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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 UsCl ' ' ' _ USPC """"" _ _ _ _ gig‘gggisg; _ _ ' ’ ’ ’ None 1, _ ?l f 1 h h_ See app lcanon e or Comp ete Seam lstory' 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) ' Int Cl 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 \u\‘6\8 P m 0‘ Time (Minutes) m mpwlv m0 53MM C9Hn t. 1 Total Protem :20 TMINm;5s .m. K69. n_4onm m 15m _ i .H .1 5K .0 F — m 0f W 4PNOn .w m m @ 5n w C a_ 3. 4 .7. ABC=26mw:=c9m5 Sx?E36Q.SE 26:.9“6583e.G53 eM m D .5PmK3 W_ rm m.B n MM0 M _ -4 am mmm Iv“ .wNn M % w .v m04 8642 m w =__.mor P 0\I NO. ePaw 0N5 n06. 0M 0O. NWTmd% F. Emission Wavelength (nm) F|GURE8 US. Patent Mar. 11,2014 Sheet 9 0f 14 US 8,669,074 B2 0.64 3 6:“ 5 0'62 5°° "M G669” ‘U5 (566983 forskoiin thapsigargin g 0.68 i ; 2 PDBu DMSO (306983 . 200 nM PDBu m 0.60 ,g 072 ‘ 5 g uEJ s g 0.64 a a § 0.58 tE g g 0.60 0.56 0 5 10 15 20 25 0 Time (Minutes) 5 10 ‘15 20 25 Time (Minutes) 1 o 2 g °-6°' “ g 0.95 2 OnM PDBu s 0 ss.‘2 E ' .3 200 nM PDBu L; 2 2 0.56‘ g E Q8 2 V = g. 0.54- Q9 E LU 0.85 C 100 nM calyculin A 3‘ 0.75 100 nM cal culin A y o - 0 0.7 5 10 15 20 0 Time (Minutes) ITI 2° 4° 60 Time (Minutes) 20' PDBu 40' Cal culin A S 5 : § 0 .: IL '17: E D. it 11 CD o 65 0.62 0 60 200 nM PDBu ‘o ' 10 pM histamine 100 nM calyculin A E g m ‘ “00/ Z 0.60 N“... Z 0 o 0 1o 20 Time (Minutes) 30 0‘550 s 10 15 2o Time (Minutes) FIGURE 9 25 US. Patent Mar. 11,2014 Sheet 10 0f 14 US 8,669,074 B2 & "w 5 U 1 0 u. M h .5 m .m e ou\I 0.0Oo2.mu6m1:t0o DvA>c2o?6=m>E1w O Mlw u.shlVwu w 4|n.|1 2 1. 9. 01 o1 O. r753211‘l1| O50 )121. m G5mMoiv59 q _ n * 0 m-w GO-0MTv D M 9M H %O H % 5PHn %M w B.mh T 2.m-OemWEF M 00u.3 5.m .m .m m. a m. ou 26 28 3) Time (Minutes) A wm .._.. m m w w m .41 m 4| 12 Time (Minutes) FIGURE 10 US. Patent Mar. 11,2014 Sheet 11 0f 14 A B w >\owmmcomwE US 8,669,074 B2 S mi4“ w. x. m. m .o.. 0o4| .m u m .m sm n w 8Ww m m E3$RE2.35 >5coz_wmEw m. m mm H E B 3Su.am2 5 o o e 10 pM histamine 40 Ss0e w m H 0.52 50 10 pM histamine 5 462 A 5. o5 S813a:m25 91 o O 12 Time (Minutes) 58 Time (Minutes) FIGURE 11 US. Patent Mar. 11,2014 Sheet 12 0f 14 0'69 " US 8,669,074 B2 10 pM histamine ' 1 .9 T“, I! > 0.67 - = c 2 g 9 .‘2 c E ‘ _ m 0.65 g g [I - 6 g 0.63 ~ 2 0.8 Ea LL l E >. 0.61 O I I 4 8 0.7 12 Time (Minutes) FIGURE 12 US. Patent Mar. 11,2014 Sheet 13 0f 14 US 8,669,074 B2 B 0 1IV . u. M -h .5 Al3 m .m e 0O.0n u 6. 2. 3. 4. 5. 7. O0 6. 4 31m35“:.95 12 Time (Minutes) 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