Session 4.1: Pessin
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This document discusses the regulation of acetyl CoA carboxylase (ACC) by AMP-activated protein kinase (AMPK) and the role of liver kinase B1 (LKB1) in this process. It shows that LKB1 interacts with and is phosphorylated by the tyrosine kinase Fyn. Fyn promotes the nuclear export and activation of LKB1 leading to increased AMPK phosphorylation and inhibition of mTORC1 signaling. Mutation of specific LKB1 tyrosine residues involved in Fyn phosphorylation disrupts this process and LKB1 localization.
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3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
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Session 4.1: Pessin
- 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. 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. LKB1 STRAD MO25 PIPPSP SH3 SH4 SH2 SH1 Fyn (Inactive) pY SH3 SH4 SH2 SH1 Fyn (Active)
- 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. 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. 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. 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. 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. 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. 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. LKB1-WT LKB1-P328A LKB1 DAPI Merge LKB1-PA mutant is nuclear excluded
- 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. Nucleus pY261/365 Cytosol pT Fyn-CA LKB1 LKB1 Fyn-KD AMPK AMP/ATP &/or NAD + /NADH PP2C
- 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. -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. LKB1 expression inhibits HeLa cell proliferation Vector LKB1-WT LKB1-P328A
- 17. Cell number x10 -3 LacZ Vector LKB1-WT LKB1-Y261/365F LKB1-P328A LKB1 expression inhibits HeLa cell growth Time (h)
- 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. Nucleus pY261/365 LKB1 LKB1 AMPK pT Fyn-CA Fyn-KD p53 Polarity Cytoskeleton -catenin Metabolism Protein Synthesis
- 20. Acknowledgements Claire Bastie Bhaskar Das Eijiro Yamada