src kinase activity upon substrate phosphorylation

Src KinaseActivity upon

substrate phosphorylation

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

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.

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

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.

Compositions of Src reporter

Fluorescent Proteins and FRET

• FPs: visualize signaling molecule– tECFP/EYFP pair

• FRET: visualize dynamical molecular activities.

How does FRET work?

• 2 chromophores are in proximity

• Overlap of excitation spectrum of donor and acceptor

• Energy transfer

Significance of flexible linker and substrate peptide

Src Kinase Structure

• Non-receptor tyrosine kinases family

• N-terminal SH4 domain• SH3 domain• SH2 domain (catalytic

domain)• C-terminal regulatory

sequence

How to activate Src Kinase?

1. Hormone binds cellular surface receptors (EGF, insulin) to generate phosphotyrosine

2. Phosphotyrosine attracts SH2 domain to activate src.

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

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

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

Designation of a robust fluorescent labeling protein

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)

Cplx1 etot

Cplx2 etot

Cplx3 etot

Cplx4 etot

Cplx5 etot

Cplx1 temperature

Cplx2 temperature

Cplx3 temperature

Cplx4 temperature

Cplx5 temperature

Cplx1 rmsd

Cplx2 rmsd

Cplx3 rmsd

Cplx4 rmsd

Cplx5 rmsd

Cplx1(left) vs. Cplx2(right)

Cplx2 (left) vs. Cplx1(right)

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

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

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

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

Conclusion

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