Download Chimeric phosphorylation indicator

US008669074B2
(12) Ulllted States Patent
(10) Patent N0.:
Violin et al.
(54)
US 8,669,074 B2
(45) Date of Patent:
CHIMERIC PHOSPHORYLATION
INDICATOR
Mar. 11, 2014
6,410,255 B1
6,465,199 B1
6/2002 Pollok et al.
10/2002 Craig et al.
6,656,696 B2 *
12/2003
Cra1g et al. .................. .. 435/76
(75) Inventors: Jonathan D. Violin, Durham, NC (U S);
Alexandra C. Newton, San Diego, CA
FOREIGN PATENT DOCUMENTS
(US); Roger Y. Tsien,~La Jolla, CA
(Us); Jill Zhang, Baltlmore, MD (Us)
W0
W0
W0 98/0257l A1
WO 00/71565 A2
(73) Assignee: The Regents of the University of
V1998
11/2000
OTHER PUBLICATIONS
California, Oakland, CA (U S)
_
_
( * ) Nonce:
_
_
_
Wells, Biochemistry, vol. 29, pp. 8509-8517, 1990.*
subleqto any dlsclalmer1 the term Ofthls
Seffernick et al. (J. Bacteriology, vol. 183, pp. 2405-2410, 2001).*
Patent 15 extended or adJusted under 35
Nagai, Yasuo et al.; “A ?uorescent indicator for visualizing cAMP
U'S'C' 154(1)) by 2722 days‘
induced phosphorylation in vivo”; 2000, Nature Biotechnology, vol.
18,
(21) Appl. N0.: 10/857,622
*
(22)
Filed:
May 28, 2004
(65)
Prior Publication Data
US 2005/0026234 A1
Feb. 3, 2005
pp
. 313-316.
't d b
C1 e
'
y exammer
Primary Examiner * Hope Robinson
(74) Attorney, Agent, or Firm * Morgan, LeWis & Bockius
LLP
Related US. Application Data
(63) Continuation-in-part of application No. 09/865,291,
(51)
?led on May 24, 2001, noW Pat. No. 6,900,304, Which
(57)
359/336 ocggmi?iznoog'gé'paig 10599 alligiilczgaogdogg'
A chimeric phosphorylation indicator (CPI) as provided
whichi’sac’ominuation sf'a ’1iCatiO’nNO 08/792 553’
?l d J 31 1997
P; t N 5 98'1 200 ’hi 1;
herem can contam a donor molecule, a phosphorylatable
domain, aphosphoaminoacid binding domain (PAABD), and
is e aon caorlll'timiation_’illll_ovin a 'OfO' a’ lic’atiol’lw NCO
an acceptor molecule. Where the phosphorylatable domain is
08/ 59 4 575 ?led on 1251 31 1996ppnOW Pat NO’
phosphorylatable by protein kinase C (PKC), the CPI is a
6 803 1’88 ’
’
’
'
c-kinase activity reporter (CKAR). Donor and acceptor mol
ecules may be, independently, ?uorescent proteins such as
'
’
’
'
non-oligomeriZing ?uorescent proteins. A CPI can contain a
(200601)
phosphorylatable polypeptide and a ?uorescent protein; the
01 e tide may be contained Within the
P hos Pho 1'y latable PYPP
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References Cited
US. PATENT DOCUMENTS
5,795,729 A
6,376,257 B1
8/ 1998 Lee
4/2002 Persechini
sequence of the ?uorescent protein, or the ?uorescent protein
may be contained Within the sequence of the phosphorylat
(58) Fleld of Classl?catlon Search
(56)
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C1 ép 1/04
(52)
ABSTRACT
able polypeptide. The spatiotemporal properties of the PKC
signal pathWay may be tested With CKAR, calcium-sensing
?uorophores and FRET-based translocation assays. Poly
nucleotides encoding such CPIs, and kits containing the indi
cators and/ or the polynucleotides, are provided. A method of
using the chimeric phosphorylation indicators to detect a
kinase or phosphatase in a sample is provided.
23 Claims, 14 Drawing Sheets
US. Patent
Mar. 11,2014
Sheet 1 0f 14
US 8,669,074 B2
kinase + ATP
VII R
phosphatase
phosphoarninoacid
binding protein
pS/DT/DY ‘Ht
Arg/Lys-rich site
kinase + ATP
Dhosphoaminoacid-phosphatasepS/pT/PY I 5'
binding protein
9
Arg/Lys-rlch Slte
weak or no
?uorescence
pS/pT/pY 1
Arg/Lys-rich site
FIGURE 1
US. Patent
Mar. 11,2014
Sheet 2 0f 14
US 8,669,074 B2
U7;NHMS
$85Q5-05
E9650
US. Patent
Mar. 11,2014
Sheet 3 0f 14
US 8,669,074 B2
less fluorescent
kinase + NP
-—_-*~
~‘—_
phosphatase
FIGURE 3
US. Patent
Mar. 11,2014
Sheet 4 0f 14
association‘
dissociation
carbostyril
342 nm
totracysleine
association
.mamw»
dissociation
FIGURE 4
US 8,669,074 B2
US. Patent
Mar. 11, 2014
biarsenicai
Sheet 5 0f 14
US 8,669,074 B2
or to Cys“
FIGURE 5A
carbostyrii
antenna
| Cys-Arg-Gln-lle-Lys-Trp-PnGin-As-Arg-Arg-Met-Lys-Trp-Lys-Lys |
membrane translocating peptide
i
Cys-Arg-Gln-IIe-Lys-Trp-Phe-GIn-Asn-Arg-Arg-Met
Lys-Trp-Lys-Lys
RFP excitation
---- -- RFP emission
INonrtmaelizsdy
.......... .. Tb3+ _ TTHAcs 124 emission
01
Wavelength (nm)
FIGURE 5B
US. Patent
Mar. 11,2014
Sheet 8 0f 14
US 8,669,074 B2
M
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US. Patent
Mar. 11,2014
Sheet 9 0f 14
US 8,669,074 B2
0.64
3
6:“
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5°° "M G669”
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FIGURE 9
25
US. Patent
Mar. 11,2014
Sheet 10 0f 14
US 8,669,074 B2
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FIGURE 10
US. Patent
Mar. 11,2014
Sheet 11 0f 14
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US 8,669,074 B2
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Time (Minutes)
FIGURE 11
US. Patent
Mar. 11,2014
Sheet 12 0f 14
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US 8,669,074 B2
10 pM histamine
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FIGURE 12
US. Patent
Mar. 11,2014
Sheet 13 0f 14
US 8,669,074 B2
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FIGURE 13
US 8,669,074 B2
1
2
CHIMERIC PHOSPHORYLATION
INDICATOR
rescent Protein (GFP) has been used to visualiZe translocation
of PKC to membranes upon generation of diacylglycerol
This application is a continuation-in-part (CIP) of US. Ser.
No. 09/865,291, ?led May 24, 2001, now US. Pat. No. 6,900,
304, Which is a CIP ofU.S. Ser. No. 09/396,003, ?led Sep. 13,
1999, Which is a continuation (CON) ofU.S. Ser. No. 08/792,
553, ?led Jan. 31, 1997 (now US. Pat. No. 5,981,200), Which
is a CIP ofU.S. Ser. No. 08/594,575, ?led Jan. 31, 1996 (now
US. Pat. No. 6,803,188), the entire contents of each of Which
(DAG) and increases in calcium in living cells. (Oancea and
Meyer, 1998; Sakai et al., 1997; Shirai et al., 1998b). These
m
clear to What extent visualiZation of PKC translocation pro
vides a measure of PKC activation or of PKC substrate phos
is incorporated herein by reference.
phorylation.
This invention Was made With government support under
Grant No. GM 62114 aWarded by the National Institutes of
Health. The government has certain rights in this invention.
Previous techniques for imaging protein heterodimeriZa
tion in single cells have included observing luminescence
resonance energy transfer (LRET) from a lanthanide donor
attached to an antibody against one member of the bet
erodimer to a red dye attached to an antibody against the other
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to reagents for
determining kinase and phosphatase activity, and more spe
ci?cally to chimeric proteins containing tWo ?uorescent pro
teins and a phosphorylatable domain, and methods of using
such chimeric proteins to detect kinase or phosphatase activ
20
partner (Root, Proc. Natl. Acad. Sci., USA 94:5685-5690,
1997). This approach has the same advantages and disadvan
tages as phosphorylation-speci?c antibodies, including it is
applicable to examining endogenous proteins in intact non
transfected tissues, but has poor time resolution and dif?culty
in generating a continuous time course. Another mode of
energy transfer is bioluminescence resonance energy trans
ity.
2. Background Information
studies have revealed a Wealth of information on the kinetics
and localiZation of PKC inside the cell. HoWever, GFP-label
ing has not proven su?icient to determine the activation state
of PKC or PKC-substrate phosphorylation. HoWever, trans
location and activation are different processes. Thus, it is not
25
fer, in Which the donor is a luciferase and the acceptor is a
Pho sphorylation is the mo st important Way that individual
proteins are post-translationally modi?ed to modulate their
GFP @(u et al., Proc. Natl. Acad. Sci., USA 96:151-156,
function, While practically all signal transduction involves
dynamics of protein-protein interaction. Phosphorylation is
be detectable, the feebleness of bioluminescence Would be
expected to make the technique dif?cult or impossible to use
catalyZed and controlled by kinases such as Calcium modu
1999). HoWever, although emission from multiple cells may
30
rylation/dephosphorylation events and interacting protein
partners involved in cell function, including, for example,
function of cardiomyocytes and B lymphocytes. HoWever,
With single mammalian cells, especially if high spatial reso
lution is desired.
lated kinase (CaM kinase, such as CaMKII), protein kinaseA
(PKA), protein kinase C (PKC), and other kinases. Various
technologies have been used to enumerate the main pho spho
Additionally, reporters have been designed that alter ?uo
rescence resonance energy transfer (FRET) betWeen ?uores
35
cent proteins (MiyaWaki and Tsien, 2000) or the intrinsic
?uorescent properties of a ?uorescent protein (Llopis et al.,
1998; Nagai et al., 2001). Reporters based on FRET betWeen
the most common currently used technologies such as tWo
?uorescent proteins can be used to glean information from
dimensional gel electrophoresis, mass spectrometry, co-im
munoprecipitation assays, and tWo-hybrid screens require
destroying large numbers of the cells or transferring genes to
heterologous organisms. As such, these methods have poor
temporal and spatial resolution, and are insu?icient to
living cells, provided that such reporters do not signi?cantly
perturb cell function (for example by buffering of cell signals
resulting from reporter overexpression), and provided
40
reporter speci?city is maintained in cells. FRET reporters for
kinase activity have been described (Sato et al., 2002; Ting et
directly probe physiological functions such as contracture or
al., 2001; Zhang et al., 2001).
chemotaxis, Which occur on the time scale of milliseconds to
minutes.
45
The most Widely used method for detecting phosphoryla
tion of speci?c proteins in single cells utiliZes antibodies that
discriminate betWeen the phosphorylated and dephosphory
probe Whose ?uorescence can be sensitive to the phosphory
lation of the peptide. For example, When acrylodan Was
attached to a peptide from myosin light chain, an approxi
mately 40% decrease in emission peak amplitude upon phos
lated forms of an antigen. Such antibodies can, in principle,
reveal the phosphorylation state of the endogenous protein
50
just prior to the time the cells Were ?xed for examination,
Without any introduction of exogenous substrates. HoWever,
the identi?cation of antibodies that can discriminate betWeen
a phosphorylated and unphosphorylated form of a protein is
time consuming and expensive. In addition, the necessary
phorylation in vitro Was observed. When microinj ected into
?broblasts, the peptide incorporated into stress ?bers, but no
dynamic changes Were observable. Substrates for CaMKII
and PKA also have been labeled With acrylodan and, after
exposure to the kinase, ?uorescence Was about 200% and
55
immunocytochemistry is tedious, and is di?icult to reas
semble into a quantitative time course.
97%, respectively, of initial values. These peptides Were
hydrophobic enough to stain live cells, and local intensity
changes of up to 10% to 20% of initial ?uorescence Were seen
PKC is knoWn to play a key role in maintaining balance
betWeen normal groWth and transformation (NishiZuka,
1995). PKC function in cells is exquisitely controlled by three
In order to achieve dynamic recording of phosphorylation
in single cells, peptides have been labeled With acrylodan, a
in some regions. The ?uorescence of the PKA substrate
simultaneously decreased in the cytosol and increased in the
60
major mechanisms: phosphorylation, required for catalytic
competence, membrane-targeting, required for conforma
nucleus by an amount that Was greater than could be
explained by the in vitro sensitivity, indicating that more
complex factors such as translocation Were dominating.
tional activation, and proteinzprotein interactions Which poise
Although the use of acrylodan-labeled peptides provides
the enZyme at speci?c intracellular locations (Mellor and
Parker, 1998; NeWton, 2002b). Pertubation of any of these
no rational mechanism for phosphorylation sensitivity, the
mechanisms disrupts cell function by altering the degree of
substrate pho sphorylation. A ?uorescent protein, Green Fluo
65
approach of developing phosphorylation-sensitive ?uores
cent substrates may be WOITh pursuing. Thus, a need exists for
phosphorylation-sensitive indicators that can be used to
US 8,669,074 B2
3
4
detect phosphorylation or dephosphorylation events in a cell.
REP, or other ?uorescent protein may be a non-oligomeriZing
The present invention satis?es this need and provides addi
?uorescent protein. A non-oligomeriZing ?uorescent protein
is a ?uorescent protein having a reduced propensity to oligo
tional advantages.
meriZe as compared to a reference ?uorescent protein. For
example, a non-oligomeriZing ?uorescent protein related to a
SUMMARY OE THE INVENTION
GEP may have a mutation of an amino acid residue corre
The present invention relates to a chimeric phosphoryla
sponding to A206, L221, E223, or a combination thereof of
SEQ ID N012, for example, an A206K mutation, an L221K
tion indicator, Which may comprise, in operative linkage, a
?rst ?uorescent protein, a pho sphoaminoacid binding domain
comprising the forkhead-associated (EHA2) sequence EEI
GRSEDCNCKIEDNRLSRVH
mutation, an E223R mutation, or an L221K and E223R muta
tion of SEQ ID NO:2; or an A206K mutation, an L221K
mutation, an E223R mutation, or an L221K and E223R muta
CEIEKKRHAVGKSMYESPAQGLDDIWYCHTGTN
tion of SEQ ID NO:6 or SEQ ID NO:10. Non-oligomeriZing
VSYLNNNRMIQGTKELLQDGDEIKII (SEQ ID NO: 57),
?uorescent proteins having one or more of these mutations
may be, for example, a monomeric GEP (mGEP), a mono
meric CEP (mCEP) or a monomeric YEP (mYEP) Where the
mutations are With respect to a corresponding GEP, CEP, or
a protein kinase C (PKC)phosphorylatable domain compris
ing the amino acid sequence RERREQTLKIKAKA (SEQ ID
NO:44), and a second ?uorescent protein, Wherein the ?rst
YEP reference sequence. A non-oligomeriZing ?uorescent
protein related to a Discosoma REP may be, for example, an
I125R DsRed mutant (SEQ ID NO:12, including an I125R
and the second ?uorescent proteins are different, at least one
of the ?rst and second ?uorescent proteins comprises a non
oligomeriZing ?uorescent protein, and the ?rst and second
?uorescent proteins are selected from the group consisting of
20
green ?uorescent proteins (GEPs), red ?uorescent proteins
In embodiments of the invention, the PKC phosphorylat
able domain in a chimeric phosphorylation indicator of the
invention may be any domain that can be phosphorylated by
(REPs), and ?uorescent proteins related to a GEP or an REP,
Wherein a ?uoresyent protein related to a GEP or related to an
REP comprises an amino acid sequence having at least 90%
sequence homology to a GEP or an REP. The ?rst and the
second ?uorescent proteins exhibit a detectable resonance
energy transfer When the ?rst ?uorescent protein is excited,
While the PKC-phosphorylatable domain and phosphoami
noacid binding domain do not substantially emit light to
excite the second ?uorescent protein. The ?uorescent protein
PKC, or that can contain a phosphate group and can be
25
30
may be a non-oligomeriZing ?uorescent protein. The chi
meric phosphorylation indicator may further comprise a
able domain. A polypeptide linker may comprise betWeen
15 amino acid residues. A polypeptide linker may comprise,
for example, GGSGG (SEQ ID NO: 45), GHGTGSTGSGSS
(SEQ ID NO: 61), RMGSTSGSTKGQL (SEQ ID NO: 62),
or RMGSTSGSGKPGSGEGSTKGQL (SEQ ID NO: 63).
dephosphorylated by a speci?c phosphatase. Thus, the phos
phorylatable domain can be a synthetic peptide, a peptide
portion of a naturally-occurring kinase or phosphatase sub
strate, a peptidomimetic, a polynucleotide, or the like. By Way
of example, a PKC phosphorylatable domain may include an
amino acid sequence such as, for example, that set forth in
SEQ ID NO:37 or SEQ ID NO:44 and SEQ ID NOsz46-55,
Where SEQ ID NO: 44 is RERREQTLKIKAKA; SEQ ID
NO: 46 is KKKKKRESEKKSEKLSGESEKKNLL; SEQ ID
polypeptide linker adjacent the PKC substrate pho sphorylat
about 3 to about 50 amino acid residues, or betWeen about 4
to about 30 amino acid resdues, or betWeen about 5 to about
mutation).
35
40
A ?uorescent protein in a chimeric phosphorylation indic
NO: 47 is KKRESEKKEKL, SEQ ID NO: 48 is KRESSKKS
EKLSGESEKKNKKEA; SEQ ID NO: 49 is KRESSKKS
EKLSGESEKKSKKEA; SEQ ID NO: 50 is KKE
SSKKPEKLSGESER;
SEQ
ID
NO:
51
is
ETTSSEKKEETHGTSEKKSKEDD; SEQ ID NO: 52 is
KLESSSGLKKLSGKKQKGKRGGG; SEQ ID NO: 53 is
EGITPWASEKKMVTPKKRVRRPS; SEQ ID NO: 54 is
EGVSTWESEKRLVTPRKKSKSKL; and SEQ ID NO: 55 is
tor can be a green ?uorescent protein (GEP), a red ?uorescent
protein (REP), or a ?uorescent protein related to a GEP or an
RTPS.
REP, including a non-oligomeriZing ?uorescent protein. An
phosphorylated by a kinase in the phosphorylatable domain
REP, for example, can be a Discosoma REP or a ?uorescent
protein related to a Discosoma REP such as Discosoma
In other embodiments, the speci?c amino acid that can be
45
DsRed (SEQ ID NO:12) or a mutant thereof(SEQ ID NO: 12,
including an I125R mutation), or a non-oligomeriZing tan
dem DsRed containing, for example, tWo REP monomers
operatively linked by a peptide linker. For example, a non
oligomeriZing tandem REP can contain tWo DsRed (SEQ ID
of a chimeric phosphorylation indicator is not phosphory
lated, such that the indicator can be used to detect the presence
of the kinase in a sample. In other embodiments, the speci?c
amino acid that can be phosphorylated by a kinase in the
pho sphorylatable domain of a chimeric phosphorylation indi
50
cator is pho sphorylated, such that the indicator can be used to
detect the presence of a phosphatase in a sample. The speci?c
NO: 12) monomers or tWo mutant DsRed-I125R monomers
amino acid can be any amino acid that can be phosphorylated
operatively linked by a peptide having an amino acid
by a kinase or dephosphorylated by a phosphatase, for
example, serine, threonine, tyrosine, or a combination
thereof.
sequence as set forth as SEQ ID NO: 13.
A GEP useful in a chimeric phosphorylation indicator can
be an Aequorea GEP, a Renilla GEP, a Phialidium GEP, or a
55
The phosphoaminoacid binding domain (PAABD) in a
?uorescent protein related to an Aequorea GEP, a Renilla
chimeric phosphorylation indicator of the invention can be an
GEP, or a Phialidium GEP. A ?uorescent protein related to an
PAABD that speci?cally binds the particular phosphoami
Aequorea GEP, for example, canbe a cyan ?uorescent protein
(CEP), or a yelloW ?uorescent protein (YEP; e.g., citrine
noacid that is present in the indicator or that can be formed
60
(SEQ ID NO: 10 With Q69M)), or a variant (e.g., a spectral
variant) of CEP or YEP, including an enhanced GEP (EGEP;
SEQ ID NO:4), an enhanced CEP (ECEP; SEQ ID NO:6), an
ECEP(1-227) (amino acids 1 to 227 of SEQ ID NO:6), an
EYEP-V68L/Q69K (SEQ ID NO: 10), an enhanced YEP
(EYEP; SEQ ID NO:8), or other variant. In particular, a
?uorescent protein related to an Aequorea GEP, a Discosoma
due to phosphorylation of the indicator by a kinase. For
example, Where the phosphorylatable domain is a C-kinase
substrate domain, the phosphoaminoacid binding domain
may be a EHAI phosphothreonine binding domain from the
yeast checkpoint protein rad53p (SEQ ID NO: 56), or a EHA2
65
phosphothreonine binding domain from the yeast checkpoint
protein rad53p (SEQ ID NO: 57), and is preferably SEQ ID
NO: 57. The forkhead-associated (EHA) domain is a small
US 8,669,074 B2
5
6
protein module shown to recognize phosphothreonine
epitopes on proteins With a striking speci?city (Durocher et
a1., EEBS Letters 513158-66 (2002)).
A chimeric phosphorylation indicator speci?c for detect
phosphorylatable polypeptide, for example, in a hinge region
or a turn, provided the ability of the polypeptide to act as a
substrate is not disrupted.
In another embodiment, a chimeric phosphorylation indi
cator containing a phosphorylatable polypeptide and a ?uo
ing activity of a C-Kinase may be termed a “C-Kinase Activ
rescent protein further contains a pho sphoaminoacid binding
domain operatively linked to the phosphorylatable polypep
tide, Wherein the ?uorescent protein comprises an N-terminal
portion and a C-terminal portion, and Wherein the phospho
ity Reporter” (CKAR) and, for example, may be composed of
a CEP and aYEP ?anking a PKC substrate sequence tethered
by a ?exible linker to an EHA2 phosphothreonine binding
domain from the yeast checkpoint protein rad53p
rylatable polypeptide and operatively linked phosphoami
SEQUENCES. The CEP may be, for example, an mCEP and
the YEP may be, for example, an mYEP, Wherein an mCEP
and an mYEP are variants of CEP andYEP respectively hav
ing a mutation of an amino acid residue corresponding to
A206, L221, E223, or a combination thereofofSEQ ID N012,
for example, an A206K mutation, an L221K mutation, an
E223R mutation, or an L221K and E223R mutation of SEQ
protein. The ?uorescent protein can be any ?uorescent pro
tein, such as a non-oligomeriZing ?uorescent protein, includ
ID N012; or an A206K mutation, an L221K mutation, an
linked phosphoaminoacid binding domain can operatively
E223R mutation, or an L221K and E223R mutation of SEQ
ID N016 or SEQ ID N0110. Alternatively, or in addition, a
noacid binding domain is operatively inserted betWeen the
N-terminal portion and C-terminal portion of the ?uorescent
ing, a GEP, an REP, or a ?uorescent protein related to a GEP
or an REP. For example, the ?uorescent protein can be an
EYEP, and the pho sphorylatable polypeptide and operatively
20
CKAR may include a red ?uorescent protein, such as a non
oligomeriZing ?uorescent protein related to DsRed, such as,
for example, an I125R DsRed mutant (SEQ ID N0112,
including an I125R mutation).
A CKAR may be exempli?ed herein by a fusion protein
inserted betWeen an amino acid sequence corresponding to
amino acid positions 145 and 146 of the EYEP or can be
substituted for amino acid 145. A ?uorescent protein that is a
non-oligomeriZing ?uorescent protein may be, for example,
mCEP or mYEP, Wherein an mCEP and an mYEP are variants
25
of CEP and YEP respectively having a mutation of an amino
acid residue corresponding to A206, L221, E223, or a com
bination thereof of SEQ ID N012, for example, an A206K
containing, in an orientation from the amino terminus to
carboxy terminus, a CEP, a linker, a phosphoaminoacid bind
ing domain, a ?exible linker GGSGG (SEQ ID N01 45), an
mutation, an L221K mutation, an E223R mutation, or an
RERREQTLKIKAKA (SEQ ID N0144) phosphorylatable
mutation, an L221K mutation, an E223R mutation, or an
domain, a GGSGG (SEQ ID N0145) linker, and aYEP. For
example, the phosphoaminoacid binding domain may be an
L221K and E223R mutation of SEQ ID N012; or an A206K
30
EHAl (SEQ ID N01 56) or an EHA2 domain (SEQ ID N01
57). The phosphoaminoacid binding domain is preferably
EHA2 (SEQ ID N01 57). In more preferred embodiments,
CKAR may be exempli?ed herein by a fusion protein con
taining, in an orientation from the amino terminus to carboxy
terminus, mCEP, a linker, a EHA2 (SEQ ID N01 57) phos
phoaminoacid binding domain, a ?exible linker GGSGG
(SEQ ID N01 45), an RERREQTLKIKAKA (SEQ ID
N0144) phosphorylatable domain, a GGSGG (SEQ ID
35
L221K and E223R mutation of SEQ ID N016 or SEQ ID
N0110. In a further example, a non-oligomeriZing ?uorescent
protein may also be a mutant DsRed, Which has an amino acid
sequence of SEQ ID N0112, and including an I125R muta
tion.
The present invention also relates to polynucleotide encod
ing chimeric phosphorylation indicator, Which contains, in
operative linkage, a donor molecule, a phosphorylatable
domain, a phosphoaminoacid binding domain, and an accep
tor molecule, Wherein the phosphoaminoacid binding domain
40
speci?cally binds to a phosphoaminoacid When present in the
phosphorylatable domain, the donor molecule and the accep
N0145) linker, and mYEP, Where an mCEP and an MYEP are
tor molecule exhibit a detectable resonance energy transfer
as discussed above.
When the donor is excited, and the phosphorylatable domain
and phosphoaminoacid binding domain do not substantially
emit light to excite the acceptor. The donor and/or acceptor
The present invention also relates to a chimeric phospho
rylation indicator, Which contains a phosphorylatable
polypeptide and a ?uorescent protein. The speci?c amino
45
molecules may be, independently, ?uorescent proteins, such
as, for example, non-oligomeriZing ?uorescent proteins. In
acid that can be phosphorylated by a kinase in the phospho
rylatable polypeptide can be unphosphorylated, such that the
addition, the present invention relates to a polynucleotide
encoding a chimeric phosphorylation indicator containing a
indicator can be used to detect a kinase activity, or can be
phosphorylated, such that the indicator can be used to detect
50
a phosphatase activity.
In one embodiment of a chimeric phosphorylation indica
tor containing a phosphorylatable polypeptide and a ?uores
and C-terminal portion of the phosphorylatable polypeptide.
cent protein, the phosphorylatable polypeptide comprises an
N-terminal portion and a C-terminal portion, and the ?uores
cent protein is operatively inserted betWeen the N-terminal
phosphorylatable polypeptide and a ?uorescent protein,
Wherein the pho sphorylatable polypeptide includes an N-ter
minal portion and a C-terminal portion, and the ?uorescent
protein is operatively inserted betWeen the N-terminal portion
portion and C-terminal portion of the phosphorylatable
The present invention further relates to a polynucleotide
encoding a chimeric phosphorylation indicator containing a
phosphoaminoacid binding domain operatively linked to a
polypeptide. The ?uorescent protein can be any ?uorescent
protein, such as a non-oligomeriZing ?uorescent protein, and
may be, for example, a GEP, an REP, or a ?uorescent protein
Wherein the ?uorescent protein includes an N-terminal por
tion and a C-terminal portion, and Wherein the phosphorylat
55
phosphorylatable polypeptide and a ?uorescent protein,
60
related to a GEP or an REP, and can be in a circularly per
able polypeptide and operatively linked phosphoaminoacid
muted form. For example, a non-oligomeriZing ?uorescent
binding domain is operatively inserted betWeen the N-termi
nal portion and C-terminal portion of the ?uorescent protein.
protein may be, for example, an mGEP, an mCEP or an mYEP.
The phosphorylatable polypeptide can be any substrate for a
kinase, for example, a tyrosine kinase or a serine/threonine
kinase, including PKC, or for a phosphatase. The ?uorescent
protein can be operatively inserted into any region of the
65
Such a ?uorescent protein may be a non-oligomeriZing ?uo
rescent protein or other ?uorescent protein.
Also provided is a vector containing a polynucleotide of
the invention, including an expression vector, as Well as host
US 8,669,074 B2
7
8
cells that contain a polynucleotide of the invention or a vector
resonance energy transfer (FRET), Which may be used, for
containing such a polynucleotide. In one embodiment, a poly
nucleotide of the invention is operatively linked to an expres
example, to monitor the activity of PKC by real time imaging
of phosphorylation resulting from PKC activation.
sion control sequence, for example, a transcription regulatory
In another embodiment, a method for detecting a kinase or
element, a translation regulatory element, or a combination
phosphatase in a sample is performed by contacting the
thereof. In another embodiment, the polynucleotide is opera
tively linked to a nucleotide sequence encoding a membrane
translocating domain or a cell compartmentaliZation domain.
The present invention also relates to kits, Which contain at
sample With a chimeric phosphorylatable indicator contain
ing a phosphorylatable polypeptide and a ?uorescent protein,
determining a ?uorescence property in the sample, Wherein
the presence of kinase or phosphatase activity in the sample
least one chimeric phosphorylation indicator of the invention,
results in a change in the ?uorescence property as compared
or a polynucleotide encoding such an indicator. A kit of the
invention also can contain a plurality of different chimeric
to the ?uorescent property in the absence of a kinase or
phosphatase activity, thereby detecting the kinase or phos
phatase in the sample. The chimeric phosphorylation indica
phosphorylation indicators, or of encoding polynucleotides,
tor can contain a phosphorylatable polypeptide that includes
an N-terminal portion and a C-terminal portion, such that the
as Well as a combination thereof. Where a kit contains a
plurality of different chimeric pho sphorylation indicators, the
different indicators can contain different pho sphorylatable
?uorescent protein is operatively inserted betWeen the N-ter
domains, or different donor molecules or acceptor molecules
or both, or different ?uorescent proteins (such as ?uorescent
minal portion and C-terminal portion of the pho sphorylatable
proteins including at least one non-oligomeriZing ?uorescent
protein, or including different non-oligomeriZing ?uorescent
proteins), as appropriate to the chimeric phosphorylatable
polypeptide; or the chimeric phosphorylation indicator can
contain a phosphoaminoacid binding domain operatively
20
indicator. Where a kit contains a polynucleotide encoding a
chimeric phosphorylatable indicator, the polynucleotide can
other ?uorescent protein.
be in a vector, or in a host cell, or can be operatively linked to
one or more expression control sequences. Where a kit con
linked to a phosphorylatable polypeptide, Which is opera
tively inserted betWeen an N-terminal portion and a C-termi
nal portion of the ?uorescent protein. Such a ?uorescent
protein may be a non-oligomeriZing ?uorescent protein, or
25
The sample to be examined for kinase activity can be any
tains a plurality of different polynucleotides, the polynucle
sample, including, for example, a sample containing a syn
otides can encode a different chimeric phosphorylation indi
cator, or each can contain different expression control
sequences, or be contained in different vectors, particularly
thetic product to be examined for kinase or phosphatase activ
different expression vectors.
ity. In one embodiment, the sample is a biological sample,
Which canbe cell, tissue or organ sample, or an extract of such
30
The present invention further relates to a method for detect
a sample. In another embodiment, the method is performed
on an intact cell, Which can be in cell culture or can be in a
ing a kinase or phosphatase in a sample. In one embodiment,
a method of the invention is performed, for example, contact
tissue sample. For such a method, the chimeric phosphory
ing the sample With a chimeric phosphorylatable indicator,
cell compartmentaliZation domain that can target the chi
meric phosphorylatable indicator to a membrane (e.g., cell
membrane or an internal membrane), cytosol, endoplasmic
Which contains, in operative linkage, a donor molecule, a
latable indicator can contain a targeting sequence such as a
35
phosphorylatable domain, a phosphoaminoacid binding
reticulum, mitochondrial matrix, chloroplast lumen, medial
domain, and an acceptor molecule, Wherein the phosphoami
noacid binding domain speci?cally binds to a phosphoami
noacid When present in the phosphorylatable domain, the
donor molecule and the acceptor molecule exhibit a detect
able resonance energy transfer When the donor is excited, and
trans-Golgi cisternae, a lumen of a lysosome, or a lumen of an
endosome. A membrane targeting domain can be a particu
40
to or near to a cell membrane. A membrane translocating
the phosphorylatable domain and pho sphoaminoacid binding
domain can be a particularly useful cell compartmentaliZa
tion domain is a membrane translocating domain, Which can
domain do not substantially emit light to excite the acceptor;
exciting the donor molecule; and determining a ?uorescence
or luminescence property in the sample, such as ?uorescent
facilitate translocation of the chimeric phosphorylation indi
45
rylation indicator comprising a ?uorescent protein and a
phosphorylatable polypeptide can be unphosphorylated or
phosphorylated at an amino acid position speci?c for a kinase
energy transfer (LRET), Wherein the presence of a kinase or
phosphatase in the sample results in a change in the degree of
FRET or LRET, thereby detecting the kinase or phosphatase
50
be an increased amount of FRET or LRET, or can be a
decreased amount of FRET or LRET, and the change can be
indicative of the presence of a kinase in the sample, or, Where
the phosphorylatable domain is phosphorylated prior to con
tacting the sample With a chimeric phosphorylatable indica
tor, can be indicative of a phosphatase in the sample. Depend
ing on the particular structure of the chimeric
phosphorylation indicator as disclosed herein, FRET or
LRET can be increased or decreased due to phosphorylation
of the indicator by a kinase, and, likeWise, can be increased or
expressed in mammalian cells causes changes in ?uorescence
or a phosphatase, depending on Whether the method is for
detecting a kinase or phosphatase. A method of the invention
also can be used to detect an absence of kinase or phosphatase
55
activity in the sample, for example, due to the presence of a
kinase inhibitor or phosphatase inhibitor.
The present invention relates to a chimeric phosphoryla
tion indicator, Which contains, in operative linkage, a donor
molecule, a phosphorylatable domain, a phosphoaminoacid
binding domain, and an acceptor molecule, Wherein the phos
phoaminoacid binding domain speci?cally binds to a phos
60
decreased due to phosphorylation of the indicator by a phos
phatase. A change in FRET or LRET can be determined by
monitoring the emission spectrum of the acceptor. Geneti
cally encoded ?uorescent reporters for protein kinase C
(PKC) activity are provided herein that reversibly respond to
stimuli activating PKC. Pho sphorylation of the reporter
cator into an intact cell.
The phosphorylatable polypeptide in a chimeric phospho
resonance energy transfer (FRET) or luminescent resonance
in the sample. The change in the degree of FRET or LRET can
larly useful to target the chimeric phosphorylation indicators
phoaminoacid When present in the pho sphorylatable domain,
the donor molecule and the acceptor molecule exhibit a
detectable resonance energy transfer When the donor is
excited, and the phosphorylatable domain and phosphoami
noacid binding domain do not substantially emit light to
65
excite the acceptor. The donor molecule or the acceptor or
both can be a ?uorescent protein, such as, e.g., a non-oligo
meriZing ?uorescent protein, or a luminescent molecule, or a
US 8,669,074 B2
9
10
combination thereof. In one embodiment, each of the donor
molecule and the acceptor molecule is a ?uorescent protein,
such as, e.g., a non-oligomeriZing ?uorescent protein. In
another embodiment, one of the donor or acceptor molecule is
a luminescent molecule and the other is a ?uorescent protein,
such as, e.g., a non-oligomeriZing ?uorescent protein. In a
kinase/phosphatase substrate peptide also indicated (“sub
strate peptide”), and includes any spacers present in the con
struct. In-pointing and out-pointing arroWs indicate excita
tion and emission maxima; respectively, for the GFPs, though
the actual spectra are broader than the speci?c numbers
shoWn in the illustration.
FIG. 1A illustrates a CFP-PAABD-substrate-YFP chi
third embodiment, each of the donor molecule and acceptor
molecule is a luminescent molecule.
meric reporter protein, in Which phosphorylation of the sub
A luminescent molecule useful in a chimeric phosphory
lation indicator can be, for example, a lanthanide, Which can
be in the form of a chelate, including a lanthanide complex
strate can increase FRET.
FIG. 1B illustrates a CFP-substrate-YFP-PAABD chi
meric reporter protein, in Which phosphorylation can
containing the chelate. Thus, the luminescent molecule can
be a terbium ion (Tb3+) chelate, for example, a chelate of Tb3 +
and triethylenetetraamine hexaacetic acid (TTHA), and can
decrease FRET.
FIG. 1C illustrates a YFP(1-144)-peptide-PAABD-YFP
further include carbostyril 124 operatively linked to the Tb3+
chelate. Where the chimeric phosphorylation indicator is to
tion can modulate the YFP protonation state and emission
(146-238) chimeric reporter protein, in Which phosphoryla
intensity (shoWn here as an increase).
FIGS. 2A to 2C shoW the structures of various phospho
be contacted With a cell, for example, to detect the presence of
a kinase or phosphatase in the cell, the luminescent molecule
can further include a membrane translocating domain such as
that set forth as SEQ ID NO:18. Such a membrane translo
aminoacid-binding domains complexed to phosphorylated
peptides, fused together and bracketed by CFP and YFP to
20
The phosphorylatable domain in a chimeric phosphoryla
tion indicator of the invention can be any molecule that can be
form chimeric indicators as illustrated in FIG. 1A. The dark
gray lines represent the protein, With a feW key residues
involved in binding the peptide shoWn in stick form, and the
phosphopeptide shoWn in ball-and-stick representation.
cating domain or other molecule to be linked to the lumines
cent molecule can be operatively linked using, for example, a
tetracysteine motif such as that set forth in SEQ ID NO:17.
25
Heavy arroWs indicate linkers that can connect the protein to
the peptide or either of them to CFP or YFP (the arroW
threonine kinase, a tyrosine kinase, or PKC, or that can con
direction indicates amino to carboxy). The required length in
real space of the linker betWeen protein and peptide is indi
tain a phosphate group and can be dephosphorylated by a
cated. By rearrangement of these linkers (not shoWn), indi
phosphorylated by a speci?c kinase, for example, a serine/
speci?c phosphatase. Thus, the phosphorylatable domain can
be a synthetic peptide, a peptide portion of a naturally-occur
ring kinase or phosphatase substrate, a peptidomimetic, a
polynucleotide, or the like. By Way of example, a serine/
30
FIG. 2A shoWs the SH2 domain from phospholipase C-y
complexed to a phosphopeptide (D-(pY)-IIPLPD; SEQ ID
NO:14) from PDGF receptor (Pascal et al., Cell 77:461-472,
threonine kinase domain can include an amino acid sequence
as that set forth in SEQ ID NO:20 or SEQ ID NO:32, and a
tyrosine kinase phosphorylatable domain can include an
amino acid sequence as that set forth in SEQ ID NO:23 or
35
1994). This mitten-shaped SH2 domain Was the model for the
PAABD shoWn in FIG. 1.
FIG. 2B shoWs the PTB domain from Shc complexed With
a phosphopeptide HIIENPQ-(pY)-F (SEQ ID NO:15) from
TrkA (Zhou et al., Nature 378:584-592, 1995).
SEQ ID NO:25 . Another example of a serine/threonine kinase
domain is a PKC phosphorylatable domain.
The present invention also relates to a method for detecting
FIG. 2C shoWs the 14-3-3% domain complexed With the
a kinase inhibitor or phosphatase inhibitor. Such a method can 40
be performed, for example, by determining a ?rst ?uores
phosphopeptide ARSH-(pS)-YPA (SEQ ID NO:16; Yaffe et
al., Cell 91:961-971, 1997).
FIG. 3 provides a schematic structure of a circularly per
cence property of a chimeric phosphorylatable indicator in
the presence of a kinase or a phosphatase, contacting the
chimeric phosphorylatable indicator With a composition sus
pected of being a kinase inhibitor or a phosphatase inhibitor,
determining a second ?uorescence property of a chimeric
cators of the generic structures illustrated in FIGS. 1B and 1C
can be constructed analogously.
muted GFP (cpGFP; cylinder) inserted Within a protein
(clamshell) Whose conformation changes upon phosphoryla
45
tion. The tube (arc) at the top of the cpGFP cylinder indicates
a spacer linking the original N-terminus and C-terminus of
GFP. Linkers connecting the neW N-ter'minus and C-terminus
of the cpGFP to the insertion site Within the phosphorylatable
protein are indicated by tubes. The N-terminus and C-termi
phosphorylatable indicator in the presence of the composi
tion, Wherein a difference in the ?rst ?uorescence property
and second ?uorescence property identi?es the composition
as a kinase inhibitor or phosphatase inhibitor. Such a method 50 nus of the chimera are the same as those of the phosphorylat
able protein alone, and also are indicated by tubes. The circle
in the protein indicates the pho sphorylated amino acid. In this
example, phosphorylation favors a closed conformation,
Which pries open a cleft in the cpGFP, diminishing cpGFP
is particularly adaptable to high throughput screening meth
ods and, therefore, provides a means to screen libraries of
compounds to identify a composition that acts as a kinase
inhibitor or a phosphatase inhibitor. Accordingly, the present
phorylation-speci?c chimeric reporter proteins, Which
change ?uorescence upon phosphorylation. The ?uorescent
?uorescence.
FIGS. 4A and 4B illustrate detection by resonance energy
transfer of heterodimer formation betWeen proteins X andY
FIG. 4A shoWs GFP fused to X and the coral RFP fused to
Y. Proximity of X and Y promotes ?uorescence resonance
energy transfer (FRET) from GFP to RFP.
FIG. 4B shoWs a terbium chelate (“Tb3+ chelate”), With a
carbostyril antenna (“carbostyril), attached to X via a
GFPs, “CFP” and “YFP”, are indicated, as are the phospho
biarsenical ligand (“biarsenical”), binding to a “tetracysteine
invention also provides a kinase inhibitor or a phosphatase
55
inhibitor identi?ed by such method.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A to 1C illustrate three generic designs for phos
aminoacid binding domain (PAABD). The larger circle in the
PAABD indicates the phosphate-binding site, Which is rich in
Arg and Lys residues, and the smaller circle Within the larger
circle indicates phosphoaminoacids (see FIG. 6B). The
60
65
motif” fused to or inserted Within X. Proximity of X andY
promotes luminescence resonance energy transfer (LRET)
from Tb3+ to RFP. Detailed structures of the chelate, antenna,
and ligand are provided in FIG. 5A (note that they are much