Björn Schuster

Lisa Hendry

Helen Byers

Steven F Lynham

Malcolm A Ward

Dr Susan John
email: susan.john@kcl.ac.uk

PURIFICATION AND IDENTIFICATION OF THE STAT5 PROTEASE IN MYELOID CELLS Running title: Generation of truncated STAT5

Keywords: STAT5, nuclear, serine protease, cathepsin G, C-terminal, truncation

Signal transducer and activator of transcription (STAT) proteins are critical regulators of cytokine-induced cell proliferation, differentiation and survival. STAT functional activity can be variably regulated by post-translational modifications, including phosphorylation, acetylation, methylation and sumoylation. Additionally, limited proteolytic digestion of full-length STAT proteins (STATα) generates C-terminally truncated forms (STATγ) in different cell lineages, which have significantly reduced transcriptional activity due to the lack of the transactivation domain. Previously, it was shown that STAT5γ, generated by an unidentified nuclear serine protease, plays an important role in myeloid-cell differentiation and is aberrantly expressed in acute myeloid leukaemia. To better understand this regulatory mechanism for STAT5 function, we have purified the STAT5 protease from the immature myeloid cell line 32D and identified it by mass spectrometric analysis as the granule-derived serine protease, Cathepsin G (CatG). We show that purified CatG can specifically cleave full-length STAT5 to generate STAT5γ, and this activity can be inhibited by AEBSF in an in-vitro protease assay. Importantly, preparation of nuclear and cytoplasmic extracts from immature myeloid cell lines, 32D and FDC-P1 in the presence of a specific inhibitor for CatG results in the identification of only STAT5α. These studies indicate that nuclear STAT5γ does not naturally exist in immature myeloid cells and is artificially generated from STAT5α during the preparation of extracts due to the abundance of CatG in these cells. Therefore, in contrast to earlier studies, our data suggest that STAT5α, rather than STAT5γ is the active form in immature myeloid cells.

INTRODUCTION

Signal transducer and activator of transcription (STAT) proteins play a major role in cell proliferation, differentiation and survival [START_REF] Levy | Stats: transcriptional control and biological impact[END_REF]. Following cytokine or growth factor stimulation, STAT proteins are tyrosine phosphorylated, and the activated dimeric form translocates into the nucleus, to induce expression of specific target genes [START_REF] Leonard | Jaks and STATs: biological implications[END_REF][START_REF] Darnell | STATs and gene regulation[END_REF][START_REF] Ihle | Cytokine receptor signalling[END_REF]. The family of STAT proteins comprises seven members, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.

There are two homologous STAT5 proteins, STAT5a and STAT5b, which are encoded by two distinct genes [START_REF] Mui | Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of STAT5[END_REF][START_REF] Mui | Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs[END_REF]. Cytokines and growth factors activating STAT5 proteins include those which regulate the proliferation and differentiation of myeloid cells such as interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin or of lymphoid cells such as IL-2, IL-7 and IL-15 [START_REF] Leonard | Jaks and STATs: biological implications[END_REF]. The important role of STAT5 in the development and survival of haematopoietic cells has been shown in STAT5 deficient mice. Mice deficient in either STAT5a [7] or STAT5b [START_REF] Udy | Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression[END_REF] were found to exhibit dramatic defects in non-haematopoietic Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society tissues, but only subtle alterations in the regulation of haematopoietic cells [START_REF] Imada | Stat5b is essential for natural killer cell-mediated proliferation and cytolytic activity[END_REF][START_REF] Feldman | STAT5A-deficient mice demonstrate a defect in granulocyte-macrophage colony-stimulating factor-induced proliferation and gene expression[END_REF][START_REF] Nakajima | An indirect effect of Stat5a in IL-2induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor alpha chain induction[END_REF]. In contrast, mice lacking both STAT5a and STAT5b showed a much stronger defect in the development of myeloid, erythroid and lymphoid lineages [START_REF] Hoelbl | Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation[END_REF][START_REF] Snow | STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells[END_REF][START_REF] Yao | Stat5a/b are essential for normal lymphoid development and differentiation[END_REF].

In immature myeloid cells, STAT5 has been implicated in both the IL-3-induced proliferation and granulocyte-colony-stimulating factor (G-CSF)-induced differentiation of 32D cells [START_REF] Ilaria | Dominant negative mutants implicate STAT5 in myeloid cell proliferation and neutrophil differentiation[END_REF]. In myeloid progenitors, IL-3 stimulates the activation of C-terminally truncated isoforms of STAT5a (77 kD) and STAT5b (80 kD), collectively called STAT5γ [START_REF] Bovolenta | Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5[END_REF][17][START_REF] Meyer | Carboxyl-truncated STAT5beta is generated by a nucleus-associated serine protease in early hematopoietic progenitors[END_REF][START_REF] Azam | Interleukin-3 signals through multiple isoforms of Stat5[END_REF][START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF][START_REF] Rothman | Cytokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors[END_REF]. In immature myeloid cells the STAT5γ isoforms are predominantly localized to the nucleus and activates only a subset of genes activated by STAT5α in mature myeloid cells [START_REF] Meyer | Carboxyl-truncated STAT5beta is generated by a nucleus-associated serine protease in early hematopoietic progenitors[END_REF][START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF]22]. It was demonstrated that STAT5γ is generated by limited proteolytic processing of STAT5α instead of expression of an alternatively spliced form of STAT5 (STAT5β) [START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF]. The protease responsible for the processing of STAT5 was characterized to be a member of the serine protease family, as demonstrated by its sensitivity to the broad-spectrum serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Similar protease activities with the capacity to cleave STAT3 and STAT5 were also found in acute myeloid leukaemia cells [START_REF] Xia | A novel serine-dependent proteolytic activity is responsible for truncated signal transducer and activator of transcription proteins in acute myeloid leukemia blasts[END_REF] and neutrophils [START_REF] Caldenhoven | Activation of a functionally distinct 80-kDa STAT5 isoform by IL-5 and GM-CSF in human eosinophils and neutrophils[END_REF][START_REF] Chakraborty | Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils[END_REF][START_REF] Epling-Burnette | Carboxy-terminal truncated STAT5 is induced by interleukin-2 and GM-CSF in human neutrophils[END_REF][27], and similarly in mast cells to cleave STAT6 [28][START_REF] Sherman | IL-4 preferentially activates a novel STAT6 isoform in mast cells[END_REF][START_REF] Suzuki | Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism[END_REF]. Mutation of the mapped cleavage site in STAT5b yielded a more cleavageresistant mutant STAT5b molecule, which when transfected into the immature myeloid cell line FDC-P1 conferred a strong defect in GM-CSF-induced differentiation of these cells [22,31].

These results suggested that STAT5γ is essential for the differentiation of immature myeloid cells. Furthermore, the protease responsible for the cleavage of STAT5 in the nucleus of immature myeloid cells was not identified.

In this study, we purified the STAT5 protease from 32D cells and identified the protease by mass spectrometry analysis. Surprisingly, the results show that the protease responsible for the conversion of STAT5α to STAT5γ in immature myeloid cells is CatG. Unexpectedly, our results also show that the predominant form of STAT5 in 32D and FDC-P1 cells is STAT5α and the truncated form is rapidly generated during the preparation of the nuclear extracts with the conventional buffers lacking CatG specific protease inhibitors.

MATERIAL AND METHODS

Cells and Reagents

293T and WEHI-3B cells were maintained in DMEM (Cambrex, Verviers, Belgium) and RPMI 1640 (PAA Laboratories, Pasching, Austria) respectively, supplemented with 10 % heat inactivated fetal bovine serum (PAA Laboratories), 2 mM glutamine and 100 units/ml penicillinstreptomycin (both from Sigma-Aldrich, Dorset, UK). The cell lines 32D and FDC-P1 (a Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society generous gift from Dr. N. J. Dibb) were grown in RPMI 1640 supplemented with 5 % WEHI conditioned medium, 10 % FBS (PAA Laboratories), glutamine and penicillin-streptomycin (both from Sigma-Aldrich) as before. pCMV-FLAG-STAT5a has been previously described [START_REF] John | The significance of tetramerization in promoter recruitment by Stat5[END_REF]. The anti-FLAG M2 antibody was purchased from Stratagene (Amsterdam, The Netherlands), and the anti pan-Stat5 antibody was from BD-Biosciences Pharmingen (San Diego, CA, USA). Specific

Cell transfection, preparation of cell extracts, SDS-PAGE and Western blotting

293T cells were transfected with FLAG-STAT5a by the calcium phosphate method of transfection as described previously [START_REF] Meyer | A single residue modulates tyrosine dephosphorylation, oligomerization, and nuclear accumulation of stat transcription factors[END_REF]. Total cell lysates were generated from transfected 293T cells to prepare N-terminal FLAG epitope-tagged STAT5a protein for use as substrate in cleavage assays. Following a PBS wash, cells were lysed on ice for 10 min in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM sodium fluoride, 3 mM sodium orthovanadate (Sigma-Aldrich) and 1 % NP-40 (Calbiochem), supplemented with 1 mM DTT, 5 µg/ml leupeptin, 5 µg/ml aprotinin and 1 µg/ml pepstatin (all from Roche, East Susses, UK) frozen in liquid nitrogen, thawed at 4°C and the supernatant clarified by centrifugation. Cytoplasmic and nuclear extracts were prepared from 32D or FDC-P1 cells according to a standard method [START_REF] Suzuki | Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism[END_REF]. After a PBS wash, cells were resuspended in approximately 10 volumes of hypotonic lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl and 1.5 mM MgCl 2 ) and incubated on ice for 10 min. NP-40 was added to a final concentration of 0.1% and the sample vortexed briefly. The supernatant was collected as a cytoplasmic fraction by centrifugation and the nuclear pellet resuspended in equal volumes of low salt lysis buffer (10 mM Hepes, pH 7.9, 20 mM KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA and 25% glycerol) followed by high salt lysis buffer (20 mM Hepes, pH 7.9, 800 mM KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA and 25% glycerol). Following a 30 minute incubation on ice, samples were centrifuged and the supernatant taken. All of the buffers used in extract preparations contained 1 mM DTT, 1 mM sodium orthovanadate (Sigma-Aldrich), 5 µg/ml leupeptin, 5 µg/ml aprotinin and 1 µg/ml pepstatin (all from Roche). Protein quantification was performed according to manufacturer's instructions, using Bio Extracts were separated by SDS-PAGE, and transferred to Immobilon PVDF membrane (Millipore, Bedford, USA). Western blots were developed using the Amersham Biosciences ECL Plus chemiluminescence kit.

In-vitro protease assay 293T cells were transiently transfected with FLAG-STAT5a and lysed in NP-40 lysis buffer. 1 µl of the lysate was mixed with nuclear extracts from 32D cells and incubated at 37°C for 15-60 minutes in the presence or absence of different serine protease inhibitors. Samples were separated on SDS-PAGE and analyzed by Western blotting using an anti-FLAG antibody.

Protein Purification

The buffer of the nuclear extracts were exchanged to a 20 mM Tris buffer, pH 7.4 using Nap-10 columns (Amersham Biosciences, Buckinghamshire, UK) and precipitated proteins were removed by centrifugation. Ion exchange was performed at room temperature using the peristaltic pump P-1 (Amersham Biosciences). The flow rate was 2.5 ml per minute. For purification, 10 mg of the proteins were fractionated on a HiPrep 16/10 DEAE FF column (Amersham Biosciences) equilibrated in 20 mM Tris buffer, pH 7.4. Proteins were eluted with a stepwise sodium chloride gradient (150-1000 mM, in 20 mM Tris buffer, pH 7.4). The protease activity was determined by the in-vitro protease assay, and purification progress was analysed on a 12 % SDS-gel stained with the Biorad silver gel kit. The peak fraction was diluted 1:2 with 20 mM Tris, pH 7.4 without sodium chloride and loaded to a HiPrep 16/10 CM FF column (Amersham Biosciences) equilibrated in 20 mM Tris, pH 7.4. The proteins were eluted and analysed as described above.

The fraction with peak activity was concentrated 15 fold by Amicon Ultra centrifuge tubes (Millipore). The concentrated proteins were separated in a 16 % silver gel, and 1 ml was applied

LC/MS/MS Identification

Enzymatic Digestion: The corresponding bands from two gels were pooled together before enzymatic digestion thereby increasing the total amount of protein present for enhanced detection by MS. In-gel reduction, alkylation and digestion with trypsin were performed prior to subsequent analysis by MS. Cysteine residues were reduced with dithiothreitol and derivatised by treatment with iodoacetamide to form stable carbamidomethyl derivatives. Trypsin digestion was carried out overnight at room temperature after an initial two hours incubation at 37 o C. LC/MS/MS: Peptides were extracted from the gel pieces by a series of acetonitrile and aqueous washes. The extract was pooled with the initial supernatant and lyophilised. Each sample was then resuspended in 23µ l of 50 mM ammonium bicarbonate and analysed by LC/MS/MS. Chromatographic separations were performed using an Ultimate LC system (Dionex, UK).

Peptides were resolved by reversed phase chromatography on a 75 µm C18 PepMap column. A gradient of acetonitrile in 0.05% formic acid was delivered to elute the peptides at a flow rate of 200 nl/min. Peptides were ionised by electrospray ionisation using a Z-spray source fitted to a QTof-micro (Waters Corp.). The instrument was set to run in automated switching mode, selecting precursor ions based on their intensity, for sequencing by collision-induced fragmentation. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the m/z and the charge state of the peptide.

Database Searching: The mass spectral data was processed into peptide peak lists and searched against the Swiss Prot or NCBI non-redundant databases using Mascot software (Matrix Science, UK). The data was searched using specific amino acid modification parameters, ie. variable cysteine carbamidomethylation modification (resulting from reduction and alkylation reaction), variable methionine oxidation modification, phosphorylation (S, T and Y), ubiquitination, Nacetylation and glycosylation (raw MS data was check for the presence of glycan modification).

The data was also searched using a non-specific enzyme parameter. This parameter enables the identification of non-tryptic peptides, which may be present if there is N or C-terminal cleavage of proteins for example. MS/MS sequence information was obtained for all the peptides included in the results.

RESULTS

Purification of the STAT5 protease

We wished to more clearly understand the molecular regulation of STAT5 by proteolytic cleavage and its importance in different cell types by identifying the protease(s) responsible for the conversion of STAT5α to STAT5γ. Previously, it was shown that immature myeloid cell lines Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society such as FDC-P1 and 32D contain nuclear STAT5γ and, therefore we used the 32D cell line to identify the STAT5-protease in these cells. We initially confirmed previous findings that 32D cells contain nuclear STAT5γ, by lysing cells in boiling SDS sample buffer or NP-40 lysis buffer, followed by western blot analysis of lysates using a pan STAT5 antibody. When cells were lysed in boiling SDS-buffer, the majority of STAT5 detected was STAT5α, and only a small amount of STAT5γ was detected (Fig. 1A, lanes 1-3). In contrast, when the lysates were prepared in NP-40 lysis buffer, most of the STAT5 proteins were STAT5γ (Fig. 1A, lanes 4-6). This confirmed previous findings that immature myeloid cells contain truncated STAT5 proteins, albeit at a considerably lower amount than STAT5α [START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF]. Using an in-vitro protease assay, with FLAGtagged STAT5a as a substrate, we detected the STAT5 protease activity in the nuclear, but not cytoplasmic extracts of 32D cells (Fig. 1B, lanes 1 and 2). To confirm that this proteolytic activity belonged to the serine protease family, as previously described, we performed the in-vitro protease assay in the presence or absence of AEBSF (Fig. 1C). The STAT5 protease activity was inhibited by 4 mM AEBSF in the in-vitro protease cleavage assay (Fig. 1C, lanes 2, 4, 6, 8). Furthermore, Stat5a cleavage activity was clearly detected using low amounts of cell extracts (5 µg-0.625 µg, lanes 1, 3, 5 and 7), and also in the presence of 4mM AEBSF (lanes 2 and 4). These results suggest that the proteolytic activity is abundantly present or highly active in these immature myeloid cells and requires higher concentrations of AEBSF to fully inhibit the activity of the protease.

Based on the results depicted in Figure 1B, we next used only the nuclear extracts for purification of the protease via ion exchange on a DEAE column followed by a CM column.

Utilizing the in-vitro assay to evaluate the STAT5-specific protease activity, maximal cleavage activity was recovered in the fraction eluted with 150 mM sodium chloride from the DEAE column (Fig. 2A, lane 4). The semi-purified protease activity was at high enough levels in this fraction so that the truncated FLAG-STAT5 product was not observed under the experimental conditions used in the in-vitro protease assay (1 hour incubation at 37°C), most likely due to removal of the FLAG-tag by over-digestion at additional secondary sites. The 150 mM fraction was next loaded and separated on the second CM column. The STAT5 protease was eluted with 350 and 450 mM sodium chloride with peak activity detected in the fraction eluted with 450 mM sodium chloride (Fig. 2B, lane 7). The silver stain gel of the eluted fractions, including the active fractions at 350 and 450 mM NaCl did not clearly reveal specific proteins (lanes 4-11). However, after a 15-fold concentration of the 450 mM fraction, two bands were visible between the 22 kD and 36 kD protein marker bands in a SDS-gel, after silver staining (Fig. 2B, lane 12). The exact size of these bands was determined by size exclusion chromatography, which yielded a single Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 peak with a light shoulder and a calculated MW of about 23.5 kD and 25.5 kD (data not shown). This is consistent with the results from a previous purification of STAT5 protease from FDC-P1 cells [22]. Because it was not clear which of the two bands corresponds to the STAT5 protease, an aliquot of the concentrated sample was mixed with the serine protease inhibitor AEBSF coupled to beads, precipitated and analysed on a silver gel. Surprisingly, both bands were found in the precipitate (data not shown), indicating that they both contain active serine proteases.

Identification of the purified proteins by LC/MS/MS

Analysis of the two bands revealed the presence of a single identical protein in each band. The previously unknown STAT5 protease was identified as murine CatG precursor (P28293) (EC 3.4.21.20) (Vimentin-specific protease) with 19 peptides matching and with sequence coverage of 56% (Fig. 2C). Searching the data with the non-specific enzyme parameter did not yield any further increase in sequence coverage. Additionally, no evidence for ubiquitination, phosphorylation, N-acetylation or glycosylation was found in these experiments (data not shown).

From the different parameters used to search the data, there is no obvious explanation as to why the protein resolves as a doublet.

Cathepsin G is responsible for the proteolytic conversion of STAT5

We incubated N-terminal tagged FLAG-STAT5a with nuclear extracts or varying amounts of purified CatG to verify that CatG was the protease that cleaves STAT5 in 32D cells. The resulting STAT5 isoforms were analysed by immunoblotting with anti-FLAG antibody (Fig. 3A). As shown in Figure 3A, STAT5γ generated by purified CatG is of the same size as STAT5 cleaved by the protease in 32D nuclear extracts (lane 1 vs. 2-4). In contrast, the cleavage pattern of STAT5 generated by purified human Leukocyte Elastase (hLE) appears similar, but the truncated form observed was subtly smaller (lane 1 vs. lanes 5-7). To exclude the possibility that hLE is also involved in the proteolytic processing of STAT5 in 32D cells, we used specific inhibitors to block the activity of these two proteases (Fig. 3B). The protease inhibitor specific for CatG blocks the activity of CatG to cleave STAT5a in the in-vitro protease assay (lane 1 and 2), while the specific hLE inhibitor does not block this activity (lanes 1and 3).

Using 32D nuclear extract instead of purified CatG or hLE, the protease activity cleaving STAT5 was strongly inhibited by 2.5 and 25 µM CatG inhibitor and completely blocked with 250 µM (Fig. 3C, lanes 2-4). By contrast, there was no reduced protease activity in the presence of 250 µM hLE specific inhibitor (lanes 6 and 7), while even 25 µM hLE inhibitor can inhibit the Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society activity of purified hLE (lanes 8 and 9). Together, these results demonstrate that CatG is the specific protease that generates STAT5γ in 32D cell lysates. Previously, we observed that incubation of N-terminal tagged FLAG-STAT5a with 32D nuclear extracts containing higher amounts of the STAT5a-protease in the in-vitro cleavage assay, resulted in undetectable levels of FLAG-STAT5a (Fig. 2A, lane 4). We hypothesized that this was due to over-digestion of FLAG-STAT5 at higher protease concentrations. To confirm this, we repeated the in-vitro cleavage assay under the same conditions as before (1 hour at 37°C), but with varying amounts of purified CatG instead of 32D cell nuclear extract. As shown in figure 3D, the amount of FLAG-STAT5a (α or γ) detected was strongly reduced with increasing amounts of CatG added (lanes 2-4) and was completely undetectable in the presence of 10 mU CatG (lane 1). This indicates that CatG can cleave at additional cleavage sites in the STAT5a protein in a dose dependent manner.

STAT5 is full-length in the nucleus of immature myeloid cells and STAT5γ is generated upon lysis of cells.

CatG is normally stored in azurophilic granules in the cytoplasm. With the surprising identification of CatG as the protease responsible for the conversion of STAT5 in 32D cell nuclear extracts, we wanted to clarify whether the detection of STAT5γ, rather than Stat5α in this compartment is due to an authentic in-vivo biologic processing event or instead due artificial exposure of STAT5 to highly active CatG during cell lysate preparation. Therefore, we prepared cytosolic and nuclear extracts by standard methods and also in the presence of 4mM AEBSF or the specific CatG inhibitor in all of the buffers. In contrast to AEBSF, which inhibits multiple serine proteases, the CatG inhibitor allowed us to specifically block CatG activity only. We also included one sample of nuclear extract prepared in boiling SDS-buffer, in which all proteases are denatured. As shown in Fig. 4, in both 32D and FDC-P1 cells, only STAT5α was detected by western blotting for STAT5 when the extracts were prepared in the presence of the specific CatG inhibitor (lanes 3 and 8). By contrast, the nuclear extracts prepared without inhibitors (lanes 1 and 5) or with the serine protease inhibitor AEBSF alone (lanes 2 and 6) contained predominantly STAT5γ. Furthermore, the addition of AEBSF and CatG specific inhibitor together did not increase the amount of STAT5α detected over the addition of the CatG-specific inhibitor alone (lane 7 and 8). The finding that AEBSF did not fully inhibit the proteolytic cleavage of STAT5 by CatG in these cells was not surprising given the results in Figure 1C, which suggests that very high concentrations of this general serine protease inhibitor would be required to fully inactivate all active serine proteases. As before, in the samples prepared with boiling SDS-buffer (lane 4 and Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society 9), the amount of STAT5γ detected was markedly reduced and the full-length form increased. Thus, nuclear STAT5γ is specifically generated by the action of CatG during cell lysate preparation and does not occur in-vivo in intact immature myeloid cells.

DISCUSSION

STAT5 is the signal transducer for the IL-3 and IL-2 family of cytokines. In contrast to mature myeloid cells, in several immature myeloid lineages STAT5γ isoforms lacking the C-terminus were found to be predominantly expressed rather than the full-length form [18-20, 22, 31]. The Cterminus of STAT5 contains the transactivation domain and plays a role in transcriptional activation, and consistent with the detection of STAT5γ, immature myeloid cells express only a subset of genes activated by STAT5α in mature cells [START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF]. Moreover, in acute myeloid leukemia patients a change in constitutive STAT3 and STAT5 expression from full-length to a C-terminally truncated form was significantly associated with disease progression and relapse [START_REF] Xia | Truncated STAT proteins are prevalent at relapse of acute myeloid leukemia[END_REF]. Therefore, unravelling the underlying mechanism that mediates STAT processing enhance our understanding of the regulation of STAT proteins in normal hematopoiesis and its potential dysregulation in abnormal malignant hematopoiesis. Therefore, we decided to study this regulation in normal myeloid cell differentiation.

Previously, it was shown that STAT5γ is generated from the full-length form by limited proteolysis [START_REF] Meyer | Carboxyl-truncated STAT5beta is generated by a nucleus-associated serine protease in early hematopoietic progenitors[END_REF][START_REF] Azam | Functionally distinct isoforms of STAT5 are generated by protein processing[END_REF]22,[START_REF] Xia | A novel serine-dependent proteolytic activity is responsible for truncated signal transducer and activator of transcription proteins in acute myeloid leukemia blasts[END_REF][START_REF] Xia | Truncated STAT proteins are prevalent at relapse of acute myeloid leukemia[END_REF]. The responsible STAT5 protease was constitutively active in the nucleus of immature myeloid cells and cleaved independently of the activation state of STAT5. This unusual nuclear STAT5 specific protease was characterised and shown to be sensitive to PMSF, a broad-spectrum serine protease inhibitor, but not to aprotinin, pepstatin, benzamidine [22] or ONO-5046, a specific inhibitor of hLE and STAT6 protease in mast cells [START_REF] Suzuki | Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism[END_REF]. The size of the STAT5 protease was predicted to be 25 kD, and activity was observed in a wide range of physiological pH values. The cleavage site in murine STAT5 was mapped between Y720 and M721 in STAT5a and Y724 and M725 in STAT5b and mutation of these residues perturbed STAT5 function in myeloid cell maturation [22,31]. We have purified the STAT5 protease from nuclear extracts prepared from the immature myeloid cell line 32D and identify it as CatG, which is expressed during the promyeloid stage of neutrophil differentiation [START_REF] Hanson | Developmental regulation of the human cathepsin G gene in myelomonocytic cells[END_REF]. The characteristics of CatG [START_REF] Wiedow | Neutrophil serine proteases: potential key regulators of cell signalling during inflammation[END_REF] match perfectly with the characteristics described for the STAT5 protease in size, sensitivity and specificity. There are several possible cleavage sites for CatG in the STAT5 protein, and consistent with this our results show a second band for STAT5γ, depending on the concentration of the protease in the extracts and the incubation time. Indeed, in some cases, N- Together with leukocyte elastase, proteinase 3 (PR3) and azurocidine, a proteolytically inactive protease homolog, CatG belongs to a superfamily of azurophil granule serine proteases. These proteases are expressed as inactive proforms (zymogens) which are activated by dipeptidylpeptidase I [START_REF] Mcguire | Generation of active myeloid and lymphoid granule serine proteases requires processing by the granule thiol protease dipeptidyl peptidase I[END_REF], and the active enzymes are stored in azurophilic granules of hematopoietic cells [START_REF] Gullberg | Biosynthesis, processing and sorting of neutrophil proteins: insight into neutrophil granule development[END_REF][START_REF] Levy | Antibiotic proteins of polymorphonuclear leukocytes[END_REF]. It is possible that the two bands we obtained after purification represent active processed forms of CatG with a size of about 23.5 kD and 25.5 kD, because both bands were precipitated with the serine protease inhibitor AEBSF coupled to beads, while CatG was the only protein identified by mass-spectroscopy. To exclude the possibility that other serine proteases from the azurophile granule are also responsible for the conversion of STAT5α we used purified hLE and specific inhibitors for CatG and hLE. The hLE is able to process STAT5 with a similar cleavage pattern to that of CatG, but with the specific inhibitors we could clearly show that only CatG is responsible for the conversion of STAT5 in 32D cell nuclear extracts.

It is most likely that CatG, which is stored in cytoplasmic granules is pelleted together with the nuclei during the standard method for preparing cytoplasmic and nuclear extracts. In that case, CatG is only a contamination of the nuclear fraction as it was clearly shown for hLE in neutrophils cleaving STAT3 [27]. However, CatG has also been found in the nucleus of cells; in the leukemic cell line NB4, Biggs et. al [START_REF] Biggs | The human brm protein is cleaved during apoptosis: the role of cathepsin G[END_REF] demonstrated that the human brm protein is cleaved by CatG during apoptosis when CatG was released from the granule of NB4 cells and became diffusely distributed. Because CatG is found in the nucleus only after induction of apoptosis, but the truncated form of STAT5 is thought to be generated in the nucleus of uninduced cells, we wanted to determine whether the proteolytic event occurs in the living cell or during the preparation of the cytoplasmic and nuclear extracts. Our results clearly show that full-length STAT5 is cleaved during the preparation of the extracts without inhibitors for CatG. With the conventional buffers only STAT5γ was detected by western blotting, and the addition of AEBSF also did not yield STAT5α. This alone would suggest that STAT5γ is generated in the nucleus of intact immature myeloid cells, because the presence of 4 mM AEBSF should block all serine protease activity in the extracts, as shown in Figure 1C. But also in this in-vitro cleavage assay, where the nuclear extract was 20x diluted in the sample mix (lane 1 and 2), the CatG activity was not completely blocked. It is possible that the concentration of 4 mM AEBSF, used in various studies, was not high enough to block the activity of all the serine proteases (including CatG) in these cells during lysis. In contrast, the STAT5 cleavage was completely blocked by the specific CatG inhibitor. This strongly indicates that the limited proteolytic conversion of STAT5α to C-Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society terminally truncated STAT5 is mediated by CatG and occurs during the preparation of the extracts from 32D and FDC-P1 cells. Similar results were shown for the conversion of STAT3 by hLE in neutrophils [27]. When the nuclear fraction was lysed in boiling SDS-buffer inconsistent results were obtained; in all cases STAT5α was significantly increased, but in some cases STAT5γ was also present. While this is a standard method to lyse cells and inactivate proteases simultaneously, the results may be misleading when the cells contain high amounts of proteases.

The identification of the STAT5 protease allowed us to use a specific inhibitor to selectively and completely block CatG activity during the preparation of the extracts, yielding more conclusive results than lysis with boiling SDS-buffer. From our results we cannot exclude the possibility that under certain normal (such as apoptosis) or disease situations, leakage of active CatG into the nucleus may cleave STAT5, but we can exclude this for IL-3 stimulated 32D and FDC-P1 cells. Thus, our results strongly suggest that full-length STAT5 rather than truncated STAT5 is essential for myeloid differentiation. (A) 2.5 µg nuclear extract (lane 1) or varying amounts of CatG (lanes 2-4) or hLE (lanes 5-7) were mixed with 1 µl 293T cell lysate containing FLAG-STAT5a in a total volume of 20 µl and incubated for 20 min. at 37°C, followed by immunoblotting with anti-FLAG antibody. As control, the 293T cell lysate was incubated for 20 min. at 37°C alone (lane 8). (B) The STAT5 protease is inhibited by an protease inhibitor specific for CatG but not by an hLE inhibitor in the in-vitro cleavage assay. 2.5 mU CatG were mixed with 1 µl 293T cell lysate containing FLAG-STAT5a alone (lane 1) or in the presence of 25 µM of the CatG inhibitor (lane 2) or 25 µM of the hLE inhibitor (lane 3) and incubated for 30 min. at 37°C, followed by immunoblotting with anti-FLAG antibody. As control, the 293T cell lysate was incubated for 30 min. at 37°C alone (lane 4). (C) 2.5 µg 32D cell nuclear extract were mixed with 1 µl 293T cell lysate containing FLAG-STAT5a alone (lanes 1, 6 and 8) or in the presence of the CatG inhibitor (2.5-250 µM) (lane 2-4) or 250 µM of the hLE inhibitor (lane 7) and incubated for 30 min. at 37°C, followed by immunoblotting with anti-FLAG antibody. As control, the 293T cell lysate was incubated for 30 min. at 37°C alone (lanes 5 and 10). The protease activity in the 32D nuclear extract was blocked in the presents of 250 µM of the CatG inhibitor, whereas the addition of an hLE inhibitor showed no effect. (D) Varying amounts of CatG (lanes 1-4) were mixed with 1 µl 293T cell lysate containing FLAG-STAT5a in a total volume of 20 µl and incubated for 60 min. at 37°C, followed by immunoblotting with anti-FLAG antibody. As control, the 293T cell lysate was incubated for

  protease inhibitors, AEBSF, cathepsin G Inhibitor I and Elastase Inhibitor IV (N-(2-(4-(2,2-Dimethylpropionyloxy) phenylsulfonylamino) benzoyl) aminoacetic acid N-(o-(p-Pivaloyloxybenzene) sulfonylaminobenzoyl) glycine) were all from Calbiochem (Darmstadt, Germany) and used at the concentrations indicated in the appropriate figure. The proteases Cathepsin G and human Leukocyte Elastase were from Sigma-Aldrich.

  to a HiLoad 16/60 Superdex 75 prep grade size exclusion (SE) column (Amersham Biosciences) equilibrated in 20 mM Tris, pH 7.4 buffer containing 150 mM sodium chloride. The SE column was calibrated with the Low Molecular Weight Gel Filtration Calibration Kit from Amersham Biosciences including Ribonuclease A (13,7 kD), Chymotrypsinogen A (25 kD), Ovalbumin (43 kD), Albumin (67 kD) and Blue Dextran 2000. The SE column run was performed with the Biologic Duo Flow FPLC system from Biorad at 4°C and a flow rate of 1 ml per minute. Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society terminal tagged STAT5 was not detected by the FLAG-antibody, indicating at least one more cleavage site near the N-terminus used by CatG (data not shown).

Figure 1 .

 1 Figure 1. Truncated STAT5 is generated by a protease, which is found in nuclear extracts.(A) 32D cells were lysed in equal volumes of hot SDS-sample buffer (lanes 1-3) or conventional NP-40 lysis-buffer (lanes 4-6). Proteins of 10, 5 and 1 µl of the lysates were separated on an SDS gel and the STAT5 proteins were analysed by immunoblotting using anti-pan STAT5 antibody, detecting full-length and C-terminal truncated STAT5. (B) 25 µg cellular (lane 1) or 5 µg nuclear (lane 2) extracts from 32D cells were mixed with FLAG-STAT5a (293T cell lysate, 5 µg) in a total volume of 40 µl and incubated for 15 min. at 37°C. 20 µl were separated on an SDS-gel followed by immunoblotting with anti-FLAG antibody. As control, FLAG-STAT5a-containing lysate were incubated for 15 min. at 37°C alone (lane 3). (C) Varying amounts of 32D cell nuclear extracts were incubated with FLAG-STAT5a (293T cell lysate, 5 µg) in a total volume of 20 µl for 20 min. at 37°C in the presence or absence of 4 mM AEBSF before separating on an SDS-gel followed by immunoblotting with anti-FLAG antibody. As control, FLAG-STAT5acontaining lysate were incubated for 20 min. at 37°C alone (lane 9).

Figure 2 .

 2 Figure 2. Purification of the STAT5 protease.To asses progress during purification of the STAT5 protease activity, 23 µl of each fraction from the ion exchange columns were separated by 12% SDS-PAGE and the proteins visualized by silver staining. STAT5 protease activity was analysed using the in-vitro cleavage assay. Therefore

Figure 3 .

 3 Figure 3. Comparison of the STAT5 cleavage pattern by the protease in 32D cell nuclear extracts with that of CatG or hLE via in-vitro cleavage assay.

  at 37°C alone (lane 5).

Figure 4 .

 4 Figure 4. Comparison of STAT5 present in nuclear extracts using different conditions during preparation of extracts. 20 µl nuclear extracts prepared from 32D (left panel) or FDC-P1 cells (right panel) were separated by SDS-PAGE followed by western blotting using anti-pan STAT5 antibody. Extracts were prepared using the standard method (lanes 1 and 5), with 4 mM AEBSF (lanes 2 and 6), with 4 mM AEBSF and 250 µM CatG inhibitor (lane 7) or with 250 µM CatG inhibitor alone (lanes 3 and 8) in all buffers. Additionally the extracts were prepared with the standard method, but the nuclei were lysed in hot SDS-buffer (lanes 4 and 9). The full-length and truncated forms of STAT5 are indicated with arrows.

Figure 1 Biochemical

 1 Figure 1

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Figure 2 Biochemical

 2 Figure 2

21

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Figure 2 Biochemical

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Figure 3 Biochemical

 3 Figure 3

Biochemical Journal Immediate Publication. Published on 14 Feb 2007 as manuscript BJ20061877 © 2007 Biochemical Society 12

  .5% 2-mercaptoethanol, and a trace amount of bromphenol blue in 375 mM Tris-HCl, pH 6.8)

	-Rad Protein Assay Dye Reagent
	Concentrate (Bio-Rad Laboratories GmbH, Muenchen, Germany). Alternatively cells or the
	nuclear fraction were lysed in boiling (95°C) 5x SDS-sample buffer (25% SDS, 25% glycerol,

TABLE OF ABBREVATIONS

 OF

	CM, carboxymethyl
	DEAE, diethylaminoethanol
	DMEM, Dulbecco's Modified Eagle Medium
	LC, liquid chromatography
	MS, mass spectrometry
	m/z, mass-to-charge-ratio
	RPMI, Roswell Park Memorial Institute

© 2007 Biochemical Society

ACKNOWLEDGMENTS

We thank Dr. N. J. Dibb for the generous gift of the FDC-P1 cells. Dr. A. Woods and Dr. S.

Gallois-Montbrun are thanked for their helpful discussions, and Dr. S. D. Barbee for critical reading of the manuscript. BS and LH were supported by grants from the MRC, UK and the Wellcome Trust respectively.


G M Findlay

L Yan

Mieulet V Lamb

Richard F Lamb
email: richard.lamb@icr.ac.uk

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

Keywords: MAP4K3, amino acid signalling, mTOR, cell growth

The mTOR signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Whilst signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K/4E-BP1 phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.

INTRODUCTION

Both growth factors such as insulin/IGF-1 and ambient nutrients such as amino acids stimulate growth via signalling to the mTOR pathway [1]. The pathway regulates growth primarily through regulation of ribosome biogenesis and protein translation, and has emerged as a promising target for therapies to combat diseases such as cancer and diabetes [2]. Recent work has indicated that the growth factor signalling pathway resulting in mTOR pathway activation involves the PI3K or MAPK signalling cascades which activate protein kinases such as PKB, ERK and p90Rsk. These kinases phosphorylate TSC2 thereby functionally inactivating the TSC1-2 tumour suppressor complex through an undefined mechanism [3]. TSC1-2 functions as a GAP inhibitor of the Rheb GTPase [4], which itself positively regulates the mTOR pathway, possibly through regulation of mTOR activity [1].

In contrast to the growth factor regulation of mTOR signalling, the role of nutrients in regulation of mTOR signalling has been relatively understudied. Although amino acids have been identified as activators of mTOR signalling, and are required for maximal activation of mTOR signalling by growth factors such as insulin [5], the signalling pathway downstream of amino acids is poorly characterized. To identify new components in this pathway that may be amenable to future therapies, we performed an RNAi screen of Drosophila protein kinases required for signalling to dS6K, a serine-threonine kinase downstream of mTOR. We report here our identification of a Ste20-related kinase, MAP4K3, which we show is required for amino acids to activate S6K and also induces phosphorylation of the mTORregulated inhibitor of eIF4E, 4E-BP1. Importantly, we show that the activity of MAP4K3 is itself regulated by amino acids but not by insulin, suggesting that MAP4K3 plays an important role primarily in the nutrient regulation of mTOR signalling.

MATERIALS AND METHODS

Chemicals and Antibodies

Rapamycin and wortmannin (Calbiochem) were dissolved in DMSO, and used at a final concentration of 50 nM and 100nM, respectively. PD184352 (a gift from Dr. Richard Marais, ICR, London), was dissolved in DMSO, and used at a final concentration of 1 or 10 µM. The anti-MAP4K3 antibody was prepared at the Hybridoma Unit (Institute of Cancer Research) by immunizing rabbits with a GST fusion of aa360-530 of human MAP4K3 after PCR cloning from a MAP4K3 cDNA (Origene) into pGEX6P-1.

Antibody was subsequently affinity-purified using the immunogen chemically-coupled to glutathione agarose. A monoclonal antibody to Rheb (3H6) was isolated at the Cancer Research UK London Research Institute by immunizing mice with a GST-fusion to Rheb aa1-167 and generating a hybridoma by standard protocols. The monoclonal antibody to S6K1 is from Transduction Laboratories. Polyclonal anti-dTsc1 antibody and anti-dS6K antibodies were provided by D. J. Pan (Johns Hopkins, Baltimore) and D. Alessi (University of Dundee), repectively. All other antibodies were purchased from Cell Signaling Technologies.

Plasmid Constructs

pRK5 S6K1-GST and pcDNA 3xHA 4E-BP1 have been described previously [6]. pRK5mycMAP4K3 was constructed using a full-length cDNA clone (Origene, Acc. No. NM_003618). A mutation of the DFG motif (to AFG) was made using a Quick-change mutagenesis kit (Stratagene) and sequenced to ensure only the correct mutation had been introduced. pRK5Msn (misshapen) and pCINeo HA-TNIK were provided by Drs. Edward Skolnik (NYU Medical Centre, USA) and K. Kariya (University of Ryukyus, Japan), respectively. pGEX GST-S6 was provided by Dr. Andrew Tee (University of Dundee) and GST-S6 produced by standard purification on glutathione-agarose.

Production of dsRNA for RNAi of kinases in Drosophila S2 cells

239 protein kinases in the Drosophila genome were identified by database searches (www.kinase.com/kinbase), and the position of each kinase dsRNA template in a whole-genome Drosophila RNAi library (MRC Geneservices, UK) determined. 1 µl of each kinase template was diluted in 50 µl TE, 5 µl of which was amplified by Expand Hi-Fidelity PCR kit (Roche Diagnostics) using T7 primers. PCR products were then analyzed by agarose gel electrophoresis, purified by ethanol precipitation, and 1 µg of DNA was used as a template for dsRNA synthesis by an in vitro transcription kit (Ambion). dsRNAs were purified by LiCl precipitation according to the manufacturer's instructions, before quantity and quality were determined by agarose gel electrophoresis; subsequently 202 kinase dsRNAs were used for screening. For addition of dsRNA, Drosophila S2 cells were diluted to a final concentration of 1 x 10 6 cells/ml in standard Schneider's media (Invitrogen). For the primary screen dsRNA additions were scaled down to a 24-well format and kinase dsRNAs added in equal combination with dsRNA targeting dTsc1 [7] (total of 30 µg/ml dsRNA). The cDNAs for positives identified in a primary screen were subsequently cloned into pGEM-T (Promega) and sequenced to verify correct annotation of the gene. Positives were confirmed in a secondary screen in 12-well dishes; 1 ml of cells was plated per well of a 12-well cell culture dish, and allowed to settled for at least 2 h. dsRNAs were added directly to the media to a final concentration of 15 µg/ml for each dsRNA, followed immediately by vigorous agitation, with addition of water serving as control. Following incubation at room temperature for 1h, 1 ml of Drosophila SFM containing 10% FCS (PAA laboratories) was added and cells were incubated for 5 days to allow for turnover of the target protein, before cell extracts were prepared in lysis buffer.

Mammalian cell treatments, transfections, siRNAs

Hela and HEK293T cells were grown in DMEM containing 10% FCS (TCS Laboratories) and antibiotics. For plasmid transfections of HEK293T, cells were plated at a density of 8 x 10 5 cells per well of a 6-well plate, and incubated overnight before transfection. Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and cell extracts prepared 40 h post-transfection in lysis buffer (50 mM Tris-HCl pH 7.5, 1% (v/v) Triton X-100, 1 mM EDTA, 1mM EGTA, 50 mM NaF, 50 mM -glycerophosphate, 1 mM Na 3 VO 4 , 0.27 M sucrose, 0.1% (v/v) -mercaptoethanol, and complete protease inhibitors (Roche)). For siRNA transfections Hela cells were plated at 1x10 5 cells/dish in a 6-well plate and transfected with Lipofectamine 2000 with the following siRNAs purchased from Dharmacon: control siRNA (scrambled S6K1 control [8]), Rheb (siRNA-2 [4]) TSC2 human siRNA Smartpool, M4K3-1 (nucleotides 847-865 of NM_003618 human MAP4K3). M4K3-2 corresponds to duplex 2 (MAP4K3 targeting Duopack, Invitrogen). All siRNAs were used singly at a final concentration of 66nM whilst for double transfections the final concentrations were 33nM. For amino acid deprivation experiments in Hela or HEK293T cells, cells in 6-well plates were serum-starved for 1h (Hela) or 16h (HEK293T), washed once in D-PBS containing 1xMEM vitamins and 10mM glucose, and maintained in this media for 5-60 minutes. To restimulate with amino acids D-PBS was replaced with DMEM (also containing 10mM glucose) lacking serum.

In vitro assay of S6K and MAP4K3 activity S6K-GST was purified from cell extracts using 10 µl glutathione-agarose (Sigma-Aldrich) for 2 h at 4°C. Beads were washed three times with lysis buffer plus 0.5M NaCl, and once with S6K kinase buffer (50 mM Tris-HCl pH 7.5, 0.1 mM EGTA, 0.1% (v/v) -mercaptoethanol, 10 mM magnesium acetate, 2.5 µM PKI (a Protein Kinase A inhibitor; Sigma-Aldrich)). Kinase assays were carried out in 50 µl S6K kinase buffer supplemented with 100 µM ATP, 3 µCi -32 P-labeled ATP, and 3 µg GST-S6. Reactions were carried out at 30°C and terminated after 30 mins by the addition of 2X SDS-PAGE sample buffer.

Proteins were separated by SDS-PAGE, transferred to PVDF membranes whereupon 32 P incorporation into GST-S6 was determined by autoradiography. Myc-tagged MAP4K3 was immunoprecipitated from cell extracts using 5 µg anti-myc 9E10 monoclonal antibody plus 20 µl protein G-sepharose for 2 h at 4°C. Immunoprecipitates were washed three times with lysis buffer plus 0.5M LiCl, and once with Ste20 kinase buffer (20 mM Tris-HCl pH 7.2, 25 mM -glycerophosphate, 5 mM EGTA, 1 mM Na 3 VO 4 , 0.1% (v/v) -mercaptoethanol, 15 mM MgCl 2 ). Kinase assays were carried out in 50 µl Ste20 kinase buffer supplemented with 100 µM ATP, 3 µCi -32 P-labeled ATP and 5 µg MBP (Sigma-Aldrich). Reactions were carried out at 30°C and terminated after 10 mins by the addition of 2X SDS-PAGE sample buffer.

Proteins were separated by SDS-PAGE, transferred to PVDF membranes whereupon 32 P incorporation into MBP was determined by autoradiography and subsequently analyzed on a phosphorimager. Phosphorimager quantitation of S6K1-GST and myc-MAP4K3 kinase assays were performed on three parallel dishes from a single experiment using ImageQuant software (Amersham Biosciences).

FACS analysis

Hela cells transfected with siRNAs and maintained in DMEM + 10% FCS (one 6-well plate per condition) were harvested after 72 hours by trypsinization and washed twice in PBS. Cells were fixed with 70% ethanol for 30mins at 4ºC. The fixed cells were centrifuged at 2000rpm for 3mins, washed twice with PBS and treated with Ribonuclease (Roche) at 100µg/ml at room temperature for 15min.

Fixed cells were stained with propidium iodide at a final concentration of 50µg/ml, and subjected to FACS analysis on a Becton Dickonson LSRII to determine the forward scatter of G1-, S-and G2/Mphase populations. Analysis of FACS and overlays of forward scatter were performed using CellQuest Pro software.

RESULTS

To activate dS6K in Drosophila S2 cells we depleted dTsc1, which activates dS6K [7] via Rheb, a small GTPase inhibited by TSC1-2 [4], and monitored dS6K phosphorylation on SDSPAGE. As in other cell types, activation of dS6K leads to a slower-migrating hyperphosphorylated species that is diminished by treating cells with the mTORC1 inhibitor rapamycin or depleting dTOR by RNAi (Fig. 1a, lanes 3 and 4).

Using dsRNA to co-suppress around 200 Drosophila protein kinases along with dTsc1 we identified three kinases not implicated previously in mTOR signalling that are required for dS6K phosphorylation in this setting (Fig. 1a, lanes 7-9). Suppression of CG1776, CG7097, or CG8767, decrease dS6K phosphorylation in S2 cells, whilst co-depletion of 196/202 of the other kinases tested (such as sev and tefu, Fig. 1a, lanes 5 and 6) do not.

BLAST homology searches reveal that the closest human orthologue of Drosophila CG7097 is MAP4K3 (also known as GLK, germinal centre-like kinase [START_REF] Diener | Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase[END_REF]), a Ste20-related MAP4K. We therefore tested whether MAP4K3 suppression by RNA interference in mammalian cells also inhibits S6K1 phosphorylation as in Drosophila cells. S6K1 activity is regulated by multi-site phosphorylation, including Thr 389 in the hydrophobic motif, a site that may be directly phosphorylated by mTORC1 [10] and is essential for enzyme activity. Using RNAi we suppressed Rheb or MAP4K3 in Hela cells and measured phosphorylation of S6K1 at Thr 389 in DMEM containing amino acids or following amino acid removal and restimulation. In DMEM media phosphorylation of S6K1 at Thr 389 is strongly inhibited by RNAi of Rheb, and partially by two RNAi sequences that suppress MAP4K3 expression (Fig. 1b). We consistently observe that more complete knock-down of MAP4K3 expression is required to elicit effective suppression of S6K1 phosphorylation at Thr 389 (data not shown), suggesting that MAP4K3 may be a very active or abundant kinase that must be very effectively suppressed to fully inhibit signalling to S6K1. To verify that the effect of MAP4K3 on maintaining S6K1 activity is not via inhibition of TSC1-2, we suppressed TSC2 by RNAi in Hela cells and co-depleted Rheb or MAP4K3. Depleting TSC2 alone stimulates S6 phosphorylation at the site phosphorylated only by S6K, (Ser 240/244 ), and this activation is suppressed by co-depletion of Rheb or MAP4K3, indicating that the effect of MAP4K3 on promoting S6K1 activity is independent of effects on TSC1-2 (Fig. 1c).

Since amino acid activation of S6K1 also appears to be independent of TSC1-2 [START_REF] Smith | The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses[END_REF] [12], we asked whether MAP4K3 is involved in the activation of S6K1 by amino acids. As previously reported [START_REF] Nobukuni | Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase[END_REF], stimulation of Hela cells with amino acids induces phosphorylation of S6K1 Thr 389 and Ser 240/244 phosphorylation of S6 that is strongly suppressed by an RNAi sequence targeting Rheb (Fig. 1d, lanes 2 and 4), but also by two siRNAs targeting MAP4K3 (Fig. 1d, lanes 6 and 8). Since MAP4K3 has been proposed to be involved in activation of JNK [START_REF] Diener | Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase[END_REF], we also tested whether it is required for JNK activation in response to two common stresses. However, both osmotic stress and treatment with the protein synthesis inhibitor anisomycin induce equivalent phosphorylation of JNK in control Hela cells or in cells in which MAP4K3 is suppressed (data not shown). Thus MAP4K3 is the likely mammalian orthologue of Drosophila CG7097, and plays an unexpected role in the phosphorylation of S6K1.

To lend support to the loss-of-function analyses we explored whether overexpression of MAP4K3 in HEK293T cells increases mTOR signalling resulting in phosphorylation of S6. Overexpression of myc epitope-tagged wild-type MAP4K3 but not a kinase-inactive mutant (of the conserved DFG motif) stimulates phosphorylation of S6 at both Ser 235/6 and Ser 240/44 to a level similar to stimulation with serum (Fig. 2a). As a control for specificity of phosphorylation, we similarly overexpressed MAP4K3 and determined the phosphorylation of the related AGC-family kinase PKB at its hydrophobic motif Ser 473 site, since increased phosphorylation of this site generally correlates with increased PKB activity. Unlike S6K, PKB activity is not regulated by amino acids [5] and is regulated by an mTOR complex (mTORC2) that is insensitive to rapamycin [START_REF] Sarbassov | Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[END_REF], unlike the mTORC1 complex upstream of S6K1. However, upon overexpression MAP4K3 does not induce PKB Ser 473 phosphorylation under conditions where it increases phosphorylation of S6 to a level similar to stimulation with insulin, whilst expression of related control Ste20-family kinases TNIK and Msn (misshapen) poorly stimulate S6 phosphorylation (Fig. 2b), indicating that MAP4K3 is unlikely to be a general regulator of mTOR activity.

To examine whether MAP4K3 regulates phosphorylation of the hydrophobic motif Thr 389 site in S6K1 and/or promotes S6K1 activity, we compared Thr 389 phosphorylation and activity of a co-expressed S6K1 reporter induced by MAP4K3 to that of two related Ste20 kinases. Overexpression of MAP4K3, but not TNIK nor Msn, leads to increased reporter S6K1-GST Thr 389 phosphorylation and activity similar to insulin stimulation (Fig. 2c, lanes 2 and 4). MAP4K3's activation of reporter S6K1 is rapamycin-sensitive (Fig. 2c, lane 8), but is relatively insensitive to concentrations of an inhibitor of PI3K (wortmannin) up to 200nM (Fig. 2c, lanes [START_REF] Diener | Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase[END_REF][10][START_REF] Smith | The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses[END_REF] or an inhibitor of MEK1-2 (PD184352) up to concentrations of 10µM (Fig. 2c, lanes 12 and 13), under conditions where these concentrations of inhibitors efficiently block activation of S6K1 by insulin and ERK1/2 by TPA (data not shown).

Lastly, in HEK293T cells we assessed the effect of overexpressed MAP4K3 on 4E-BP1, an inhibitor of the translation initiation factor eIF4E which is similarly regulated by nutrients and mTORC1 [START_REF] Hara | Regulation of eIF-4E BP1 phosphorylation by mTOR[END_REF][START_REF] Gingras | Regulation of translation initiation by FRAP/mTOR[END_REF]. Stimulation of cells with insulin (Fig. 2d, lane 2) or overexpression of MAP4K3 (Fig. 2d, lane 6) also leads to an accumulation of slower-migrating forms of 4E-BP1 that are hyperphosphorylated at Thr 70 , an inhibitory site thought to be regulated by mTORC1 [START_REF] Gingras | Hierarchical phosphorylation of the translation inhibitor 4E-BP1[END_REF]. Similar to the results with S6K1, increased phosphorylation of 4E-BP1 induced by MAP4K3 overexpression is inhibited by treatment with rapamycin (Fig. 2d, lane 7), and is relatively insensitive to 100nM wortmannin (Fig. 2d, lane 8).

Because MAP4K3 is required for the activation of S6K1 by amino acids, we next sought to determine whether ectopic MAP4K3 overexpression rescues S6K1 from inactivation during amino acid withdrawal. Amino acid withdrawal leads to a rapid inactivation of S6K1 activity [5] associated with dephosphorylation at Thr 389 . Following stimulation of serum-starved HEK293T with insulin or overexpression of MAP4K3 to activate S6K1, we removed amino acids and at varying times measured S6K1 activity and phosphorylation at the Thr 389 site. We find that when overexpressed MAP4K3 significantly delays dephosphorylation of Thr 389 and inhibition of S6K1 activity (S6K1 remaining around 50% active in cells overexpressing MAP4K3 compared to >10% for control insulin-stimulated after 15 minutes of amino acid withdrawal, Fig. 3a,b), indicating that ectopic MAP4K3 overexpression partly uncouples S6K1 from the inhibitory effect of amino acid withdrawal.

These data suggested to us that amino acid sufficiency might itself regulate MAP4K3 activity. To test this possibility, we transiently transfected myc-tagged MAP4K3 in HEK393T cells at a level of expression that does not significantly activate S6K1, and removed amino acids, or readded them after deprivation for 30 minutes. Like S6K1, we find that MAP4K3 activity measured against the generic substrate MBP is strongly inhibited (to around 16% of initial activity) after 30 minutes of amino acid withdrawal (Fig. 3c, lane 2). Importantly, MAP4K3 is rapidly restimulated by readdition of amino acids and is activated 3.8-fold within 5 minutes, with peak activity observed 15 minutes after amino acid readdition (an 8.8-fold activation compared to the amino acid-deprived condition, Fig. 3d). Interestingly, phosphorylation of S6K1 at Thr 389 is somewhat delayed relative to MAP4K3 activation and is detectable only 15-30 minutes after amino acid readdition (Fig. 3c, compare lanes 3 and 4 of panels 1 and 3). As controls, we assessed MAP4K3 activity following either insulin stimulation or mTORC1 inhibition with rapamycin. However, unlike S6K1, myc-MAP4K3 is not activated by insulin nor inhibited by rapamycin (Fig. 3c, panel 1, lanes 9 and 10) under conditions where these treatments respectively stimulate or inhibit S6K1 Thr 389 phosphorylation (Fig. 3c, panel 3, lanes 9 and 10). Thus, like S6K1, MAP4K3 activity is regulated by amino acid sufficiency, but unlike S6K1 is independent of mTORC1 and not activated by insulin stimulation.

The mTOR pathway regulates cell growth at least in part by regulation of S6K and eIF4E activities [START_REF] Fingar | Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E[END_REF]. To determine whether MAP4K3 regulates cell growth we again used RNAi to downregulate MAP4K3 or Rheb in Hela cells growing in serum, or treated cells with rapamycin for a sustained (48h) period to block mTORC1 activity, and analysed the size of populations in G1, S and G2/M cell cycle phases by FACS analysis. The results (Fig. 3e) show that suppression of either MAP4K3 (using either the M4K3-1 or M4K3-2 siRNAs, bottom panels) or Rheb (top right panel) markedly decreases cell size in G1 (top four panels) and at all cell cycle phases (bottom table) in a similar manner to sustained mTORC1 inhibition caused by treating cells with rapamycin. Thus MAP4K3 function promotes both mTOR signalling to S6K1 and 4E-BP1 and cell growth in Hela cells. On the basis of these observation we propose a model in which MAP4K3 is downstream of a signal provided by amino acids but upstream of mTORC1 (Fig. 3f).

DISCUSSION

In metazoans both nutrient amino acids and mitogens are required to promote mTOR signalling and cell growth [1]. Activation of mTOR signalling by mitogens involves activation of protein kinases such as PKB [3], p90Rsk [START_REF] Roux | Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase[END_REF] and Erk [START_REF] Ma | Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis[END_REF] that have been reported to somehow inhibit the function of the TSC1-2 complex leading to increased GTP loading of the Ras-like GTPase Rheb [4]. In contrast, amino acids do not appear to regulate Rheb GTP loading [START_REF] Nobukuni | Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase[END_REF], and although a class III PI3K (Vps34) has been implicated upstream of mTORC1 [20] [START_REF] Nobukuni | Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase[END_REF], no analogous protein kinases have been identified that respond to amino acids and activate mTOR signalling.

We find, both in Drosophila and in mammalian cells, that suppression of MAP4K3 inhibits mTOR signalling to S6K in the context of activation of the pathway induced by deficiency in TSC1-2. Since a variety of oncogenic signalling pathways inhibit TSC1-2 [3, [START_REF] Roux | Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase[END_REF][START_REF] Ma | Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis[END_REF], and TSC1-2 is predicted also to be inactivated by loss of function of the tumour suppressor PTEN via activation of PKB [3], MAP4K3 may represent a promising new candidate for inhibition of the pathway in diseases of TSC1-2-or PTEN-loss of function. Although we have not defined the mechanism of MAP4K3 action on mTOR signalling, our data indicates that; 1) kinase activity is required; 2) both S6K1 and phosphorylation of 4E-BP1 are induced; 3) these effects are rapamycin-sensitive, point to the mTORC1 complex as the likely target of MAP4K3 action. Interestingly, addition of excess amino acids (relative to the normal concentrations found in DMEM) has been reported to fully activate S6K in the absence of growth factors [5], similar to overexpression MAP4K3, suggesting that MAP4K3 may be activated further by amino acid excess. Future studies will be required to clarify these points. However, having defined the action of a new protein kinase in the mTOR pathway, delineating the signals downstream of amino acids that activate MAP4K3, and determining its mechanism of action will likely shed further light on nutrient regulation of cell growth.

CONCLUSIONS

In conclusion, our data indicate that amino acids stimulate the activity of a Ste20-family kinase, MAP4K3, with maximal kinase activation occurring concordant with phosphorylation of the Thr 389 site of S6K1. In Hela cells MAP4K3 activity is required for amino acid-induced activation of S6K1 and mediates rapamycin-senstive signalling to two effectors of mTORC1, but is not itself stimulated by insulin or inhibited by rapamycin. Lastly, MAP4K3 promotes cell growth in human Hela cells in culture in a similar manner to Rheb and mTORC1. HEK293T cells were transfected with 2.5 µg of pRK5myc vector or pRK5myc MAP4K3 wild-type or AFG kinase-dead MAP4K3 (KD). After 24 hours cells were serum-starved for 16 hours and controls stimulated with 10% FCS for 30 minutes (FCS 30') or pretreated with 100nM wortmannin for 60 minutes prior to stimulation (FCS 30'+Wort) and lysates prepared. Panel 1, endogenous S6 phosphorylation detected with a phospho-specific antibody to Ser 235/6 ; panel 2, S6 phosphorylation detected with a phospho-specific antibody to Ser 240/44 ; panel 3, total S6; panel 4, anti-myc (9E10) to detect expression of myc-epitope tagged kinase. b) Overexpression of MAP4K3 does not activate phosphorylation Ser 473 of PKB. HEK293T cells were transfected as in a with pRK5myc vector, pRK5mycMAP4K3, pRK5mycMsn or pCI HA TNIK and lysates probed with antibodies as in a, or with antibodies to PKB Ser 473 (panel 1), total PKB (panel 2) or HA epitope to detect HA-TNIK (panel 6). As a control for PKB activation vector-transfected cells were stimulated with 1µM insulin (Ins) for 30 minutes or pretreated with 100nM wortmannin (Wt) for 60 minutes prior to stimulation. c) Activation of S6K1 by MAP4K3 is rapamycin-sensitive, but independent of MEK and PI3K signalling. HEK293T cells were transfected as in Fig. 2a with 2.5 µg pRK5myc vector or pRK5mycMAP4K3 and 0.1µg pRK5 S6K-GST reporter and serum-starved for 16 hours. Vector-transfected cells were subsequently stimulated for 30 minutes with 1µM insulin (Ins, time=0) and transferred D-PBS as in Fig. 1d for 5-60 minutes prior to lysis, or if expressing MAP4K3 left unstimulated and transferred to D-PBS for 5-60 minutes (MAP4K3). Panel 1, Thr 389 phosphorylation (arrow) of S6K-GST reporter; panel 2, S6K-GST reporter detected with a monoclonal antibody to S6K1; panel 3, autoradiograph of in vitro kinase assay measuring S6K1-GST activity with S6 substrate; panel 4, detection of MAP4K3 expression with 9E10 antibody to myc epitope. b) Quantitation of 32 P-GST-S6 shown in a from triplicate assays from a single experiment repeated three times with similar results. Arbitrary values for 32 P-S6 were measured using a phosphorimager and results expressed separately for the MAP4K3 and insulin-stimulated samples as a % (+/-SD) of the initial activity (100% at time 0, prior to amino acid withdrawal) of each group. c) MAP4K3 is regulated by amino acids, but not insulin or mTORC1. HEK293T cells were transfected with 0.1µg pRK5myc vector (+AA Vec), 0.1 µg pRK5mycMAP4K3 AFG kinase-dead mutant (+AA KD) or 0.1µg pRK5mycMAP4K3 wild-type (MAP4K3) and serum-starved for 16 hours. Right panels; serum-starved cells (+AA) were either stimulated for 30 mins with 1µM insulin (+AA+ Ins 30') or treated with 50nM rapamycin for 30 minutes (+AA+Rapa 30'); left panels, serum-starved cells were transferred to D-PBS (-AA) for 30 minutes and lysates prepared or were subsequently restimulated with amino acids by transferring to DMEM lacking serum for 5-30 minutes (-AA+AA). Panel 1, autoradiograph of in vitro kinase assay of anti-myc immunoprecipitates with MBP substrate; panel 2, MAP4K3 levels in 1/5th of the 9E10 immunoprecipitate prior to in vitro kinase assay detected with polyclonal anti-MAP4K3 antibody; panel 3, phosphorylation of S6K1 at Thr 389 detected with phospho-specific antibody; panel 4, reprobing of panel 2 with monoclonal antibody to total S6K1. d) Quantitation of 32 P-MBP shown in c from triplicate assays from a single experiment repeated three times with similar results. Arbitrary values for 32 P -MBP were measured using a phosphorimager and processed with ImageQuant software. Results are expressed as fold difference of the activity in the presence of amino acids (+AA). Quantitation reveals an 6.3-fold inhibition of MAP4K3 activity following 30 minutes of amino acid withdrawal and an 8.8-fold activation after 15 minutes of amino acid readdition to amino acid-deprived cells. In the presence of amino acids both insulin stimulation and treatment with rapamycin lead to a 1.3-fold activation of MAP4K3 activity. e) MAP4K3 regulates cell size in Hela cells. Hela cells maintained in DMEM with 10% FCS were transfected with control siRNA and left untreated or treated with 50nM rapamycin for 48 hours, or transfected with siRNAs against Rheb or MAP4K3 (M4K3-1 or M4K3-2) and analyzed by FACS analysis after propidium iodide staining. G1 cells analyzed by forward scatter from untreated control siRNA-transfected cells (black) are shown overlaid with the rapamycin-treated cells, and Rheb siRNAor MAP4K3 siRNA-transfected cells (grey). In the table the differences in values of median FSC of G1-, S-and G2/M-phase cells (marked by *) are highly statistically significant by z-test (p<0.001) compared to control siRNA vehicle-treated cells. f) Model for the involvement of MAP4K3 in mTOR signalling. Growth factors activate the mTORC1 pathway primarily via activation of protein kinases such as PKB [3] and/or ERK/Rsk [START_REF] Roux | Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase[END_REF][START_REF] Ma | Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis[END_REF] leading to inactivation of the TSC1-2 tumour suppressor and increased Rheb.GTP levels. In turn Rheb.GTP has been proposed to activate mTORC1 kinase activity, although uncertainty exists on this mechanism as binding of Rheb to mTOR is independent of bound nucleotide [START_REF] Long | Fig. 3e G1: c-siRNA Rapamycin G1: c-siRNA vehicle G1: Rheb siRNA G1: c-siRNA vehicle G1: M4K3 siRNA-1 G1: c-siRNA vehicle G1: M4K3 siRNA-3 G1: c-siRNA vehicle c-siRNA[END_REF], unlike other small GTPase/effector interactions (question mark). Since Rheb.GTP levels are insensitive to amino acid withdrawal [START_REF] Nobukuni | Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase[END_REF], but both MAP4K3 and S6K activities are sensitive (this study and [5]), we suggest that MAP4K3 may provide a specific link between amino acids and S6K1 activity. The involvement of Vps34 in amino acid signalling to S6K is also indicated, and since it is currently unclear whether this lipid kinase acts upstream or downstream of MAP4K3, both possibilities are indicated [Vps34?]. Since phosphorylation of 4E-BP1 (at Thr 70 , a site regulated by mTORC1 [START_REF] Gingras | Hierarchical phosphorylation of the translation inhibitor 4E-BP1[END_REF]) is also regulated by MAP4K3, and MAP4K3 activity is insensitive to rapamycin, we suggest that MAP4K3 may regulate both S6K and 4E-BP1 activities by acting upstream of mTORC1.

Legends to Figures

Fig. 1

 1 Fig.1 Identification of MAP4K3 as an activator of mTOR signalling to S6K. a) Co-depletion of dTsc1 with Drosophila kinases by dsRNA addition to S2 cells. Upper panels, immunoblots of dS6K; lower panels, immunoblots of dTsc1 following dsRNA addition. Depletion of dTsc1 leads to increased abundance of a hyperphosphorylated species of dS6K (arrowhead) compared to control untreated cells (UN) that is reversed by treatment with 50nM rapamycin for 1 hour (Rapa) or codepletion of dTOR, CG1776, CG7097 or CG8767. The ratio of hyperphosphorylated dS6K (top) to hypophosphorylated dS6K (bottom) is indicated above panels following quantitation of the autoradiographs with ImageQuant software. NS=non-specific band detected with dTsc1 antibody. BLAST homology of the predicted Drosophila proteins indicates that the closest human orthologues of CG1776, CG7097, and CG8767 respectively are: myosin light chain kinase (MYLC, E value 3e -77); MAP4K3 (E value 4e -131); and c-Mos (E value 1e -18). b) Suppression of S6K1 Thr 389 by RNAi of MAP4K3 in Hela cells. Hela cells were transfected in DMEM containing 10mM glucose and 10% FCS with control siRNA, Rheb siRNA and two distinct siRNA duplexes (M4K3-1 and M4K3-2) targeting MAP4K3. After 72 hours cells were serum-starved for 1 hour and lysates analyzed by immunoblotting. Panel 1, phosphorylation of S6K1 at Thr 389 detected with phospho-specific antibody; panel 2, reprobing of panel 1 with monoclonal antibody to total S6K1; panel 3, MAP4K3 levels detected with polyclonal anti-MAP4K3 antibody, panel 4, Rheb levels detected with monoclonal anti-Rheb antibody. Note: the blots in these panels were spliced to remove a lane corresponding to treatment with a MAP4K3 siRNA that did not effectively knock down MAP4K3 expression. The ratio of T 389 -P/total S6K following siRNA treatments shown was quantified from scanned autoradiographs using ImageQuant software, relative to control siRNA treatment which was assigned a value of 1. c) Depletion of MAP4K3 by RNAi inhibits phosphorylation of S6 induced by depletion of TSC2. Hela cells were transfected with control siRNA alone or TSC2 siRNA together with control siRNA or siRNAs targeting Rheb or MAP4K3 (M4K3-1) and lysates prepared as in b. Upper panel, phosphorylation of S6at Ser 240/44 detected with phospho-specific antibody; middle panel, duplicate gel probed with antibody to S6; bottom panel, TSC2 detected with polyclonal anti-TSC2 antibody. The ratio of S 240/44 -P/total S6 following siRNA treatments shown was quantified from scanned autoradiographs using ImageQuant software, relative to control siRNA treatment which was assigned a value of 1.

Figure 2 .

 2 Figure 2. MAP4K3 activates mTOR signalling to S6K and 4E-BP1. a) Overexpression of MAP4K3 activates phosphorylation of S6 and is dependent upon kinase activity.

  HEK293T cells were transfected with the indicated plasmids as in a and b together with 0.1µg pRK5 S6K-GST. Prior to lysis cells were pretreated with 50nM rapamycin for 60 minutes where indicated (Rapa) or treated with rapamycin and stimulated with 1µM insulin (Ins) for 30 minutes. For wortmannin and PD184352 treatments cells were treated with 50nM (lane 9), 100nM (lane 10) or 200nM (lane 11) wortmannin, or 1µM (lane 12) or 10µM (lane 13) PD184352 for 60 minutes prior to lysis. Panel 1, Thr 389 phosphorylation of S6K-GST reporter; panel 2, S6K-GST reporter detected with a monoclonal antibody to S6K1; panel 3, autoradiograph of in vitro kinase assay measuring S6K1-GST activity with S6 substrate; panels 4 and 5, detection of kinase expression with 9E10 antibody to myc epitope (panel 4) and Biochemical Journal Immediate Publication. Published on 26 Jan 2007 as manuscript BJ20061881 HA epitope (panel 5). Values of S6K activity were determined by quantitation of the phosphorimage using ImageQuant software. d) Overexpression of MAP4K3 activates phosphorylation of 4E-BP1. HEK293T cells were transfected as in a with 2.5 µg pRK5myc vector or pRK5mycMAP4K3 together with 1µg pcDNA-3xHA-4E-BP1 reporter. Treatments with 100nM wortmannin (Wt) and 50nM rapamycin (Rapa) and insulin stimulation were as in c. Top panel, HA-4E-BP1 phosphorylation at Thr 70 detected with a phospho-specific antibody; middle panel, level of HA-4E-BP1 detected with an antibody to 4E-BP1; bottom panel, level of MAP4K3 detected with anti-myc 9E10 antibody.

Figure 3 .

 3 Figure 3. Involvement of MAP4K3 in amino acid signalling and growth. a) Ectopic MAP4K3 overexpression delays inactivation of S6K1 induced by amino acid withdrawal.

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Fig. 2b

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© 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 26 Jan 2007 as manuscript BJ20061881 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank Drs. E. Skolnick, K. Kariya and A. Tee for plasmids, Dr. Buzz Baum (Ludwig Institute for Cancer Research, London) for advice on RNAi screening in Drosophila cells, and Pascal Meier and Chris Marshall for critical reading of the manuscript. Work in this study was funded by grants from Cancer Research UK and the Tuberous Sclerosis Association (UK), and an Institute of Cancer Research Studentship to G. M. Findlay.