A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling - PubMed (original) (raw)

Greg M Findlay et al. Biochem J. 2007.

Abstract

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although 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 (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] 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.

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Figure 1

Figure 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 with control untreated cells (UN) that is reversed by treatment with 50 nM rapamycin for 1 h (Rapa) or co-depletion of dTOR, CG1776, CG7097 or CG8767. The ratio of hyperphosphorylated dS6K (upper panel) to hypophosphorylated dS6K (lower panel) is indicated above the immunoblots following quantitation of the autoradiographs with ImageQuant software. NS indicates a non-specific band detected with the 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 (E value 3e -77), MAP4K3 (E value 4e -131) and c-Mos (E value 1e -18). (B) Suppression of S6K1 Thr389 by RNAi of MAP4K3 in HeLa cells. HeLa cells were transfected in DMEM containing 10 mM glucose and 10% FCS with control siRNA, Rheb siRNA and two distinct siRNA duplexes (M4K3-1 and M4K3-2) targeting MAP4K3. After 72 h cells were serum-starved for 1 h and lysates analysed by immunoblotting. Panel 1, phosphorylation of S6K1 at Thr389 detected with a phospho-specific antibody; panel 2, reprobing of panel 1 with a monoclonal antibody to total S6K1; panel 3, MAP4K3 levels detected with a polyclonal anti-MAP4K3 antibody; panel 4, Rheb levels detected with a 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 knockdown MAP4K3 expression. The ratio of Thr389 phosphorylated (T389-P) to total S6K following siRNA treatments is shown and 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 S6 at Ser240/244 detected with a phospho-specific antibody; middle panel, duplicate gel probed with an antibody to S6; lower panel, TSC2 detected with a polyclonal anti-TSC2 antibody. The ratio of Ser240/244 phosphorylated (S240/44-P) total S6 following siRNA treatments is shown and was quantified from scanned autoradiographs using ImageQuant software, relative to control siRNA treatment which was assigned a value of 1. (D) Depletion of MAP4K3 suppresses S6K1 Thr389 phosphorylation induced by amino acid restimulation. HeLa cells were transfected and serum-starved for 1 h as in (B). For amino acid depletion/restimulation, duplicate wells transfected with control siRNA, Rheb siRNA or MAP4K3 siRNAs were transfered into DPBS containing 10 mM glucose and 1×MEM with vitamins (−AA) for 30 min or the same treatment followed by transfer into serum-free DMEM (containing 10 mM glucose) for 30 min (+AA). Panel 1, phosphorylation of S6K1 at Thr389 (arrow) detected with a phospho-specific antibody; panel 2, duplicate gel probed with an antibody to total S6K1; panel 3, phosphorylation of S6 at Ser240/244 detected with a phospho-specific antibody; panel 4, duplicate gel probed with an antibody to total S6; panel 5, MAP4K3 levels detected with a polyclonal anti-MAP4K3 antibody; panel 6, Rheb levels detected with a monoclonal anti-Rheb antibody.

Figure 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. HEK-293T cells were transfected with 2.5 μg of pRK5myc vector or pRK5myc MAP4K3 wild-type or AFG kinase-dead MAP4K3 (KD). After 24 h, cells were serum-starved for 16 h and controls stimulated with 10% FCS for 30 min (FCS 30′) or pretreated with 100 nM wortmannin for 60 min prior to stimulation (FCS 30′+Wort) and lysates prepared. Panel 1, endogenous S6K phosphorylation detected with a phospho-specific antibody against Ser235/236; panel 2, S6 phosphorylation detected with a phospho-specific antibody against Ser240/244; panel 3, total S6; panel 4, an anti-Myc (9E10) antibody to detect expression of Myc-epitope tagged kinase. (B) Overexpression of MAP4K3 does not activate Ser473 phosphorylation of PKB. HEK-293T cells were transfected as in (A) with pRK5myc vector, pRK5mycMAP4K3, pRK5mycMsn or pCI HA TNIK and lysates were probed with antibodies as in (A), or with antibodies to PKB Ser473 (panel 1), total PKB (panel 2) or a HA epitope to detect HA-TNIK (panel 6). As a control for PKB activation, vector-transfected cells were stimulated with 1 μM insulin (Ins) for 30 min or pretreated with 100 nM wortmannin (Wt) for 60 min prior to stimulation. (C) Activation of S6K1 by MAP4K3 is rapamycin-sensitive, but independent of MEK and PI3K signalling. HEK-293T cells were transfected with the indicated plasmids as in (A) and (B) together with 0.1 μg of pRK5 S6K–GST. Prior to lysis, cells were pretreated with 50 nM rapamycin for 60 min where indicated (Rapa) or treated with rapamycin and stimulated with 1 μM insulin (Ins) for 30 min. For wortmannin and PD184352 treatments, cells were treated with 50 nM (lane 9), 100 nM (lane 10) or 200 nM (lane 11) wortmannin, or 1 μM (lane 12) or 10 μM (lane 13) PD184352 for 60 min prior to lysis. Panel 1, Thr389 phosphorylation of the S6K–GST reporter; panel 2, S6K–GST reporter detected with a monoclonal antibody to S6K1; panel 3, autoradiograph of an in vitro kinase assay measuring S6K1–GST activity with S6 substrate; panels 4 and 5, detection of kinase expression with the 9E10 antibody to the Myc epitope (panel 4) and 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. HEK-293T cells were transfected as in (A) with 2.5 μg of pRK5myc vector or pRK5mycMAP4K3 together with 1 μg of pcDNA-3xHA-4E-BP1 reporter. Treatments with 100 nM wortmannin (Wt) and 50 nM rapamycin (Rapa) and insulin stimulation were as in (C). Upper panel, HA–4E-BP1 phosphorylation at Thr70 detected with a phospho-specific antibody; middle panel, level of HA–4E-BP1 detected with an antibody to 4E-BP1; lower panel, level of MAP4K3 detected with an anti-Myc 9E10 antibody.

Figure 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. HEK-293T cells were transfected as in Figure 2(A) with 2.5 μg of pRK5myc vector or pRK5mycMAP4K3 and 0.1 μg of pRK5 S6K-GST reporter and serum-starved for 16 h. Vector-transfected cells were subsequently stimulated for 30 min with 1 μM insulin (Ins, time 0) and transferred to DPBS as in Figure 1(D) for 5–60 min prior to lysis, or if expressing MAP4K3 left unstimulated and transferred to DPBS for 5–60 min (MAP4K3). Panel 1, Thr389 phosphorylation (arrowhead) of S6K–GST reporter; panel 2, S6K–GST reporter detected with a monoclonal antibody to S6K1; panel 3, autoradiograph of an in vitro kinase assay measuring S6K1–GST activity with S6 substrate; panel 4, detection of MAP4K3 expression with the 9E10 antibody against the Myc epitope. (B) Quantitation of [32P]GST-S6 shown in (A) from triplicate assays of a single experiment repeated three times with similar results. Arbitrary values for [32P]GST-S6 were measured using a phosphorimager and results expressed separately for the MAP4K3 and insulin-stimulated samples as a percentage (means±S.D.) 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. HEK-293T cells were transfected with 0.1 μg of pRK5myc vector (+AA Vec), 0.1 μg of pRK5mycMAP4K3 AFG kinase-dead mutant (+AA KD) or 0.1 μg of pRK5mycMAP4K3 wild-type (MAP4K3) and serum-starved for 16 h. Right panels: serum-starved cells (+AA) were either stimulated for 30 mins with 1 μM insulin (+AA+ Ins 30′) or treated with 50 nM rapamycin for 30 min (+AA+Rapa 30′); left panels: serum-starved cells were transferred to DPBS (−AA) for 30 min 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 an in vitro kinase assay of anti-Myc immunoprecipitates with MBP substrate; panel 2, MAP4K3 levels in one-fifth of the 9E10 immunoprecipitate prior to the in vitro kinase assay detected with a polyclonal anti-MAP4K3 antibody; panel 3, phosphorylation of S6K1 at Thr389 detected with a phospho-specific antibody; panel 4, reprobing of panel 2 with a monoclonal antibody against total S6K1. (D) Quantitation of [32P]MBP shown in (C) from triplicate assays of a single experiment repeated three times with similar results. Arbitrary values for [32P]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 min of amino acid withdrawal and an 8.8-fold activation after 15 min of amino acid re-addition to amino acid-deprived cells. In the presence of amino acids both insulin stimulation and treatment with rapamycin led 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 50 nM rapamycin for 48 h, or transfected with siRNAs against Rheb or MAP4K3 (M4K3-1 or M4K3-2) and analysed by FACS analysis after propidium iodide staining. G1 cells analysed by forward scatter from untreated control siRNA-transfected cells (black) are shown overlaid with the rapamycin-treated cells, and Rheb siRNA- or MAP4K3 siRNA-transfected cells (grey). In the Table the differences in values of median FSC (forward scatter) of G1-, S- and G2/M-phase cells (marked by *) are highly statistically significant by _z_-test (P<0.001) compared with 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 [17,18] 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 [20], unlike other small GTPase/effector interactions (indicated by the question mark). Since Rheb.GTP levels are insensitive to amino acid withdrawal [12], but both MAP4K3 and S6K activities are sensitive (the present 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. Since phosphorylation of 4E-BP1 (at Thr70, a site regulated by mTORC1 [15]) 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.

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