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-calpastatin proteolytic 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? 1.Hormone binds cellular surface receptors (EGF, insulin) to generate phosphotyrosine 2.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

16. Designation of a robust fluorescent labeling protein

17. 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)

18. Cplx1 etot

19. Cplx2 etot

20. Cplx3 etot

21. Cplx4 etot

22. Cplx5 etot

23. Cplx1 temperature

24. Cplx2 temperature

25. Cplx3 temperature

26. Cplx4 temperature

27. Cplx5 temperature

28. Cplx1 rmsd

29. Cplx2 rmsd

30. Cplx3 rmsd

31. Cplx4 rmsd

32. Cplx5 rmsd

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

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

35. 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

36. GBSA of Cplx 2 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 EGB 595.6172 35.9889 1.1381 ESURF -9.1868 0.6921 0.0219 DELTA G gas -646.4749 39.7427 1.2568 DELTA G solv 586.4304 35.7000 1.1289 DELTA G binding = -60.0445 +/- 7.9898 0.2527

37. 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

38. 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

39. Conclusion Substrate phosphorylation by Src Kinase would enhance binding affinity, and yield lower FRET response.