Src Kinase Biosensor

Outline

1. Src Kinase Introduction2. Impacts of Src3. 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 (sarcoma virus) consists of 3 simple genes: gag, pol, and env for replication and encapsulation

• 4th gene (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.

Impacts of Src activation• Impacts on cell polarity, adhesion, focal adhesion

assembly/disassembly, lamellipodia formation, and migration.• 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

• 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.

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 (homologous structure of src) to activate src.

Src inactive state vs. active stateSH3 and SH2 couple, catalytic domain masked by C-terminal tail, prevent substrate binding.

1. Interaction between integrin and Src changes Src conformation, thus activate it

2. Integrin recruit RPTPα to dephosphorylate Y527 on C-terminal tail of Src and release it from kinase domain, thus make it active

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