Fluorescent Indicators for Imaging
Protein Phosphorylation in Single
To study protein phosphorylation, investigators
have used electrophoresis, immunocytochemistry, and in vitro
kinase assays. However, these methods do not
provide enough information about spatial and temporal
dynamics of protein phosphorylation in each living
cell. To overcome this limitation, we have developed
genetically encoded novel fluorescent indicators and
visualized signal transduction based on protein phosphorylation
in living cells (Sato et al.
, 2002) (Fig. 1).
Within the fluorescent indicator, a substrate domain
for a protein kinase of interest is fused with a phosphorylation
recognition domain via a flexible linker
sequence. The tandem fusion unit consisting of the
substrate domain, linker sequence, and phosphorylation
recognition domain is sandwiched with two
different color fluorescent proteins, cyan fluorescent
protein (CFP) and yellow fluorescent protein (YFP),
which serve as the donor and acceptor fluorophores
for fluorescence resonance energy transfer (FRET). As
a result of phosphorylation of the substrate domain
and subsequent binding of the phosphorylated substrate
domain with the adjacent phosphorylation
recognition domain, FRET is induced from CFP to YFP
when CFP is excited at 440 ± 10nm. Upon activation
of phosphatases, the phosphorylated substrate domain
is dephosphorylated and the FRET signal is decreased.
This FRET change is represented by the change in the
fluorescence emission ratio of CFP at 480 ± 15 nm and
YFP at 535 ± 12.5 nm, both of which were monitored
continuously by a dual-emission fluorescence microscope.
We named this indicator "phocus" (a fluorescent
indicator for protein phosphorylation that can be
custom-made). Until now, by using suitable substrates
and phosphorylation recognition domains, we have
developed a large number of phocus variants for
several key protein kinases, such as a receptor tyrosine
kinase, insulin receptor, and a serine/threonine
protein kinase, Akt/PKB (Table I). In addition, these
phocus variants were further tailored to visualize subcellular
local activity of the respective protein kinases
in living cells (Table I). For example, the phocus
variant for Akt protein kinase was tethered to the cytoplasmic
surface of mitochondria or Golgi membranes
by connecting each appropriate sequence/domain.
This membrane tethering prevented the free diffusion
of the indicator and avoided the resulting loss of
spatial information as to phosphorylation by the activated
Akt. We thus found that the activated Akt is not
in the cytosol but is localized at subcellular membranes,
including Golgi and mitochondria membranes,
when the cells were stimulated.
II. MATERIALS AND
|FIGURE 1 Schematic representation of the present fluorescence
indicator for protein phosphorylation, which was named phocus.
Upon phosphorylation of the substrate domain within phocus by the
protein kinase, the adjacent phosphorylation recognition domain
binds with the phosphorylated substrate domain, which changes the
efficiency of FRET between the GFP mutants within phocus. By tethering
a localization domain with phocus, the phocus can be localized
in the specific intracellular locus of interest to visualize the local
phosphorylation event there. LocD, localization domain; CFP, cyan
fluorescent protein; SubD, substrate domain; PhosRD, phosphorylation
recognition domain; YFP, yellow fluorescent protein; P in an
open circle, the phosphorylated residue.
ECFP (Cat. No. 6900-1) and EYFP (Cat. No. 6006-1)
expression vectors are from Clonthech. Ham's F-12
medium (Cat. No. 21700), fetal bovine serum (Cat. No.
10099-141), and LipofectAMINE 2000 (Cat. No. 11668-
019) reagent are from Invitrogen. Other chemicals used
are all of analytical reagent grade. Glass-bottom dishes
are from Asahi Techno Glass (Cat. No. 3911-035).
Cells are observed with a 40× oil-immersion objective
(Carl Zeiss) on a Axiovert 135 microscope (Carl
Zeiss) with a cooled CCD camera MicroMAX (Roper
Scientific Inc.) controlled by MetaFluor (Universal
Imaging). An excitation filter (440AF21), a dichroic
mirror (455DRLP), and emission filters for CFP
(480AF30) and YFP (535AF26) are from Omega
- Culture medium: Add fetal bovine serum to Ham's
F-12 medium to give a final concentration of 10%.
- Serum starvation medium: Add bovine serum
albumin to Ham's F-12 medium to give a final concentration
- Hank's balanced salt solution (HBSS): Dissolve
0.35 g of NaHCO3, 0.06 g of KH2PO4, 0.048 g of
Na2HPO4, 0.14g of CaCl2, 0.40g of KCl, 0.10g of MgCl2·6H2O, 0.10g of MgSO4·7H2O, 8.00g of NaCl,
and 1.00g of D-glucose in 800ml of Milli-Q water,
adjust the pH to 7.4, and then adjust the volume to
- Plate CHO-IR cells that express human insulin
receptor onto a glass-bottom dish with culture
medium and incubate the cells at 37°C under 5%
CO2 for 1 day.
- Transfect the cells with 0.8µg of cDNA (phocus-
2 or phocus-2pp) using Lippofecetamine 2000
reagent according to the manufacturer' s
- Incubate the cells at 37°C under 5% CO2 for 1 to 2
- Replace the culture medium with serum starvation
medium and incubate the cells at 37°C under 5%
CO2 for 2 to 4 h.
- Replace the serum starvation medium with HBSS.
- Set the glass-bottom dish onto the 40x oil immersion
objective equipped on the fluorescence microscope.
- Observe the cells with a 440-nm excitation filter
(440AF21), 455-nm dichroic mirror (455DRLP),
and 535-nm emission filter (535AF26).
- By browsing the cells on the dish, choose moderately
bright cells in which the fluorescence is well
distributed in the cytosol.
- Determine the desired observation field in which
only the cells of interest are covered.
- Select several regions of interest within the cells to
examine time courses of CFP/YFP emission ratio
during the following image acquisition.
- Start to acquire images every 5 to 10s for 10 to
30min with the 440-nm excitation filter (CFP),
455-nm dichroic mirror, 480-nm emission filter
(CFP), and 535-nm emission filter (YFP).
- During image acquisition, add insulin to give a
final concentration of 100nM.
- After finishing all the experiments, wipe the residual
immersion oil out of the objective.
Substrates for protein kinases and phosphatases
often exhibit each unique localization, including mitochondria,
Golgi, nucleus, and plasma membrane in
living cells, which is thought to be critical for specific
signal transduction in the respective intracellular loci
(Hunter, 2000). Thus, we further tailored our phocuses
to analyze the phosphorylation events in such particular
locations in single living cells. Here we exemplify
phocus for insulin receptor and that for Akt/PKB; the
latter was named Aktus.
A. Phocus for Imaging Phosphorylation
by Insulin Receptor
IRS-1 is one of the major substrates of insulin receptor.
It contains a peckstrin-homology (PH) domain and
a phosphotyrosine-binding (PTB) domain in its Nterminal
end. The PH and PTB domains bind, respectively,
with the phosphoinositides at the plasma
membrane and with the juxtamembrane domain of
insulin receptor, which is immediately tyrosine phosphorylated
by insulin stimulation (Paganon et al.
1999). Thus, the concentration of IRS-1 is thought to be
increased around the insulin receptor at the plasma
membrane upon insulin stimulation. These PH and
PTB domains were fused with phocus, named phocus-
2pp, to locate the phocus around the insulin receptor
like the IRS-1 and to measure the local phosphorylation
event there. When phocus-2pp was expressed in CHO-IR cells, fluorescence was observed throughout
the cells (Fig. 2, time 0s). Upon insulin stimulation,
the CFP/YFP emission ratio, which is expressed with
pseudocolor, was decreased in the cytosol due to
phosphorylation-induced FRET from CFP to YFP
within phocus-2pp. Three hundred seconds after
insulin stimulation, membrane ruffles, in which a large
extent of phocus-2pp were accumulated, appeared
around the plasma and disappeared in 1000s (Fig. 2).
In these membrane ruffles, phocus-2pp has been found
to colocalize with the insulin receptor accumulated
there by insulin stimulation. Interestingly, in these
membrane ruffles, the extent of phocus-2pp phosphorylation
was visualized to be ~2-fold greater than that
in the cytosol. This difference in phosphorylation
levels between intracellular loci could be due to a different
balance of kinase and phosphatase activities
between intracellular loci. Phocus-2pp should contribute
to reveal the biological significance of such a
characteristic domain for tyrosine kinase signaling in
the membrane ruffles, which were formed upon
insulin stimulation, with high spatial and temporal
B. Aktus for Imaging Phosphorylation
|FIGURE 2 Fluorescence imaging with phocus-2pp upon insulin stimulation. Pseudocolor images of the
CFP/YFP emission ratio are shown before (time 0s) and 60, 300, and 1000s after the addition of 100nM insulin
at 25°C, obtained from CHO-IR cells expressing phocus-2pp. Insulin-induced accumulation of phocus-2pp
at the membrane ruffles is indicated by white arrows in the image at 300s.
Akt/PKB is a serine/threonine kinase that regulates
a variety of cellular responses, such as cell proliferation,
cell survival, and angiogenesis (Marte and
Downward, 1997). To provide information on the
spatial and temporal dynamics of the Akt activity in
single living cells, we have developed a genetically
encoded fluorescent indicator for Akt, named Aktus
(Sasaki et al.
, 2003). Almost all Akt substrates are localized
to subcellular regions. For example, eNOS (Fulton et al.
, 2001), which mediates a vasodilatory effect by
nitric oxide production, is localized predominantly to the Golgi apparatus, whereas Bad (Chao and
Korsmeyer, 1998), which is related to apoptosis promotion,
is present in mitochondrial outer membranes.
By fusing the Aktus with the respectively subcellular
localization domains within the eNOS and Bad, eNOSAktus
and Bad-Aktus, which are respectively localized
to the Golgi apparatus and mitochondrial outer membrane,
were developed as shown in Table I and compared
with the cytosolic diffusible indicator Aktus. We
have shown that in vascular endothelial cells, the
Golgi-localized indicator, eNOS-Aktus, was phosphorylated
upon stimulation with insulin and with 17β-
estradiol, whereas the mitochondria-localized
Bad-Aktus was phosphorylated by 17β-estradiol but
not by insulin (Table II). However, the diffusible indicator
Aktus was not phosphorylated efficiently upon
both insulin or 17β-estradiol stimulation (Table II).
From these results, it is suggested that the activated
Akt is localized to subcellular compartments, including
the Golgi apparatus and/or mitochondria, rather
than diffusing in the cytosol, thereby efficiently phosphorylating
its substrate proteins. Different observation
with the mitochondria-localized indicator
indicates that localization of the activated Akt to mitochondria
is directed differently between insulin and
17β-estradiol via distinct mechanisms. The present indicators and their applications are thus expected to
contribute to the studies of a whole range of dynamics
of the activated Akt in living cells.
- Avoid too bright cells to reproducibly obtain quantitative
FRET signals. Also avoid too dim cells
because they often exhibit noisy images.
- Our measurement conditions do not significantly
affect the stability of phocus, but if photobleaching
of fluorescent proteins, particularly YFP, is still
observed, we recommend using ND filters and/or
to irradiate the cells less. We usually irradiate the
cell for 50 to 100 ms.
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