Src Kinase
Biosensor
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 (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 state
SH3 and SH2 couple, catalytic
domain masked by C-terminal tail,
prevent substrate binding.
1.
2.
Interaction between integrin and Src
changes Src conformation, thus activate it
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 Ntermini and Citrine at C-termini
Designation of a robust fluorescent
labeling protein