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Src Kinase Activity upon substrate phosphorylation

Src Kinase Activity upon substrate phosphorylation. Outline. 1.Src Kinase Introduction 2.Impacts of Src 3.Src reporter components FPs (tECFP/EYFP) SH2 Flexible linker Substrate peptide 4. Fluorescent Proteins and FRET 5. Src Kinase Inactive and Active State

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Src Kinase Activity upon substrate phosphorylation

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  1. Src Kinase Activity upon substrate phosphorylation

  2. Outline 1.Src Kinase Introduction 2.Impacts of Src 3.Src reporter components • FPs (tECFP/EYFP) • SH2 • Flexible linker • Substrate peptide 4. Fluorescent Proteins and FRET 5. Src Kinase Inactive and Active State 6. How Src influence dynamical image of molecule in live cell 7. Linker, Substrate designation for a robust labeling protein

  3. Introduction of Src Kinase • 1911 Peyton Rous isolated a virus from a chicken, which causes tumor in healthy bird, aka Rous sarcoma virus • v-src codes for a protein which induces tumor cells. • c-src (cellular counterpart of v-src) affect signal transduction pathway to regulate cell-growth • Despite external signals, v-src activates internal control mechanism, hence induce oncogenic characterization.

  4. Significant Impacts of Src activation • Impacts on cell polarity, adhesion, focal adhesion assembly/disassembly, lamellipodia formation, and migration, survival of both normal cells and cancer cells. • Inhibition of Src results in impaired polarization toward migratory stimuli • Src phosphorylate cortactin. The phosphorylated cortactin associate and activate Arp2/3 to induce the growth of cortical actin network

  5. Significant impacts of Src • Src activates the calpain-calpastatinproteolytic system to cleave FAK and disrupt focal adhesion complex => cell adhesion to ECM is reduced and cell motility is enhanced. • Src can phosphorylate p190RhoGAP and induce its binding to p120RasGAP => inhibition of RhoA, and subsequent dissolution of actin filaments. • Because of Src’s prominent roles in invasion and tumor progression, epithelial-to-mesenchymal transition, angiogenesis, and the development of metastasis, Src is a promising target for cancer therapy.

  6. Compositions of Src reporter

  7. Fluorescent Proteins and FRET • FPs: visualize signaling molecule • tECFP/EYFP pair • FRET: visualize dynamical molecular activities.

  8. How does FRET work? • 2 chromophores are in proximity • Overlap of excitation spectrum of donor and acceptor • Energy transfer

  9. Significance of flexible linker and substrate peptide

  10. Src Kinase Structure • Non-receptor tyrosine kinases family • N-terminal SH4 domain • SH3 domain • SH2 domain (catalytic domain) • C-terminal regulatory sequence

  11. How to activate Src Kinase? • Hormone binds cellular surface receptors (EGF, insulin) to generate phosphotyrosine • Phosphotyrosine attracts SH2 domain to activate src.

  12. FRET effect of Src reporter upon the actions of Src Kinase and Phosphatase

  13. Emission Spectra of Src reporter before(Red) and after(black) phosphorylation by Src • When Src is inactivated, higher FRET is observed. • When Src is activated, emission intensity drops, thus yields lower FRET efficiency

  14. Various Src biosensors with tECFP at N-termini and Citrine at C-termini

  15. Designation of a robust fluorescent labeling protein

  16. Objectives • To compare the binding affinity(using MMPBSA/GBSA) between phosphorylated complex (SH2 + phosphorylated peptide) vs. non-phosphorylated complex (SH2 + non-phosphorylated peptide) • Create 5 prmtop files • Cplx1: SH2+linker+ phosphorylated peptide. • Cplx2: SH2+linker+peptide • Cplx3: linker+peptide • Cplx4: linker+ phosphorylated peptide • Cplx 5: SH2 • Run 20ns md-production • Plot Temperature, Energy, RMSD • Use MMPBSA to measure binding energy (delta G)

  17. Cplx1 etot

  18. Cplx2 etot

  19. Cplx3 etot

  20. Cplx4 etot

  21. Cplx5 etot

  22. Cplx1 temperature

  23. Cplx2 temperature

  24. Cplx3 temperature

  25. Cplx4 temperature

  26. Cplx5 temperature

  27. Cplx1 rmsd

  28. Cplx2 rmsd

  29. Cplx3 rmsd

  30. Cplx4 rmsd

  31. Cplx5 rmsd

  32. Cplx1(left) vs. Cplx2(right)

  33. Cplx2 (left) vs. Cplx1(right)

  34. GBSA of Cplx1 • Differences (Complex - Receptor - Ligand): • Energy Component Average Std. Dev. Std. Err. of Mean • ------------------------------------------------------------------------------- • BOND -0.2565 0.6121 0.0194 • ANGLE -0.1049 0.4385 0.0139 • DIHED 1.3343 0.2205 0.0070 • VDWAALS -65.7397 5.9783 0.1891 • EEL -1256.0463 39.3857 1.2455 • 1-4 VDW 0.0000 0.0000 0.0000 • 1-4 EEL 2.3653 1.2266 0.0388 • EGB 1209.3920 34.8056 1.1007 • ESURF -11.2648 0.5569 0.0176 • DELTA G gas -1318.4478 39.5743 1.2515 • DELTA G solv 1198.1272 34.5976 1.0941 • DELTA G binding = -120.3205 +/- 9.1496 0.2893

  35. GBSA of Cplx 2 • Differences (Complex - Receptor - Ligand):Energy Component Average Std. Dev. Std. Err. of Mean-------------------------------------------------------------------------------BOND -0.2331 0.6273 0.0198ANGLE -0.1340 0.4137 0.0131DIHED 1.4480 0.1877 0.0059VDWAALS -58.8694 4.9718 0.1572EEL -590.7593 40.2792 1.27371-4 VDW 0.0000 0.0001 0.00001-4 EEL 2.0730 1.2441 0.0393EGB 595.6172 35.9889 1.1381ESURF -9.1868 0.6921 0.0219DELTA G gas -646.4749 39.7427 1.2568DELTA G solv 586.4304 35.7000 1.1289 DELTA G binding = -60.0445 +/- 7.9898 0.2527

  36. PBSA of Cplx1 • Differences (Complex - Receptor - Ligand): • Energy Component Average Std. Dev. Std. Err. of Mean • ------------------------------------------------------------------------------- • BOND -0.2565 0.6121 0.0194 • ANGLE -0.1049 0.4385 0.0139 • DIHED 1.3343 0.2205 0.0070 • VDWAALS -65.7397 5.9783 0.1891 • EEL -1256.0463 39.3857 1.2455 • 1-4 VDW 0.0000 0.0000 0.0000 • 1-4 EEL 2.3653 1.2266 0.0388 • EPB 1210.4326 35.0238 1.1075 • ECAVITY -7.6109 0.2776 0.0088 • DELTA G gas -1318.4478 39.5743 1.2515 • DELTA G solv 1202.8217 34.9004 1.1036 • DELTA G binding = -115.6261 +/- 12.3976 0.3920

  37. PBSA of Cplx2 • Differences (Complex - Receptor - Ligand): • Energy Component Average Std. Dev. Std. Err. of Mean • ------------------------------------------------------------------------------- • BOND -0.2331 0.6273 0.0198 • ANGLE -0.1340 0.4137 0.0131 • DIHED 1.4480 0.1877 0.0059 • VDWAALS -58.8694 4.9718 0.1572 • EEL -590.7593 40.2792 1.2737 • 1-4 VDW 0.0000 0.0001 0.0000 • 1-4 EEL 2.0730 1.2441 0.0393 • EPB 615.1288 37.0373 1.1712 • ECAVITY -6.7999 0.4564 0.0144 • DELTA G gas -646.4749 39.7427 1.2568 • DELTA G solv 608.3289 36.8231 1.1644 • DELTA G binding = -38.1460 +/- 7.9809 0.2524

  38. Conclusion • Substrate phosphorylation by SrcKinase would enhance binding affinity, and yield lower FRET response.

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