Session 4.1 Pessin


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Jeffrey Pessin

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Session 4.1 Pessin

  1. 1. Regulation of acetyl CoA carboxylase (ACC) AMP AMPKK (LKB1) + AMPK Pi Pi - Acetyl CoA Malonyl-CoA Fatty acid oxidation ACC Palmitoyl-CoA + - Citrate - PP2C
  2. 2. p59 Fyn: structure, regulation SH3 NH2 COOH SH4 SH2 SH1 P-tyr Activation Inhibited Y528 SH4 SH3 SH2 SH1 Activated Y417 SH4 SH3 SH2 SH1 PTP P-tyr Inhibition Csk
  3. 3. LKB1 STRAD MO25 PIPPSP SH3 SH4 SH2 SH1 Fyn (Inactive) pY SH3 SH4 SH2 SH1 Fyn (Active)
  4. 4. GST GST- Fyn-WT GST- Fyn-W119A His-LKB1 GST Input Fyn A) B) GST GST- Fyn-WT GST GST- Fyn-WT His-LKB1-WT His-LKB1-P328A The Fyn SH3 domain interacts with the LKB1 proline domain
  5. 5. Fyn co-immunoprecipitates and tyrosine phosphorylates LKB1 C) Lysate IB: Fyn IB: LKB1 pcDNA3 Fyn D) IP: LKB1 IB: PY100 IB: LKB1 pcDNA3 Fyn IB:LKB1 IgG Gastrocnemius IP:IgG IP:Fyn A) Mock IB: PY100 IB: LKB1 Fyn E) In vitro phosphorylation 3T3L1 Adipocytes IP:IgG IP:Fyn B)
  6. 6. LKB1 Y261 and Y365 are candidate Fyn substrates sites WT Y60 Y156 Y166 Y261 Y365 IP: Flag PY100 Flag IP:LKB1 WT Y261/365F Lysate IB: Flag-LKB1 IB: Fyn-CA WT Y261/365F IB: PY100 IB: Flag-LKB1
  7. 7. Nucleus Cytoplasm export export import passive diffusion acetylated deacetylated LKB1 STRAD MO25 STRAD MO25 MO25 STRAD STRAD LKB1 STRAD MO25 STRAD Fyn ? LKB1 LKB1 MO25 MO25
  8. 8. LKB1 Fyn Merge DAPI c) b) a) d) f) e) g) h) Kinase inactive Fyn recruits LKB1 out of the 3T3L1 adipocyte nucleus Fyn-CA (Y528F) Fyn-KD (K299M) Fyn-CA % Nuclear LKB1 100 80 60 40 20 0 Fyn-KD
  9. 9. LKB1 Y261 and Y365 are required for nuclear localization LKB1 DAPI Merge LKB1- WT a) b) c) LKB1- Y60F d) e) f) LKB1- Y365F d) e) f) LKB1- Y261/365F g) h) i) LKB1- Y261F a) b) c) LKB1 DAPI Merge
  10. 10. LKB1 Y261/365F increases AMPK phosphorylation Endo-AMPK P (T172) -AMPK Flag LKB1- WT LKB1- Y261/365F B) SKM GST-AMPK Flag LKB1- WT LKB1- Y261/365F A) HeLa P (T172) -AMPK
  11. 11. LKB1-WT LKB1-P328A LKB1 DAPI Merge LKB1-PA mutant is nuclear excluded
  12. 12. LKB1 Y261/365F but not P328A prevents Fyn- CA induced nuclear localization DAPI Merge Fyn-CA LKB1- Y261/365F LKB1-P328A DAPI Merge Fyn-CA a) c) d) b) e) g) h) f)
  13. 13. Nucleus pY261/365 Cytosol pT Fyn-CA LKB1 LKB1 Fyn-KD AMPK AMP/ATP &/or NAD + /NADH PP2C
  14. 14. 4E-BP1 eIF4E Translation S6K S6 eIF4B TSC1/2 Rheb mTORC1 (mTOR/Raptor/LST8/PRAS40) PI-3K I R IRS PIP3 mTORC2 (mTOR/Rictor/LST8/mSin1) PDK1 Akt AMPK LKB1 AMP PP2C MARKS Par1, SIK p53
  15. 15.  -Actin AMPK S6K pT389-S6K pT172-AMPK Vector WT Y261/365F P328A LKB1 over expression activates AMPK and inhibits mTORC1 signaling LKB1 Raptor pS792-Raptor
  16. 16. LKB1 expression inhibits HeLa cell proliferation Vector LKB1-WT LKB1-P328A
  17. 17. Cell number x10 -3 LacZ Vector LKB1-WT LKB1-Y261/365F LKB1-P328A LKB1 expression inhibits HeLa cell growth Time (h)
  18. 18. LKB1 proline domain peptide increases pT172-AMPK phosphorylation TAT = FITC-YGRKKRRQRRR-G- ALVPIPPSPDTK 11R = FITC-RRRRRRRRRRR-G- ALVPIPPSPDTK p 172 T-AMPK Total-AMPK 20 20 40 40 11R (nM) TAT (nM) AICAR (2 mM) Con
  19. 19. Nucleus pY261/365 LKB1 LKB1 AMPK pT Fyn-CA Fyn-KD p53 Polarity Cytoskeleton  -catenin Metabolism Protein Synthesis
  20. 20. Acknowledgements Claire Bastie Bhaskar Das Eijiro Yamada