Src Kinase Activity upon substrate phosphorylation.
-
Upload
abigail-adams -
Category
Documents
-
view
222 -
download
0
Transcript of Src Kinase Activity upon substrate phosphorylation.
![Page 1: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/1.jpg)
Src KinaseActivity upon
substrate phosphorylation
![Page 2: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/2.jpg)
Outline 1.Src Kinase Introduction 2.Impacts of Src 3.Src reporter components
FPs (tECFP/EYFP) SH2 Flexible linker Substrate peptide4. Fluorescent Proteins and FRET5. Src Kinase Inactive and Active State6. How Src influence dynamical image of molecule in live cell7. Linker, Substrate designation for a robust labeling protein
![Page 3: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/3.jpg)
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.
![Page 4: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/4.jpg)
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
![Page 5: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/5.jpg)
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.
![Page 6: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/6.jpg)
Compositions of Src reporter
![Page 7: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/7.jpg)
Fluorescent Proteins and FRET
• FPs: visualize signaling molecule– tECFP/EYFP pair
• FRET: visualize dynamical molecular activities.
![Page 8: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/8.jpg)
How does FRET work?
• 2 chromophores are in proximity
• Overlap of excitation spectrum of donor and acceptor
• Energy transfer
![Page 9: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/9.jpg)
Significance of flexible linker and substrate peptide
![Page 10: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/10.jpg)
Src Kinase Structure
• Non-receptor tyrosine kinases family
• N-terminal SH4 domain• SH3 domain• SH2 domain (catalytic
domain)• C-terminal regulatory
sequence
![Page 11: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/11.jpg)
How to activate Src Kinase?
1. Hormone binds cellular surface receptors (EGF, insulin) to generate phosphotyrosine
2. Phosphotyrosine attracts SH2 domain to activate src.
![Page 12: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/12.jpg)
FRET effect of Src reporter upon the actions of Src Kinase and Phosphatase
![Page 13: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/13.jpg)
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
![Page 14: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/14.jpg)
Various Src biosensors with tECFP at N-termini and Citrine at C-termini
![Page 15: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/15.jpg)
![Page 16: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/16.jpg)
Designation of a robust fluorescent labeling protein
![Page 17: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/17.jpg)
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)
![Page 18: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/18.jpg)
Cplx1 etot
![Page 19: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/19.jpg)
Cplx2 etot
![Page 20: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/20.jpg)
Cplx3 etot
![Page 21: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/21.jpg)
Cplx4 etot
![Page 22: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/22.jpg)
Cplx5 etot
![Page 23: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/23.jpg)
Cplx1 temperature
![Page 24: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/24.jpg)
Cplx2 temperature
![Page 25: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/25.jpg)
Cplx3 temperature
![Page 26: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/26.jpg)
Cplx4 temperature
![Page 27: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/27.jpg)
Cplx5 temperature
![Page 28: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/28.jpg)
Cplx1 rmsd
![Page 29: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/29.jpg)
Cplx2 rmsd
![Page 30: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/30.jpg)
Cplx3 rmsd
![Page 31: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/31.jpg)
Cplx4 rmsd
![Page 32: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/32.jpg)
Cplx5 rmsd
![Page 33: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/33.jpg)
Cplx1(left) vs. Cplx2(right)
![Page 34: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/34.jpg)
Cplx2 (left) vs. Cplx1(right)
![Page 35: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/35.jpg)
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
![Page 36: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/36.jpg)
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 0.0219
DELTA 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
![Page 37: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/37.jpg)
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
![Page 38: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/38.jpg)
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
![Page 39: Src Kinase Activity upon substrate phosphorylation.](https://reader035.fdocuments.in/reader035/viewer/2022062423/5697c01f1a28abf838cd1eae/html5/thumbnails/39.jpg)
Conclusion
• Substrate phosphorylation by Src Kinase would enhance binding affinity, and yield lower FRET response.