1. PP2AT61ɛ Is an Inhibitor of MAP4K3 in Nutrient Signaling to mTOR
Lijun Yan, Virginie Mieulet, Darren Burgess, Greg M. Findlay, Katherine Sully, Julia Procter, Jozef Goris, Veerle Janssens, Nick A. Morrice, Richard F. Lamb 
Molecular Cell 
Volume 37, Issue 5, Pages (March 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of MAP4K3 Phosphorylation Sites
(A) Colloidal Coomassie-stained gel (left panel) and anti-Myc immunoblot (right panel) of 9E10 purifications of wild-type and MAP4K3 D-A derived from stably transfected HEK293T cells.
(B) LC/MS/MS analysis using a precursor ion scan of m/z −79 to identify phosphorylated peptides. Masses and sequence of phosphorylated peptides are shown in text. Ion at 880 (2−) is the TFA (trifluroacetic acid) adduct of the 823(2−) ion.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
MAP4K3 Ser170 Phosphorylation and Rag GTPases Are Required for Activation of mTORC1 Signaling to S6K1
(A) Top panel, in vitro kinase (IVK) assays with MBP substrate of wild-type MAP4K3 and Ser170-Ala and Ser398-Ala MAP4K3 mutants; bottom panel, anti-Myc immunoblot of 1/10 of the immunoprecipitate used for IVK.
(B) Immunoblots of phosphorylation of endogenous S6K1 (T389-P) and S6 (S240/44-P) following transient transfection of HEK293T cells with 2 μg pRK5myc (VECTOR) or 2 μg wild-type or mutant MAP4K3 expression constructs. Membranes were also reprobed with antibodies detecting the level of total S6K1 and S6. An experiment representative of three independent experiments is shown.
(C) Immunoblots of phosphorylation of reporter S6K1-GST (T389-P) and total S6K-GST (S6K1) following transient transfection of HEK293T cells (−), or cells transduced with lentiviral shRNA constructs suppressing Rag C and Rag D (Rag C1/D1 and Rag C2/D2), with pRK5myc or pRK5 mycMAP4K3. Immunoblots were subsequently reprobed with 9E10 and anti-Rag C antibodies to detect expression of mycMAP4K3 and the level of Rag C after shRNA knockdown. Results are representative of three independent experiments. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Regulation of MAP4K3 Ser170 Phosphorylation
(A) Immunoblot of wild-type myc-MAP4K3, Ser170-Ala, and Ser398-Ala MAP4K3 mutants from transiently transfected HEK293T cells probed with anti-Ser170-P antibody (top panel) and reprobed with a 9E10 antibody to Myc (bottom panel).
(B) Tetracycline-inducible T-Rex293 cells (HEK293) expressing wild-type and D-A myc-MAP4K3 induced for various times with tetracycline. Upper panel, anti-Ser170-P, middle panel anti-Myc; lower panel, total MAP4K3 immunoblots.
(C) Regulation of Ser170 phosphorylation by amino acids in HEK293 cells expressing myc-MAP4K3 induced by tetracycline. Immunoblots of cell lysates (lysates) probed with Ser170-P and anti-Myc antibodies. Lower panels, IVK of anti-Myc immnoprecipitates (32P-MBP); anti-Myc immunoblot of 1/10 of the immunoprecipitate used for IVK.
(D) Levels of Ser170 phosphorylation after treatment of HEK293 cells inducibly expressing myc-MAP4K3 with wortmannin (100 or 500 nM for 60 min), insulin (1 μM for 30 min), or rapamycin (50 nM for 30 min). Immunoblots were probed with Ser170-P and anti-T389 S6K antibodies and reprobed with anti-Myc and anti-S6K1 antibodies. An experiment representative of three independent experiments is shown. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Ser170 Is an Autophosphorylation Site that Can Be Transphosphorylated by MAP4K3
(A) Kinase-inactive D-A MAP4K3 is not phosphorylated in transiently transfected HEK293T cells. GFP- and myc-tagged MAP4K3 (wild-type, lanes 2 and 4; D-A, lanes 3 and 5) were expressed in HEK293T and immunoblots probed with Ser170-P, anti-GFP, and anti-Myc.
(B) Coexpression of wild-type MAP4K3 increases Ser170 phosphorylation of MAP4K3 D-A in trans. Immunoblots of lysates from HEK293T transiently expressing GFP- or Myc-tagged MAP4K3 probed Ser170-P (top panel), anti-Myc (middle panel), and anti-GFP (lower panel) antibodies. The position of Ser170-phosphorylated myc-MAP4K3 is indicated with an arrowhead and the position of Ser170-phosphorylated GFP-MAP4K3 with an arrow.
(C) Phosphorylation of a peptide containing Ser170 by wild-type MAP4K3 in vitro. Immunoprecipitated wild-type or D-A myc-MAP4K3 was used to phosphorylate peptides in vitro, and 32P-peptide incorporation after 10 min was measured by Cerenkov counting.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
PP2A Regulates MAP4K3 Ser170 Phosphorylation and mTORC1 Signaling
(A) Inhibition of Ser170 phosphorylation during amino acid withdrawal. Immunoblots of HEK293 cell lysates were probed with an antibody to Ser170-P and reprobed with the 9E10 antibody to the Myc epitope (upper panels) or probed with an antibody detecting S6K1 T389-P and reprobed with an antibody to S6K1 (bottom panels).
(B) Immunoblots of cell lysates from untreated HEK293 (+AA), treated with 100 nM OA for 30 min in normal media (+AA/OA), deprived of amino acids for 30 min (−AA), or deprived of amino acids for 30 min in the presence of OA (−AA/OA) were probed with antibodies to Ser170-P, T389-P, and S6-P (Ser240/244) and reprobed with antibodies to Myc, S6K1, and S6. Lysates were subjected to immunoprecipitation with 9E10 and immunoprecipitates used in IVK (lower panels) together with an anti-Myc immunoblot of 10% of the immunoprecipitate used for IVK. An experiment representative of three independent experiments is shown.
(C) Immunoblots of 9E10 anti-myc immunoprecipitates of myc-MAP4K3 induced in HEK293 cells after no treatment (−, 0 time), mock incubation (−, 60 min), or treatment with 0.05U PP2A (30 and 60 min), probed with an antibody to Ser170-P (upper panel) and reprobed with an anti-Myc antibody (lower panel). See also Figures S3A and S3B.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Role of PP2AT61ɛ in Regulation of MAP4K3 and mTORC1 Signaling
(A) Association of endogenous PR61ɛ with MAP4K3. Immunoblots of 9E10 anti-Myc immunoprecipitates from lysates of HEK293T cells transfected with pRK5myc Msn, pRK5myc MAP4K3 wild-type, or MAP4K3 D-A mutant. Upper panel, anti PR61ɛ; lower panel, anti-Myc.
(B) Overexpression of PR61ɛ stimulates MAP4K3 Ser170-P dephosphorylation. Immunoblot of Ser170-P from cell lysates of HEK293T cells. Cells were transfected for 40 hr in 3 cm dishes in normal growth media containing amino acids with pRK5myc (VECTOR), 1 μg of pRK5 mycMAP4K3 alone (MAP4K3), or together with 2 μg HA-PR61ɛ (MAP4K3 + PR61ɛ) or HA-PR72 (MAP4K3 + PR72) constructs. Immunoblots were subsequently probed with anti-Myc 9E10 and anti-HA antibodies to detect the transfected proteins. Quantitation of relative S170-P phosphorylation from a representative experiment of three performed was determined using ImageQuant software (Bio-Rad) from scanned autoradiographs. The Ser170-P/Myc ratio of MAP4K3 alone was assigned an arbitrary value of 1.
(C) Overexpression of PR61ɛ inhibits activation of mTORC1 signaling by MAP4K3. HEK293T cells were transfected as in (B) with 100 ng pRK5S6K1-GST reporter and 1 μg of pRK5myc MAP4K3 alone (MAP4K3) or pRK5myc MAP4K3 together with 2 μg HA-PR61ɛ (MAP4K3 + PR61ɛ) or HA-PR72 (MAP4K3 + PR72) constructs. After 24 hr, cells were serum starved overnight prior to cell lysis. Quantitation of relative Thr389 phosphorylation from an experiment representative of two performed was determined using ImageQuant software (Bio-Rad) from scanned autoradiographs by assigning the T389/S6K1 ratio of MAP4K3 expressed alone an arbitrary value of 1. Immunoblots were probed with anti-Thr389 antibody and anti-Myc 9E10 and subsequently reprobed with antibodies to S6K1 and HA.
(D) Lentiviral shRNAs suppress PR61ɛ expression. HEK293 cells transduced with lentiviruses expressing a nontargeting sequence (NT) or shRNAs directed against PR61ɛ. Five days after lentiviral transduction, cells were lysed and immunoblots probed with antibodies detecting PR61ɛ (top panel) or α-tubulin (lower panel).
(E) Suppression of PR61ɛ inhibits MAP4K3 Ser170 dephosphorylation after amino acid withdrawal. HEK293 cells transduced with nontargeting or PR61ɛ-directed shRNAs were induced to express myc-tagged MAP4K3 by addition of tetracycline. Cells were deprived of amino acids for 30 min (−AA) or deprived of amino acids for 30 min prior to the readdition of amino acids for 15 min (+AA) before lysis. Immunoblots were probed with antibody to Ser170-P (upper panel) and reprobed with 9E10 to detect the myc-tagged MAP4K3 (lower panel). Quantitation of relative Ser170 phosphorylation was determined in an experiment representative of two performed using ImageQuant software (Bio-Rad) from scanned autoradiographs by separately assigning the Ser170/Myc ratio of NT, PR61ɛ-1, and PR61ɛ-2 in the presence of amino acids (+AA) an arbitrary value of 1.
(F) Histogram of FACS analysis of forward scatter (FSC-A) of G1-sorted HEK293 cells (control, black) or HEK293 cells transduced with PR61ɛ-1 (light gray) or PR61ɛ-2 (dark gray) lentiviruses from a representative experiment of three performed.
(G) Relative mean FSC-A for three independent experiments normalized to cells transduced with nontargeting (control), assigned the value of 100%. Error bars represent standard deviation from the mean.
(H) Suppression of PR61ɛ inhibits mTORC1 signaling after amino acid withdrawal. HEK293 cells transduced with nontargeting (NT) or PR61ɛ-directed shRNAs were deprived of amino acids for 30 min (−AA) or deprived of amino acids for 30 min prior to the readdition of amino acids for 15 min (+AA) before lysis. Immunoblots were probed with antibody to S6-P (Ser240/244, upper panel) and reprobed with an antibody detecting total levels of S6 protein (lower panel). Quantitation of relative S6 phosphorylation was determined in an experiment representative of two performed using ImageQuant software (Bio-Rad) from scanned autoradiographs by separately assigning the S6-P/S6 ratio in the presence of amino acids (+AA) the arbitrary value of 1. See also Figure S3C.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Role of Amino Acid Sufficiency in Dephosphorylation of MAP4K3 by PP2AT61ɛ
(A) In vitro dephosphorylation (IVD) of Flag-MAP4K3 by PP2AT61ɛ. Immunoblots are shown detecting phosphorylation of Ser170 (top panel) and Flag-MAP4K3 substrate. Protein G beads alone (Control, 15 min) or PR61ɛ/protein G immunoprecipitates from HEK293 cells cultured in the presence (+AA) or absence (−AA) of amino acids were added to Flag-MAP4K3 and IVD allowed to proceed for the indicated times. PP2A∗ represents a commercial PP2A preparation used as a positive control.
(B) Quantitation of relative S170-MAP4K3 shown in (A) from triplicate assays from a representative experiment of three performed. Error bars represent standard error of the mean.
(C) Microcystin-agarose pull-down of PP2A-associated MAP4K3 after amino acid deprivation. Myc-MAP4K3 was induced in HEK293 cells and cells maintained in DMEM/10% dFCS or shifted to D-PBS/10%dFCS to remove amino acids for 5–30 min. Immunoblots of MAP4K3, PP2Ac, and PR65 bound to microcystin-agarose are shown in top panels and expression in lysates in bottom panels. Immunoblots were also probed with an antibody to endogenous S6K1 that was not detected in microcystin pull-downs compared to the cell lysate. An experiment representative of four independent experiments is shown.
(D) PR61ɛ interaction with MAP4K3 increases following amino acid deprivation. Immunoblots of HA-PR61ɛ immunoprecipitates (IP: HA) probed with antibodies to MAP4K3, PR61ɛ, and PP2A-C. HEK293 cells were transfected with HA-PR61ɛ and after 24 hr induced to express myc-MAP4K3 with tetracycline for a further 24 hr. Cells were serum starved for 60 min, prior to amino acid starvation for 5–30 min by transferring cells to DPBS. To readd amino acids, after 30 min cells were transferred to DMEM for a further 5 or 15 min, prior to crosslinking with DSP. Quantitation of relative MAP4K3 binding was determined in an experiment representative of two performed using ImageQuant software (Bio-Rad) from scanned autoradiographs by assigning the binding of MAP4K3 in the presence of amino acids (+AA) the arbitrary value of 1. Lysates (lower panels) were also probed with antibodies to phospho-S6 (S6-P, Ser240/244) and total S6.
(E) Schematic of MAP4K3 truncations. The polyproline motif implicated in binding to SH3 domain of endophilin 1 (Ramjaun et al., 2001) is indicated as a hatched box. IVK activity and Ser170-P immunoblots of wild-type and truncated MAP4K3 expressed in transiently transfected HEK293T cells. Cell lysates were also immunobloted with 9E10 to detect expression of Myc-tagged MAP4K3 polypeptides.
(F) Activation of S6K1-GST reporter by MAP4K3 truncations. HEK293T cells were transfected with 2 μg pRK5myc MAP4K3 constructs and 100 ng S6K-GST reporter and level of T389-phosphorylated reporter and reporter level determined by immunoblotting with anti-Thr389 and anti-S6K1 antibodies. Expression of Myc-tagged MAP4K3 (bottom panel) was determined by immunoprecipitation with 9E10 antibody followed by immunoblotting with rabbit anti-Myc antibody and protein A-HRP. The ratio of T389/total S6K1 from a representative experiment of three performed, expressed relative to activation by wild-type MAP4K3 assigned a value of 1, is also shown.
(G) Microcystin-agarose pull-down of PP2A-associated MAP4K3 polypeptides. HEK293T cells were transfected with MAP4K3 constructs and lysates subject to binding to microcystin-agarose for 2 hr. Associated MAP4K3 and PP2A were detected by immunoblotting with 9E10 for the Myc epitope and with antibodies detecting PP2Ac and PR65. A portion of the cell lysate was also probed with 9E10 to detect expression levels of Myc-tagged MAP4K3 polypeptides. Association of MAP4K3 polypeptides relative to wild-type MAP4K3 assigned a value of 1, from a representative experiment of three independent experiments performed, is shown.
(H) Activity of MAP4K3 polypeptides following amino acid withdrawal. IVK activity of D-A, wild-type, and aa 1–431 MAP4K3 expressed in transiently transfected HEK293T cells. Top panel indicates 32P-MBP after IVK, bottom panel indicates expression level of MAP4K3 polypeptides detected by immunoblotting cell lysates with 9E10 anti-Myc antibody. Arrowheads indicate myc-tagged polypeptides.
(I) Models depicting the role of PP2AT61ɛ in the regulation of MAP4K3 and mTORC1 signaling by amino acids. In the model depicted on the left, PP2AT61ɛ provides a conditional inhibitory input to MAP4K3 Ser170 phosphorylation and kinase activity but is itself not regulated by amino acid sufficiency. Removal of amino acids therefore permits tonic dephosphorylation of Ser170 by PP2AT61ɛ. In the model depicted on the right, PP2AT61ɛ activity is itself regulated by amino acid sufficiency. In both models a putative MAP4K3 substrate (substrate-P) connects MAP4K3 to mTORC1, potentially via Rag GTPases. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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