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 6. How Src influence dynamical image of molecule in live cell 7. 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 ANGLE DIHED VDWAALS EEL VDW EEL EGB ESURF DELTA G gas DELTA G solv DELTA G binding = /

GBSA of Cplx 2 Differences (Complex - Receptor - Ligand): Energy Component Average Std. Dev. Std. Err. of Mean BOND ANGLE DIHED VDWAALS EEL VDW EEL EGB ESURF DELTA G gas DELTA G solv DELTA G binding = /

PBSA of Cplx1 Differences (Complex - Receptor - Ligand): Energy Component Average Std. Dev. Std. Err. of Mean BOND ANGLE DIHED VDWAALS EEL VDW EEL EPB ECAVITY DELTA G gas DELTA G solv DELTA G binding = /

PBSA of Cplx2 Differences (Complex - Receptor - Ligand): Energy Component Average Std. Dev. Std. Err. of Mean BOND ANGLE DIHED VDWAALS EEL VDW EEL EPB ECAVITY DELTA G gas DELTA G solv DELTA G binding = /

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