Ulk λ PPase. 32 P-Ulk1 32 P-GST-TSC2. Ulk1 GST (TSC2) : Ha-Ulk1 : AMPK. WB: Ha (Ulk1) : Glu. h CON - Glu - A.A WB: LC3 AMPK-WT AMPK-DKO

Size: px

Start display at page:

Download "Ulk λ PPase. 32 P-Ulk1 32 P-GST-TSC2. Ulk1 GST (TSC2) : Ha-Ulk1 : AMPK. WB: Ha (Ulk1) : Glu. h CON - Glu - A.A WB: LC3 AMPK-WT AMPK-DKO"

Dorcas Wilcox
5 years ago
Views:

DOI: 10.1038/ncb2152 C.C + - + - : Glu b Ulk1 - - + λ PPse c AMPK + - + + : ATP P-GST-TSC2 WB: Flg (Ulk1) WB Ulk1 WB: H (Ulk1) GST (TSC2) C.

1 DOI: /ncb2152 C.C : Glu b Ulk λ PPse c AMPK : ATP P-GST-TSC2 WB: Flg (Ulk1) WB Ulk1 WB: H (Ulk1) GST (TSC2) C.C d e WT K46R : H-Ulk1 : AMPK AMPK H-Ulk1 WB: H (Ulk1) : Glu WB: H (Ulk1) f NH 4Cl WB: LC3 h CON - Glu - A.A AMPK-WT g AMPK-WT AMPK-DKO - G A - G A - G A - G A Strvtion NH 4Cl AMPK-DKO WB: LC3 L.E. Figure S1 Ulk1 is ctivted by glucose strvtion nd by AMPK. () AMPK inhibitor blocks glucose strvtion induced Ulk1 phosphoryltion. Flg-Ulk1 ws trnsfected into HEK293 cells nd the cells were strved with glucose for 4hrs in the presence or bsence of 20 μm Compound C (C.C) before lysis. Totl cell lystes were exmined for Ulk1 mobility by Phos-tg gel, which produced bigger mobility shift of phosphorylted protein. (b) Endogenous Ulk1 kinse is ctivted by phosphoryltion. ULK1-WT MEFs were strved with glucose for 4hrs nd endogenous Ulk1 protein ws immunoprecipitted. Purified Ulk1 immune-complex ws treted with lmbd phosphtse (λ PPse) before Ulk1 utophosphoryltion rection s described in Methods. P-pre-lbeled GST-TSC2 ws lso dded to Ulk1 utophosphoryltion rection mixture to monitor the possible phosphtse contmintions fter RIPA buffer wshing. Ulk1 utophosphoryltion level ws determined by P- utordiogrm. Totl protein levels for Ulk1 nd GST-TSC2 were determined by western blots. (c) AMPK directly stimultes Ulk1 utophosphoryltion ctivity in vitro. H-Ulk1 ws immuno-purified from the trnsfected HEK293 cells nd pre-incubted with purified AMPK complex (Cell signling) for 15 min under the KA buffer supplemented with 0.2 mm AMP in the presence or bsence of 0.1 mm cold ATP s indicted. Also, AMPK inhibitor (Compound C, 10 μm, denoted s C.C) ws dded to the rection contining 0.1 mm ATP to confirm the rection specificity towrd AMPK. After in vitro AMPK rection, Ulk1 immunecomplex ws extensively wshed with RIPA buffer to remove AMPK nd the Ulk1-bed ws recovered by centrifugtion. The resulting Ulk1 immunecomplex ws used for Ulk1 utophosphoryltion ssy. (d) P-incorportion in Ulk1 utophosphoryltion is medited by Ulk1 kinse. H-Ulk1 wild-type (WT) or kinse inctive (K46R) mutnt ws immunoprecipitted from the trnsfected cells under glucose-rich medium. The Ulk1 immune complex ws pre-incubted with AMPK in vitro for 15 min nd then, wshed to remove AMPK. Ulk1 utophosphoryltion ctivity ws mesured s described in Fig. S1b.. P-incorportion in Ulk1 utophosphoryltion ws brely detected in Ulk1 K46R mutnt even treted with AMPK. (e) AMPK co-trnsfection ctivtes Ulk1 ctivity. H-Ulk1 nd AMPK (α, β, nd γ) were co-trnsfected into HEK293 cells. The cells were strved with glucose for 4hrs s indicted nd then H-Ulk1 ws immunoprecipitted to mesure the Ulk1 utophosphoryltion ctivity. (f) Glucose strvtion nd rpmycin stimulte utophgy. MEFs were strved with glucose (Glu) for 4hrs in the presence or bsence of 20 μm Compound C (C.C). In prllel, cells were lso treted with 50 nm rpmycin (Rp) or 2 mm Metformin (Met) for 4hrs. To exmine the utophgic flux, 10 mm NH 4 Cl ws dded s indicted. The cell lystes were probed for LC3 ntibody nd α-tubulin, respectively. (g-h) Glucose strvtion induces utophgic mrkers, LC3 lipidtion nd LC3GFP-LC3 punctute formtion, in n AMPK-dependent mnner. (g) AMPK-WT nd DKO MEFs were incubted in either glucose-free (G) or mino cid-free (A) medium for 4hrs with or without 10 mm NH 4 Cl s indicted. LC3 lipidtion ws monitored by LC3 western. (h) AMPK-WT nd DKO MEFs stbly expressing GFP-LC3 were incubted in either glucose-free (-Glu) or mino cid free (-A.A) medi for 4hrs nd GFP-positive utophgosome ws nlyzed by confocl microscopy. Br, 20 μm. 1

2 b GST-mUlk1 WB: S317 WT S317/777A : H-Ulk : AMPK WB: GST AMPK WB: H (Ulk1) GST-mUlk1 WB: S777 WB: GST AMPK c S317/S777A WB: H (Ulk1) P-GST-Atg13 Coomssie (GST-Atg13) 6 Fold AMPK Figure S2 AMPK cn ctivte Ulk1 by phosphorylting Ulk1 t S317 nd S777 in vitro. () Muttion of S317 nd S777 in Ulk1 decreses Ulk1 phosphoryltion by AMPK in vitro. H-Ulk1 WT or S317/777A mutnt were immunoprecipitted from the trnsfected HEK293 cells nd used s substrtes for in vitro AMPK phosphoryltion. Phosphoryltion level ws determined by P- utordiogrm. (b) Chrcteriztions of Ulk1 S317 nd S777 phospho-specific ntibodies. Recombinnt GST-mUlk1 frgments were purified from bcteri nd 500 ng of the indicted recombinnt frgments were used s substrte for in vitro AMPK phosphoryltion. After rection, 5 ng of the phosphorylted GST-mUlk1 frgments were used to test specificity of S317 nd S777 phospho-ntibodies by western blot. Two phosphoryltion defective mutnt frgments, ( )/S317A nd ( )/S777A, were used s negtive controls. (c) S317 nd S777 re the mjor sites importnt for Ulk1 ctivtion by AMPK. Seven puttive AMPK consensus sites in Ulk1 were individully mutted in the S317/777A bckground nd Ulk1 proteins were prepred by immuno-purifiction from the trnsfected HEK293 cells. The immunopurified Ulk1 ws subjected to in vitro ctivtion by AMPK nd then used in Ulk1 kinse ssys. Ulk1 ctivity ws determined by utophosphoryltion ( ) nd Atg13 phosphoryltion ( P-GST-Atg13) nd normlized to Ulk1 protein levels. Western/Coomssie stining nlyses were performed on duplicte gel to tht used for utordiogrm nlysis. The quntifiction dt were obtined from three-independent experiments nd one representtive result ws shown (men ± S.D). 2

b Flg-mUlk1 IP GST-mUlk1 (711-828) WB: Myc (mtor) P-utordiogrm Coomssie Stin IP: Flg (Ulk1) Lystes WB: Flg WB: H (Rptor) c + - + + - + - - + - - + Glu Rp d Flg-mUlk1 WB: S757 WB: Ulk1 IP: Flg WB:H

3 b Flg-mUlk1 IP GST-mUlk1 ( ) WB: Myc (mtor) P-utordiogrm Coomssie Stin IP: Flg (Ulk1) Lystes WB: Flg WB: H (Rptor) c Glu Rp d Flg-mUlk1 WB: S757 WB: Ulk1 IP: Flg WB:H (AMPKα) WB:Flg (Ulk1) WT DKO : AMPK-MEF Lystes WB:H (AMPKα) Figure S3 mtorc1 phosphoryltes S757 in Ulk1. () mtorc1, but not mtorc2, phosphoryltes Ulk1 in vitro. mtorc1 (by Rptor) nd mtorc2 (by Rictor) were immuno-purified from the trnsfected HEK293 cells. The immune complexes were incubted with bcterilly purified GST-mUlk1 ( ) nd phosphoryltion of the GST-Ulk1 frgment ws determined by P-utordiogrm. Protein levels for mtor nd GST-mUlk1 ( ) were shown by western blot nd Coomssie stining, respectively. (b) Determintion of Ulk1 domin responsible for Rptor interction. H-Rptor ws co-trnsfected with vrious Flg-Ulk1 deletion constructs. Ulk1 proteins were immunoprecipitted nd Co-IP of Rptor ws exmined by western blot. (c) Glucose strvtion fils to inhibit Ulk1 S757 phosphoryltion in AMPK- DKO MEFs. AMPK WT n DKO MEF cells were strved with glucose (4hrs) or treted with 50 nm rpmycin (Rp, 1hr), s indicted. Endogenous Ulk1 proteins were immunoprecipitted nd the phosphoryltion of S757 ws exmined. (d) S757 is importnt for Ulk1-AMPK interction. The indicted Flg-Ulk1 mutnts nd H-AMPKα were co-trnsfected into HEK293 cells. Flg-Ulk1 proteins were immunoprecipitted nd Co-IP of H-AMPKα ws exmined by western blot. 3

4 CBP/SB b Ulk1 KO WT : ULK1-MEF WT KO : ULK1-MEF WB: Ulk1 WB: GFP (GFP-LC3) c Reltive vibility (%) ULK2 expression (mrna) Dy ULK1-KO/WT ULK1-KO/ULK2-KD ULK1-KO ULK1-WT d Glu NH 4 Cl Glu NH 4 Cl ULK1-WT ATG5-KO ULK1-KO KO KO-WT KO- S317/777A WB: LC3 WB: LC3 WT KO KO : MEF-ULK1 ULK2-KD e f % of GFP positive ULK1-WT Vec WT S317/777A : Ulk1 ULK1-KO AV/Cell ULK1-MEF KO KO-WT KO S317/777A Figure S4 Anlyses of Ulk1 reconstituted ULK1 -/- MEFs. (A) Expression levels of ectopic Ulk1 proteins in ULK1 -/- (KO) MEFs. The ULK1-KO MEFs stbly expressing wild-type Ulk1 or S317/777A mutnt were prepred s described in Methods. Expression levels of Ulk1 proteins were exmined by western blot using n Ulk1 ntibody. The levels of ectopic Ulk1 expression were comprble to tht of the endogenous Ulk1 protein in the ULK1-WT MEFs. Protein levels were normlized ginst α-tubulin (α-tub). (b) Expression levels of GFP-LC3 in the ectopic Ulk1 expression cell lines. GFP-LC3 ws introduced to the cells by retrovirl infection nd the cells stbly expressing GFP-LC3 were obtined by puromycin selection. The expression levels of GFP-LC3 were exmined by western blot using nti-gfp ntibody. (c) Ulk1 plys pivotl roles in cell survivl under strvtion. ULK1-WT, ULK1-KO, ULK1-KO/WT, nd ULK1 -/- with ULK2 knockdown (ULK1-KO/ULK2-KD) MEFs were strved with glucose for the indicted time. Knock-down efficiency of ULK2 ws determined by quntittive RT-PCR nd shown in the lower pnel (men ± S.D, n=2). Dt ws normlized by GAPDH. Cell vibility ws determined by tryphn blue stining (men ± S.D, n=3). Cell vibility is represented s % of corresponding MEFs before strvtion, which is set s 100%. (d) The Ulk1 S317/777A mutnt is compromised in supporting glucose strvtion-induced utophgy. ULK1-WT, KO, nd KO expressing Ulk1-WT (KO-WT) or Ulk1 S317/777A mutnt (KO-S317/777A) MEFs were strved with glucose (Glu) for 4hrs. Also, 10 mm NH4Cl ws dded to determine the utophgic flux in these cells. Autophgy induction ws monitored by LC3-II ccumultion by LC3 western. (e) Quntifiction of GFP-LC3 punt formtion for Fig.7C. The cells displying strong GFP positive dots on confocl microscopy were counted nd quntified (men ± S.D., n=30-40 cells) s described in Supplementry Methods. (f) Expression of wild type but not the S317/777A mutnt restores utophgosome/utolysosome-like structures in ULK1-/- cells. The numbers of utophgosome/utolysosome-like structures (AV) from 5-7 AV positive cells were counted nd men ± S.D. re shown. This is the quntifiction for Fig.7d. 4

Fig.1 Fig.1b Fig.1d Fig.1e 95 72 P-GST- Atg13 Fig.

5 Fig.1 Fig.1b Fig.1d Fig.1e P-GST- Atg13 Fig.1c P-GST- Atg13 Short exposure Figure S5 Full scns of originl blots for dt in Fig. 1, 2, 3, 4, 5, nd 6. Pnels corresponding to the figures in the pper re indicted. 5

WB: Flg (Ulk1) P-utordiogrm Fig.2 Fig.2 Fig.2c g.2b Fig.2b 95 72 Fig.

6 WB: Flg (Ulk1) P-utordiogrm Fig.2 Fig.2 Fig.2c g.2b Fig.2b Fig.2 P-utordiogrm WB: Flg (Ulk1) Fig.2 H (Ulk1) Fig.2d Figure S5 continued 6

7 Fig.3 P-S317 Fig.3 Fig.3f WB: Ulk1 Fig.3 g.6c WB: S317 Fig.3b Fig.3e WB: H (ulk1) Fig.3d H (Ulk1) Fig.3d Figure S5 continued 7

Fig.4 3b 95 72 WB: H (AMPKα) WB: H (AMPKα) WB: Flg (Ulk1) Fig.4c Fig.

8 Fig.4 3b WB: H (AMPKα) WB: H (AMPKα) WB: Flg (Ulk1) Fig.4c Fig.4b Flipped WB: H (AMPKα) WB: Ulk1 Fig.4e Figure S5 continued 8

9 Fig.5b (S757) Ulk1 Fig.5e 0 0 S757 Fig.5d Fig.5c H (Ulk1) WB: H (AMPKα) L.E. IP Long exposure Lystes Fig.6b Figure S5 continued 9

10 Fig.6d P-GST-Ulk1 Fig.6e Figure S5 continued 10

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys.