Lys-C produced phosphorylation-indicative peptides (Fig. 3A). The recorded peptide ions from the MALDI (MS) analysis show that the main phosphorylation is located in the KS⁴⁸⁶K sequence stretch, suggesting serine as the phosphorylation site. The stoichiometry of S6KßII phosphorylation by PKCßII was found to be approximately 1 mol of phosphate per mol of S6KßII.

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FIG. 3. Identification of PKC phosphorylation site and characterization of phosphospecific S6Kß antibody. (A) Mass spectroscopy analysis of PKC phosphorylation site in S6KßII. The amino acid sequence of His-S6KßC is shown on top. (B and C) Analysis of specificity of anti-pS486 antibody. Bacterially expressed His-S6KßC was incubated with [-32P]ATP in the presence (+) or absence -) of recombinant PKCßII. Samples were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and analyzed by autoradiography (B) or immunoblotting with anti-pS486 antibody (C). HI, histone H1; PVDF, polyvinylidene difluoride; WB, Western blot.

Phosphospecific antibodies are a powerful tool for investigating the physiological importance of protein phosphorylations. We therefore generated an antibody that specifically recognizes S6Kß phosphorylated at S486. The antibodies were raised in rabbits and affinity purified on Actigel beads coupled with antigenic peptide. Recombinant His-S6KßC prephosphorylated with PKCßII was used to test the specificity of the antibodies generated. As shown in Fig. 3B and C, affinity-purified anti-pS486 antibody specifically recognized His-S6KßC only when it was prephosphorylated by PKCßII. Furthermore, the recognition of phosphorylated His-S6KßC by anti-pS486 antibody was abolished by preincubation with the phosphorylated form, but not with the nonphosphorylated form, of the antigenic peptide (data not shown).

Phosphorylation of S6KßII at S486 in cellular responses to mitogenic stimuli. The availability of a phosphospecific antibody has allowed us to study the phosphorylation status of S6KßII at S486 in response to various extracellular stimuli. We found that treatment of HEK 293 cells transiently overexpressing EE-S6KßII with PMA induced a significant (up to 15-fold) increase in S486 phosphorylation (Fig. 4A). A time course stimulation of cells with PMA demonstrates that phosphorylation of S486 is very rapid and reaches a peak at 30 min but is still detectable even 24 h after induction (Fig. 4B). Noticeably, phosphorylation at S486 parallels the activation profile of S6KßII, as seen from the mobility shift of activated forms of the kinase (Fig. 4B).

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FIG. 4. S6KßII is phosphorylated at Ser486 in response to different mitogenic stimuli. HEK 293 cells were transiently transfected with wild-type EE-S6KßII, serum starved, and stimulated with 10% FCS, 1 µM PMA, 100 nM insulin, 50 ng of PDGF/ml, or vehicle alone (-). Recombinant S6KßII was immunoprecipitated with anti-EE antibody and used for the in vitro S6K assay (B) or analyzed by Western blotting (WB) with anti-pS486 antibody (A). (C) Time course phosphorylation of S6KßII at Ser486 in PMA-treated HEK 293 cells. HEK 293 cells were transiently transfected with wild-type EE-S6KßII, serum starved for 24 h, and stimulated with 1 µM PMA for the indicated period of time. Cell lysates were analyzed by Western blotting with anti-pS486 or anti-EE antibodies. (D) Phosphorylation of endogenous S6Kß at Ser486 in phenylephrine (PE)-stimulated cardiomyocytes. Isolated cardiomyocytes were treated with 10 µM phenylephrine, 10 nM insulin, or vehicle alone for 30 min. Native S6Kß was immunoprecipitated (IP) from lysed cells with anti-C-terminal antibodies. Immune complexes were separated by SDS-PAGE and immunoblotted with anti-pS486 antibody. The results presented have been reproduced in three independent experiments. IgG, immunoglobulin G.

We consistently observed an increase (1.5- to 3-fold) in S486 phosphorylation when starved HEK 293 cells were treated with FCS, insulin, or PDGF (Fig. 4A). In the case of FCS stimulation, the changes in S486 phosphorylation followed a time course similar to that seen for PMA (http://www.ludwig.ucl.ac.uk/cellreg-html/research.htm). When we compared the increase in the S6K activity of exogenously expressed S6KßII with the extent of S486 phosphorylation in response to PMA, FCS, insulin, and PDGF, no obvious correlation was observed (Fig. 4A and C). These results suggested that phosphorylation of S6Kß at S486 might not affect its kinase activity or its activation by other kinases.

We have recently demonstrated that S6KßII is expressed at high levels in cardiomyocytes (ARVC) and is activated by treatment with insulin or phenylephrine (61). In contrast to S6K, which is known to be activated in cardiomyocytes via the PI3-K and mTOR signaling pathways, the activity of S6Kß can also be regulated in a MEK-dependent manner. Moreover, studies from other laboratories show that treatment of cardiomyocytes with insulin and phenylephrine induces rapid activation of PKC (43, 46).

Therefore, this cellular model was used to investigate whether endogenous S6Kß is phosphorylated at S486 in response to insulin and phenylephrine. We treated ARVC with 20 nM insulin or 10 µM phenylephrine for 30 min, and the endogenous S6Kß was immunoprecipitated with the C-terminal polyclonal antibodies. Western blot analysis of immune complexes, resolved by SDS-PAGE, with anti-pS486 antibodies indicated that S6Kß is specifically phosphorylated at S486 in cardiomyocytes treated with insulin and phenylephrine (Fig. 4D). Thus, endogenous S6Kß in primary cells undergoes phosphorylation at S486 in response to a physiological agonist that activates PKC.

PKC mediates phosphorylation of S6KßII at S486 and rpS6 in vivo. The in vitro phosphorylation studies and the ability of PMA to induce S6KßII phosphorylation at S486 strongly suggested the involvement of PKC. In order to examine whether PKC could mediate phosphorylation of S6KßII at S486 in vivo, EE-S6KßII was transiently coexpressed with various Myc-tagged PKCs in HEK 293 cells. Two days after transfection, S6KßII was immunoprecipitated with anti-EE antibodies, resolved by SDS-PAGE, and immunoblotted with anti-pS486 antibodies. The results indicated that coexpression of any PKC isoform with S6KßII induces strong phosphorylation of S486 (Fig. 5A). Coomassie staining of the polyvinylidene difluoride membrane showed that an equal amount of EE-S6KßII was immunoprecipitated from all transfected cells. Western blotting of total cell lysates with anti-Myc antibodies confirmed that all PKC isoforms were expressed at approximately equal levels (Fig. 5A, bottom section).

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FIG. 5. In vivo phosphorylation of S6KßII at Ser486 and rpS6 phosphorylation are mediated by PKC. (A) Coexpression of various PKCs with S6KßII induces phosphorylation at Ser486 in HEK 293 cells. HEK 293 cells were cotransfected with EE-S6KßII and various Myc-PKCs. Recombinant S6Kß was immunoprecipitated with anti-EE-tag antibody and analyzed by Western blotting (WB) with anti-pS486 antibody. Expression levels of transiently expressed PKCs were analyzed in whole-cell extracts with anti-Myc antibody. (B) Effect of PKC inhibitor GF109203X on Ser486 phosphorylation. HEK 293 cells were transiently transfected with wild-type EE-S6KßII, serum starved, and stimulated with 1 µM PMA. A 1 µM concentration of GF109203X was added for 30 min prior to stimulation. (C) Effect of GF109203X on PMA-stimulated phosphorylation of rpS6. MCF7 cells were serum starved for 24 h and then treated with 1 µM PMA or vehicle alone for 30 min. A 1 µM concentration of GF109203X was added for 30 min prior to stimulation. Phosphorylation of S6 protein was analyzed in whole-cell extracts with anti-phospho-rpS6 (Ser235) antibody. IgG, immunoglobulin G; +, present; -, absent.

To further establish that S6KßII is a target for PKC-mediated phosphorylation in vivo, we tested the effect of a PKC inhibitor, GF109203X, on S486 phosphorylation in response to PMA. As shown in Fig. 5B, treatment of HEK 293 cells expressing EE-S6KßII with a 1 µM concentration of GF109203X completely eliminated PMA-induced phosphorylation at S486. Collectively, the results presented above strongly suggest that PKCs mediate the in vivo phosphorylation of S6KßII at S486.

rpS6 is known to be a physiological substrate for both S6K and S6Kß (53). Phosphorylation of rpS6 is one of the earliest events detected following mitogenic stimulation, and it correlates with polysome formation and the initiation of protein synthesis (55). Multiple studies have shown that different mitogenic stimuli employ distinct signaling pathways to mediate rpS6 phosphorylation and the initiation of protein synthesis. Taking this into account, it was interesting to examine the contribution of PKC signaling to in vivo phosphorylation of rpS6. MCF7 cells were chosen for this study since they express large quantities of both S6KII and S6KßII, as determined by immunoblot and Northern blot analysis (data not shown). The treatment of serum-starved MCF7 cells with PMA induces a fivefold increase in the level of rpS6 phosphorylation at S235 (Fig. 5C). This increase was completely inhibited by 1 µM GF109203X, strongly indicating that signaling via PKC is important for rpS6 phosphorylation in response to PMA.

PKC-mediated phosphorylation of S6KßII at Ser486 does not affect S6K activity. Since S6K is activated by multiple Ser/Thr phosphorylations, it was important to investigate the effect of S486 phosphorylation on S6KßII activity. In order to explore the upstream regulation of S486 phosphorylation, we used two indirect inhibitors of S6K, rapamycin (mTOR pathway) and wortmannin (PI3-K pathway).

The treatment of serum-starved HEK 293 cells with PMA induced a fourfold increase in the activity of recombinant S6KßII towards rpS6 (Fig. 6). As expected, pretreatment of cells with rapamycin or wortmannin blocked PMA-induced activation of S6KßII. Noticeably, rapamycin did not exert any obvious effect on PMA-induced phosphorylation of S486 while wortmannin showed a slight inhibition at very high concentrations (Fig. 6).

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FIG. 6. PKC-mediated phosphorylation of S6KßII at Ser486 is insensitive to specific TOR/FRAP and PI3-K inhibitors and does not effect S6K activity. HEK 293 cells were transiently transfected with wild-type EE-S6KßII or EE-S6KßII S486A and incubated in the presence (+) or absence (-) of 1 µM PMA for 30 min after 24 h of starvation. Rapamycin (R) or wortmannin (W) was added for 30 min before cell stimulation. Recombinant S6KßII was immunoprecipitated with anti-EE-tag antibody and used for the in vitro S6K assay or analyzed by immunoblotting with anti-pS486 antibody. IgG, immunoglobulin G; PVDF, polyvinylidene difluoride; WB, Western blot.

These results have also been confirmed by in vitro studies. In these experiments, EE-S6KßII was immunoprecipitated from serum-starved HEK 293 cells and phosphorylated with different PKC isoforms in the presence of cold ATP. After washing, S6K activity towards rpS6 was measured. These experiments revealed that prephosphorylation of S6KßII by PKCs does not affect its S6K activity (http://www.ludwig.ucl.ac.uk/cellreg-html/research.htm).

To gain further insight into the importance of PKC-mediated phosphorylation of S6KßII, we mutated serine 486 to alanine. It is important to note that anti-pS486 antibodies did not recognize the mutated form of S6KßII overexpressed in HEK 293 cells, confirming their specificity (http://www.ludwig.ucl.ac.uk/cellreg-html/research.htm). Moreover, the activity of the S486A mutant was found to be similar to that of thewild-type kinase in HEK 293 cells treated or not treated with PMA (Fig. 6). Taken together, the results demonstrate that PKC-mediated phosphorylation of S6KßII at S486 does not effect the activity of the kinase in response to mitogenic stimuli.

Effect of PMA and LMB on subcellular localization of S6Kß. Since S486 is located within the C-terminal nuclear localization sequence, it was reasoned that PKC-mediated phosphorylation of this site might modulate the subcellular localization of S6Kß. To test this possibility, we initially examined the subcellular localization of EE-S6Ks in transiently transfected HEK 293 cells stimulated with PMA. As shown in Fig. 7A, EE-S6KII was mainly localized in the cytoplasm of serum-starved cells and PMA stimulation did not change its pattern of distribution. However, pretreatment of cells with LMB leads to accumulation of EE-S6KII in the nucleus, suggesting dynamic nucleocytoplasmic shuttling. These data are in agreement with studies carried out by other groups (27, 28).

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FIG.7. Analysis of subcellular localization of S6K and S6Kß by confocal microscopy. (A) HEK 293 cells were transiently transfected with wild-type EE-S6KII, EE-S6KßI, or EE-S6KßII, serum starved for 24 h, and stimulated with 1 µM PMA (+PMA) for 30 min or vehicle alone (-PMA). Treatment of cells with LMB (10 ng/ml) was carried out for 16 h before the stimulation with PMA. Cells were fixed, probed with anti-EE antibody and fluorescein isothiocyanate-labeled anti-mouse immunoglobulin G, and analyzed by confocal microscopy. (B) Subcellular localization of pSer486-S6KßII in HEK 293 cells treated with PMA and LMB. HEK 293 cells were transfected with EE-S6KßII and treated in the same way as described above. After fixation and probing with anti-pS486 antibody, confocal microscopy analysis was carried out. (C) Subcellular localization of pSer486-S6KßII in NIH 3T3 cells treated with PMA, EGF, IGF-1, insulin, or PDGF. Transient transfection of NIH 3T3 cells and confocal microscopy were performed as described in Materials and Methods.

By contrast, EE-S6KßII is found predominantly in the nucleus of serum-starved cells and shifts to the cytoplasm after PMA treatment (Fig. 7A). Moreover, LMB prevents PMA-stimulated accumulation of EE-S6KßII in the cytoplasm as seen by the retention of the kinase in the nucleus. This finding indicates that S6KßII may shuttle between the nucleus and the cytosol during the course of activation. In contrast, the nuclear localization of EE-S6KßI, which contains two NLS sequences, one at the N terminus and another at the C terminus, is not affected by treatment with PMA or LMB (Fig. 7A).

Taken together, these results suggest the existence of nucleocytoplasmic shuttling for S6KßII, which is LMB sensitive and could be regulated by the PKC signaling pathway.

Phosphorylation at S486 eliminates the function of the NLS in S6KßII. The data presented above prompted us to study the subcellular distribution of pS486-S6KßII in PMA-stimulated cells with phosphospecific antibodies. Confocal immunofluorescence microscopy clearly indicated that pS486-S6KßII is localized exclusively in the cytoplasm of PMA-treated cells (Fig. 7B). No signal was detected in serum-starved cells, confirming once again the specificity of the phosphospecific antibodies. It was interesting to study whether blocking nuclear export with LMB affected the subcellular localization of pS486-S6KßII. As shown in Fig. 7B, the pattern of pS486-S6KßII distribution did not change when cells were treated with both PMA and LMB. No changes in the subcellular localization of pS486-S6KßII were observed when cells were treated with PMA in the presence of rapamycin (data not shown). Altogether, these results strongly suggest that PMA-induced phosphorylation of S6KßII at S486 takes place in the cytoplasm and prevents translocation of the kinase to the nucleus. Moreover, we have analyzed the phosphorylation and subcellular localization of pS486-S6KßII in NIH 3T3 cells stimulated with other mitogenic stimuli. Figure 7C shows that S6KßII is phosphorylated at S486 in response to EGF, IGF-1, insulin, or PDGF, and the phosphorylated protein is localized in the cytoplasm. However, the immunofluorescent signal is significantly weaker when compared to that of PMA stimulation. These data are in agreement with anti-pS486-S6Kß immunoblot analysis presented in Fig. 4A.

Substitution of the phosphorylation site with acidic amino acids mimics, in many cases, the phosphorylation of that site in the protein of interest and therefore provides an excellent model for functional studies. To this end we have generated an S486E mutant of S6KßII and analyzed its subcellular localization with the expectation that it would be present only in the cytoplasm of transfected cells. Unexpectedly, the S486E mutant behaved similarly to the wild-type protein in serum-starved and PMA-stimulated cells (Fig. 8). However, we found that the S486A mutant is predominantly localized in the nucleus of serum-starved cells and does not accumulate in the cytoplasm in response to PMA. A likely explanation of the observed differences in the subcellular localization of S486E and S486A mutants is that regulation of nucleocytoplasmic shuttling by phosphorylation of S6KßII at this site requires the kinase to be in an activated state. To test this hypothesis, we created a double mutant of S6KßII bearing T401D and S486E substitutions. It has previously been demonstrated that a T401D (equivalent to T412 in S6K) mutant is constitutively active and possesses fourfold-higher S6K activity than the wild-type S6KßII (60). The results of immunofluorescence analysis unambiguously demonstrate that the T401D/S486E mutant is retained in the cytoplasm of serum-starved and PMA-treated cells (Fig. 8). The importance of S486 phosphorylation in controlling nuclear shuttling of the activated form of S6KßII was further confirmed with the use of a T401D/S486A mutant. This mutant was found to be localized predominantly in the nucleus of serum-starved cells and did not accumulate in the cytoplasm in response to PMA.

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FIG. 8. Subcellular localization of S6KßII mutants in HEK 293 cells. Plasmids carrying EE-S6KßII, EE-S6KßII S486E, EE-S6KßII S486A, EE-S6KßII T401D/S486E, or EE-S6KßII T401D/S486A were transfected into HEK 293 cells. After 24 h, cells were serum starved and stimulated for 30 min with 1 µM PMA (+PMA) or vehicle alone (-PMA). Fixed cells were incubated with anti-EE antibody and analyzed by immunofluorescence.

Taken together, the results of immunofluorescence microscopy clearly demonstrate that PMA-mediated phosphorylation of S6KßII at S486 regulates nucleocytoplasmic shuttling of an activated form of the kinase. Since S486 is located in the middle of the C-terminal NLS, we propose that phosphorylation of this residue eliminates its function.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we have addressed the role of PKC signaling in the regulation of nucleocytoplasmic shuttling of S6KßII. We found that S6Kß, but not S6K, is phosphorylated by PKCs in vitro and in vivo. The site of phosphorylation was identified by MS as S486, which is located in the C-terminal regulatory domain. Furthermore, the use of phosphospecific antibodies indicated that S486 phosphorylation is induced by various mitogenic stimuli, including PMA, FCS, EGF, IGF-1, insulin, and PDGF.

Studies from different laboratories demonstrated that activation of S6K is a multistep phosphorylation process, involving at least nine sites and various S/T kinases (14, 64). Most of these sites are conserved in S6Kß, with the exception of one (equivalent to T444 in S6K), indicating a very similar mode of activation. In contrast, this study clearly demonstrates that PKC-mediated phosphorylation of S6KßII at S486 is not involved in the regulation of its kinase activity.

What is the importance of S486 phosphorylation for the cellular functions of S6KßII? In agreement with the results of previous studies, we detected S6KII mainly in the cytoplasm, whereas S6KßII was predominately nuclear (27, 28). The presence of a functional NLS at the C terminus of S6KßII has been recently reported by Koh et al. The authors also found that mutation of Lys487 to Met in the KKSK⁴⁸⁷RGR sequence of S6KßII relocates the kinase from the nucleus to the cytoplasm. Since S486 is located in the middle of the C-terminal NLS, we focused our efforts on elucidating the effect of PKC-mediated phosphorylation of this site on the subcellular localization of S6KßII. Following this assumption, we found that treatment of cells with PMA induced rapid translocation of S6KßII from the nucleus to the cytoplasm, whereas no changes in the subcellular localization of S6KII were observed. Furthermore, this translocation was blocked completely by LMB, a specific inhibitor of CRM1-mediated nuclear export, indicating the existence of nucleocytoplasmic shuttling for S6KßII. Interestingly, PMA and LMB do not affect the subcellular localization of S6KßI, whose exclusive nuclear distribution is determined by the presence of two NLSs.

A continuous shuttling of S6KßII between the nucleus and the cytoplasm may require the presence of both the NLS and nuclear export signal (NES) sequences in S6KßII. During the last few years, a short leucine-rich consensus sequence was identified in a variety of signaling molecules and shown to possess nuclear export properties (18, 39). Inspection of the amino acid sequence allowed us to identify a potential NES located at the N terminus of S6KßII (Fig. 1A). This sequence resembles the Crm1 consensus sequence, which is known to be LMB sensitive. The nuclear export receptor for S6KßII remains to be identified. We are currently investigating the function of this potential NES by mutational analysis and confocal microscopy. Preliminary data indicate that the N-terminal region of S6KßII possesses a functional NES which is LMB sensitive (T. Valovka, unpublished data).

Many proteins are transported constitutively into and out of the nucleus by members of the ß-importin family of nuclear transport receptors (17). In contrast to constitutive transport, regulated transport occurs only in response to specific cellular signals and involves a specific NLS receptor, usually -importin (26). The docking of proteins that contain classical or bipartite types of NLS to the cytoplasmic side of the nuclear pore is mediated by an -importin/ß-importin heterodimer and Ran GTPase. The formation of this multiprotein complex can be influenced directly by posttranslational modifications, such as phosphorylation, acetylation, and methylation (24). Is nuclear transport of S6KßII driven by this mode of regulation?

Mutational analysis of the S486 site allowed us to gain insight into the regulation of S6KßII nucleocytoplasmic shuttling by PKCs. We observed that an S486A mutant of S6KßII does not accumulate in the cytoplasm in response to PMA, indicating that phosphorylation of S486 might be necessary for this event to occur. However, when we tested the subcellular localization of the S486E mutant, we found unexpectedly that it behaves similarly to the wild-type S6KßII. Therefore, phosphorylation of S6KßII at S486 is not sufficient on its own to confer cytoplasmic localization of the kinase. Further mutational studies of S486 and T401 (equivalent to T412 in S6KII) uncovered the dependence of nucleocytoplasmic shuttling of S6KßII on the activated state of the kinase.

A possible explanation of these findings is that S6KßII has to be in an activated state, in which the structure unfolds, making both NES and NLS operational. The structure of S6K has not been solved, and in the absence of crystallographic data, the primary structure of S6K has been functionally dissected into four domains. Based on these studies, a model for S6K activation has been proposed which implies that active conformation of the kinase is achieved by coordinated phosphorylations at three regions: the C-terminal autoinhibitory domain, by Ser-Pro-directed kinases; the activation loop in the kinase domain, by PDK1; and the conserved hydrophobic site in the kinase-extension domain (48). It is believed that in unstimulated cells, the interaction between the N- and C-terminal regulatory domains keeps the kinase domain in a locked conformation. Following mitogen stimulation, multiple phosphorylations open the structure by initially unlocking the N-terminal domain and subsequently releasing the C-terminal autoinhibitory domain. In agreement with this model, PMA-induced activation of S6KßII may release the N-terminal domain, making the NES operational. This may shift the steady-state constants for nuclear export and import, establishing an altered equilibrium in the nucleocytoplasmic shuttling of S6KßII.

Using pS486 phosphospecific antibodies, we discovered that pS486-S6KßII is exclusively localized in the cytoplasm of PMA-treated cells and that LMB does not alter its localization. These data strongly suggest that phosphorylation of S6KßII at S486 occurs in the cytoplasm of PMA-stimulated cells. Moreover, phosphorylation of S6KßII at S486 coincides with the depletion of the kinase from the nucleus and subsequent accumulation in the cytoplasm (Valovka, unpublished). We propose that phosphorylation of S6KßII at S486 eliminates the function of its sole NLS, and as a result, the kinase is confined to the cytoplasm. This mode of regulation (NLS masking) is common among signaling molecules and has been reported for DAG kinase , Ca²⁺/calmodulin-dependent protein kinase II, and the forkhead transcription factor AFX (5, 23, 59).

What is the physiological relevance of S6KßII translocation from the nucleus to the cytoplasm in response to mitogenic stimuli? One possible explanation is that it brings the kinase in close proximity to its substrate(s), such as rpS6. Knockout studies of the S6K gene in mice showed that the S6 protein is a physiological substrate for S6Kß (53). Mitogen-induced phosphorylation of rpS6 is associated with the initiation of protein synthesis of a specific pool of mRNA whose gene products are involved in ribosomal biogenesis (25).

Based on the data presented here and current knowledge of signaling via S6Ks, we propose a model to explain nucleocytoplasmic shuttling of S6KßII in response to mitogenic stimuli, such as PMA (Fig. 9). In unstimulated cells, S6KßII adopts an inactive conformation and is mainly localized in the nucleus. In this state, S6KßII import must be faster than export or the kinase may be in complex with an anchoring protein in the nucleus. Treatment of cells with PMA or other mitogenic stimuli triggers the activation of classical and novel PKCs and downstream signaling molecules, including S6KßII. The fact that exclusive nuclear forms of S6K, S6KI and S6KßI, are activated in response to mitogenic stimuli suggests that all components required for multistep phosphorylation and/or activation of S6KßII are present in the nucleus (35, 49). Activation of S6KßII may unfold the kinase, releasing the N-terminal NES from its intramolecular interactions. In this state, the kinase may be transported to the cytoplasm by Crm1-facilitated nuclear export. Phosphorylation of S6KßII by activated forms of PKC may be essential for inactivating the function of its C-terminal NLS. The addition of negative charges within the NLS or flanking regions may eliminate the interaction with the NLS receptor. Given that negatively charged sequences of the NLS receptor are thought to bind to the positively charged NLS of nuclear-targeted proteins for nuclear import to occur (54), it is not surprising that the presence of a negative charge within the NLS may inhibit this interaction. Retention of the activated form of S6KßII in the cytoplasm could be required for phosphorylation of rpS6 and initiation of protein synthesis. It is well documented that PMA-activated protein synthesis is a key event for the induction of cell growth and proliferation (3, 37, 40, 41). Dephosphorylation of S486 in response to environmental changes can unmask the C-terminal NLS, making it available for importin-dependent nuclear import.

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FIG. 9. Subcellular localization of S6KßII is regulated by PKC. See the text for details.

In conclusion, this report describes for the first time mitogen-regulated nucleocytoplasmic shuttling of S6KßII and deciphers a critical role of PKC signaling in this process.

 
We thank M. Griffin for excellent technical assistance.

This work was supported in part by grants from the Wellcome Trust (055427/Z/98), the British Heart Foundation (PE99/004), and The Royal Society (FSU/CEE/JP). T.V. was supported by the Overseas Research Students Awards Scheme (ORS/2000061024).

 
* Corresponding author. Mailing address: Ludwig Institute for Cancer Research, 91 Riding House St., London W1W 7BS, United Kingdom. Phone: 0044-207-8784088. Fax: 0044-207-8784040. E-mail: ivan{at}ludwig.ucl.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, February 2003, p. 852-863, Vol. 23, No. 3
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.3.852-863.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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