Patent application title: EMISSION RATIOMETRIC INDICATORS OF PHOSPHOINOSITIDES
Inventors:
Jin Zhang (Rosedale, MD, US)
Bharath Ananthanarayanan (Scotch Plains, NJ, US)
Assignees:
Johns Hopkins University
IPC8 Class: AC12Q148FI
USPC Class:
435 15
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving transferase
Publication date: 2011-07-07
Patent application number: 20110165602
Abstract:
Emission ratiometric indicators of phosphoinositides comprise a fusion
protein comprising a pleckstrin homology (PH) domain of Akt (also known
as protein kinase B) and a "pseudoligand" containing acidic amino acid
residues, which is sandwiched between resonance energy transfer (RET)
pairs, such as cyan and yellow mutants of GFP (a FRET pair). Such
indicators can be used, inter alia, to monitor spatiotemporal dynamics of
phosphoinositides and in high throughput assays for inhibitors of PI3K,
including drug screening assays.Claims:
1. A phosphoinositol (PI) indicator, wherein the PI indicator comprises:
(a) a first polypeptide comprising a polypeptide donor moiety of a FRET
pair; (b) a second polypeptide comprising a pleckstrin homology domain of
protein kinase B (SEQ ID NO:2), wherein the N terminus of the second
polypeptide is linked to the C terminus of the first polypeptide; (c) a
third polypeptide comprising a pseudoligand peptide sequence (SEQ ID
NO:3) for the protein kinase B, wherein the N terminus of the third
polypeptide is linked to the C terminus of the second polypeptide; and
(d) a fourth polypeptide comprising a polypeptide acceptor moiety of the
FRET pair, wherein the N terminus of the fourth polypeptide is linked to
the C terminus of the third polypeptide and wherein the polypeptide donor
moiety and the polypeptide acceptor moiety exhibit a detectable resonance
energy transfer when the donor moiety is excited.
2. The PI indicator of claim 1 wherein the FRET pair is selected from the group consisting of (1) a cyan fluorescent protein and a yellow fluorescent protein; and (2) a green fluorescent protein and a red fluorescent protein.
3. The PI indicator of claim 1, further comprising (e) a fifth polypeptide comprising a subcellular targeting peptide sequence linked to either the N terminus or the C terminus of the PI indicator.
4. A nucleic acid molecule encoding the PI indicator of claim 1.
5. A cell comprising the PI indicator of claim 1.
6. A method for measuring spatiotemporal phosphoinositide dynamics, comprising: (i) detecting a first resonance energy transfer of the PI indicator of claim 1 at a first time point or place; (ii) detecting a second resonance energy transfer of the PI indicator at a second time point or place; and (iii) comparing the first and the second resonance energy transfers, wherein a difference between the first and the second resonance energy transfers reflects a change in spatiotemporal phosphoinositide dynamics.
Description:
[0001] This application is a continuation of Ser. No. 11/826,519 filed on
Jul. 16, 2007, which claims the benefit of co-pending application Ser.
No. 60/830,811 filed Jul. 14, 2006. Both applications are incorporated
herein by reference in their entireties.
[0002] This application incorporates by reference a 25.8 kb text file created on Feb. 23, 2011 and named "sequencelisting.txt," which is the sequence listing for this application.
[0003] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIELD OF THE INVENTION
[0004] The invention relates to the monitoring of phosphoinositides in living mammalian cells.
BACKGROUND OF THE INVENTION
[0005] Ligand activation of the receptors in the plasma membrane initiates cell signaling which propagates into various intracellular compartments through multi-step processes that depend on cellular second messengers. It has been shown that these short lived small molecules not only amplify the signal in the relay but also confer specificity by their temporal and spatial distribution in the cell.
[0006] Although a relatively minor component of cellular membranes, phosphoinositides (PIs) are emerging to be a crucial component and a diverse family of lipid second messengers (1). The family's diversity stems from the existence of multi-phosphorylated states at the D-3, -4 and -5 positions, yielding seven distinct signaling molecules identified to date (2). In particular, phosphatidylinositol 3-kinase (PI3K) synthesizes four species of D-3 phosphorylated inositides, namely phosphatidylinositol 3,4,5 triphosphate (PIP3; also known as "PI(3,4,5)P3"), PI(3,4)P2, PI(3,5)P2 and PI(3)P (3). On the other hand, they are dephosphorylated by the 3'-phosphatase called phosphatase and tensin homologue deleted on chromosome ten (PTEN) and some by 5'-phosphatases (4, 5). PIP3 and PI(3,4)P2 are responsible for the recruitment of the serine/threonine kinase Akt, also known as and referred to herein as protein kinase B (PKB), to the plasma membrane where phosphorylation at two sites, T308 and S473 (in Akt1), fully activates the kinase (6, 7).
[0007] Upon activation Akt plays important roles in various cellular processes such as proliferation, differentiation, survival, and tumorigenesis (8). For example, activation of Akt by insulin or growth factors is prevented if the cells are preincubated with inhibitors of PI 3-kinase, the best known being Wortmannin or LY 294002, or by overexpression of a dominant negative mutant of PI 3-kinase (44). Further, mutation of the tyrosine residues in the PDGF receptor that when phosphorylated bind to PI 3-kinase also prevent the activation of PKB.sub.α, an isoform of PKB. Recent reports have shown the PKB is itself activated by another kinase also downstream of PIP3 (45). This kinase, termed PKB kinase, or phosphatidylinositide (PtdIns) 3-kinase (PDK1), requires PIP3 for activation (46).
[0008] Akt also is involved in regulating cell growth. It has been implicated in certain human cancers; for instance, it is known to be amplified in a percentage of ovarian carcinomas, breast carcinomas, and pancreatic carcinomas (47; 48). The amplification of the enzyme affords tumor cells a mechanism to circumvent apoptosis. Drugs that inhibit PKB activity are useful for treating diseases involving inappropriate cell growth, including cancer.
[0009] Due to the involvement of these PIs in such diverse functions, a cell must precisely control their spatial and temporal dynamics to avoid abnormalities, yet there are no reliable methods available for measuring PIP3, PI(3,4)P2 dynamics within subcellular compartments with high spatiotemporal resolution. Previously employed indicators cause artifacts due to cell movements and additionally cannot be targeted to subcellular locations. Biochemical methods of radiolabeling and cell fractionation measure total PI levels and have poor spatial and temporal resolution, and use of specific antibodies (31) to immunodetect PIs in fixed cells provides only snapshots at different time points. For dynamic monitoring of the lipid molecules, live-cell imaging using PH domains fused with fluorescent proteins (10-13, 32, 33) has been widely used, however their dependency on translocation limits their abilities to examine different pools of PIs.
[0010] There are ratiometric sensors for monitoring PI dynamics. On such sensor, termed "fllip" (34), employs a PH domain of GRP1 and required membrane anchoring to facilitate a PIP3 binding induced conformational change via rotation of rigid linkers around a diglycine hinge engineered within the construct, which limits the targetability of the reporter and generalizability of the design. Another ratiometric sensor, termed "CAY," is based on a peptide from Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding (35). CAY is a sensor for polyphosphorylated PIs showing some preference for PIP3 in cells.
[0011] There is a need in the art for sensitive, targetable indicators that would permit direct measurement and assessment of specific signaling of 3'-phosphoinositides.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIGS. 1A-G. Development of InPAkt. FIG. 1A, cartoon depicting the conformational change upon PI binding, yielding a FRET response. FIG. 1B, domain structure of the construct showing the restriction sites linking individual components. These are a SphI site (GCATGC), shown in the sequence GCATGCAT; a PstI site (CTGCAG), shown in the sequence TCTGCAGGCGGTAGC (SEQ ID NO:22, encoding SEQ ID NO:23); and a Sac I site (GAGCTC), shown in the sequence GGCGGCAGCGAGCTC (SEQ ID NO:24, encoding SEQ ID NO:25). FIG. 1C, anti-GFP Western blot of InPAkt expressed in HEK293 cells indicating the right size of the reporter. FIG. 1D, FRET response of InPAkt expressed in NIH3T3 cells. Yellow fluorescence images show membrane translocation of InPAkt upon PDGF stimulation. Pseudocolor images indicate the emission ratio change at various time points after PDGF stimulation. FIG. 1E, representative emission ratio time courses of InPAkt (n=9) and the PH domain mutant R23A/R25A (n=2), both stimulated with 50 ng/ml PDGF. InPAkt showed a response of 25.4±3.7% [average±std] (n=9). FIG. 1F, representative emission ratio time course from two independent experiments showing that the response of InPAkt is PI3K specific. NIH3T3 cells were pretreated with 20 μM of PI3K inhibitor LY294002, followed by stimulation with 50 ng/ml PDGF, gentle washing, and treatment with PDGF again. FIG. 1G, representative emission ratio time course shows the reversibility of the reporter. NIH3T3 cells expressing InPAkt were stimulated with PDGF, followed by treatment with 20 μM of LY294002 (n=5).
[0013] FIGS. 2A-C. Comparison of the cellular responses stimulated by various growth factors. FIG. 2A, representative time courses of InPAkt responses in cells stimulated by 50 ng/ml PDGF (blue), 50 nM IGF-1 (yellow), 100 ng/ml insulin (green) (n=3). FIG. 2B, representative emission ratio time courses showing the FRET response of a NIH3T3 cell expressing InPAkt sequentially stimulated by insulin, IGF-1 and PDGF (n=3). FIG. 2C, pseudocolor images showing InPAkt responses with sequential stimulation by insulin, IGF-1 and PDGF.
[0014] FIGS. 3A-D. Fusions of InPAkt targeted to various subcellular locations. FIG. 3A, domain structure of the fusion constructs. FIG. 3B, localization of plasma membrane targeted InPAkt (pm InPAkt) is shown in the fluorescence image (YFP). Pseudocolor images show colocalization of nuclear targeted reporter (NLS InPAkt) with a cell-permeable DNA dye, Hoechst 3342. FIG. 3C, representative emission ratio time course from four independent experiments showing the response of plasma membrane targeted InPAkt stimulated with 50 ng/ml PDGF (9.25±0.4%), followed by addition of 20 μM LY294002. FIG. 3D, representative emission ratio time course from 3 different trials for NLS InPAkt in NIH3T3 cells stimulated with PDGF.
[0015] FIGS. 4A-D. Simultaneous imaging of plasma membrane targeted InPAkt (pm InPAkt) and nuclear targeted BKAR (NLS BKAR). FIG. 4A, domain structure of NLS BKAR.
[0016] FIG. 4B, pseudocolor images show colocalization of NLS BKAR with Hoechst 3342. FIG. 4C, cellular distribution of the two reporters in a HEK293 cell. FIG. 4D, representative emission ratio time courses from four different experiments for pm InPAkt and NLS BKAR in the same cell stimulated with 50 nM IGF-1.
[0017] FIGS. 5A-B. Responses of InPAkt indicator for phosphoinositides based on Akt, upon microinjection of IP4 and PI(3,4,5)P3. FIG. 5A, Microinjection of IP4 into REF-52 cells expressing InPAkt. Pseudocolor images and emission ratio time courses show the change in the yellow-to-cyan emission ratio of InPAkt upon 1 mM IP4 microinjection. IP4 (AG Scientific) was dissolved in water to a final concentration of 1 mM, and this solution was used to load the microinjection tip. Shown in the graph are the responses of two individual cells imaged simultaneously but injected at two different time points (cell 1 in black and cell 2 in red). InPAkt showed 6.6+0.64% (n=5) increase in yellow/cyan emission ratio upon IP4 injection. REF-52 cells were used for ease of microinjection, particularly nuclear injection. FIG. 5B, microinjection of PI(3,4,5)P3 into REF-52 cells expressing InPAkt. Pseudocolor images and emission ratio time courses show the FRET response of the reporter upon 5 mM dioctanoyl PI(3,4,5)P3 microinjection. Dioctanoyl PI(3,4,5)P3 (Echelon Biosciences) was dissolved in water, and solutions with final concentrations of 1-5 mM were loaded in the microinjection tip. Higher concentrations of PIP3 could not be used for microinjection experiments because of aggregation of the lipid thus clogging of the tip. InPAkt showed 4.1+0.40% (n=3) increases in yellow-to-cyan emission ratio upon PIP3 injection. Control experiments injecting water or buffers showed no change in the emission ratios. In some microinjection experiments, rhodamine B dextran (MW 10,000) was mixed with phosphoinositides, and the presence of red fluorescence in the cells after microinjection indicated successful injection.
[0018] FIG. 6. Basal levels of PI(3,4)P2 and PI(3,4,5)P3 at the plasma membrane. Shown is a representative emission ratio time course of membrane-targeted InPAkt treated with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 from two independent experiments. NIH 3T3 cells expressing plasma membrane-targeted InPAkt (pm InPAkt) were directly treated with PI3K inhibitor without prior stimulation with platelet-derived growth factor (PDGF). The decrease in emission ratio upon LY294002 addition confirmed the presence of high basal levels of PI(3,4)P2 and PI(3,4,5)P3 at the plasma membrane.
[0019] FIGS. 7A-B. Responses of nuclear-targeted InPAkt upon microinjection of IP4 and PI(3,4,5)P3. FIG. 7A, microinjection of IP4 into REF-52 cells expressing nuclear localization signal (NLS) InPAkt. Pseudocolor images and representative emission ratio time courses show the response of the nuclear-localized InPAkt (NLS InPAkt) upon nuclear injection of 1 mM IP4 (from AG Scientific). NLS InPAkt showed 9.1+0.42% (n=2) increases in yellow/cyan emission ratio upon IP4 injection. FIG. 7B, microinjection of PI(3,4,5)P3 into REF-52 cells expressing NLS InPAkt. Pseudocolor images and representative emission ratio time courses show the response of NLS InPAkt upon nuclear injection of 5 mM dioctanoyl PI(3,4,5)P3 (Echelon Biosciences). NLS InPAkt showed 5.4+2.2% (n=3) increases in yellow/cyan emission ratio upon PI(3,4,5)P3 injection.
[0020] FIGS. 8A-C. Binding measurement of targeting sequence-tagged fusion constructs. FIG. 8A, domain structure of PH Akt fusion constructs containing pseudoligand and the targeting sequences. All of the constructs were cloned into pRSET B bacterial expression vector with a N-terminal His6-tag. Proteins were purified by using Ni-NTA affinity chromatography (Qiagen) according to the manufacturer's protocol. The purified proteins were further dialyzed in 20 mM Tris.HCl buffer (pH 7.4) containing 0.16 M KCl. The purity of all of the proteins was checked by SDS protein gel electrophoresis. Concentrations of the proteins were determined by using the BCA assay. FIG. 8B, representative sensograms obtained from Biacore 2000 surface plasmon resonance. All of the four flow channels of the Pioneer L1 sensor chip were coated with large unilamellar vesicles containing four different lipid mixtures. Vesicles containing phospholipid compositions of mainly 70% of dioleoyl phosphatidylcholine (PC) and 30% diacyl phosphatidyl serine (PS) from porcine brain (Avanti Polar lipids) were made with additional 3% of the three different dipalmitoyl phosphatidylinositol ligands, namely PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3 (Echelon Biosciences). Large unilamellar vesicles were made by using an extruder in buffer containing 20 mM Tris.HCl (pH 7.4) and 0.16 M KCl. Experiments were performed at 25° C. After each injection, the immobilized vesicle surface on all of the flow channels was regenerated by using 10 ml of 50 mM NaOH for subsequent measurements. For data acquisition, four or more different concentrations of the protein were used. (Each sensogram shown in this figure has been normalized to zero to facilitate comparison between the responses of different lipid surfaces.) FIG. 8C, dissociation constants for PH Akt domain fusion constructs. Dissociation constants in the table represent the mean and standard deviation from four different measurements. All measurements were performed in buffer containing 20 mM Tris.HCl (pH 7.4) and 0.16 M KCl. Data were analyzed by using BIAEVALUATION 3.2 software (BIACORE®). Surface immobilized with vesicles containing PC:PS but no phosphatidylinositol was used as a control surface for background subtraction to eliminate any refractive index changes due to injections or buffer changes. Binding parameters for the PH domain constructs for PI(4,5)P2 could not be evaluated in that no discernable difference was detected between binding to the PI(4,5)P2 containing lipid surface and the control surface containing no phosphoinositides.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention provides targetable phosphoinositide (PI) indicators. PI indicators of the invention have some unique advantages over previous methods for assessing phosphoinositide dynamics inside cells. They offer dual readout of RET change and translocation in their untargeted forms. The conformational change does not rely on any membrane anchoring, which provides the flexibility of targeting the reporters to different subcellular regions or tethering them to signaling components to track specific pools of PIs. They do not require large amounts of cells or tissue, provides high spatial and temporal resolution and eliminates artifacts caused by cell shape changes. In addition, they can be targeted to different subcellular sites or fused to signaling components and have the flexibility of monitoring specific pools of PIs and measuring PI changes within various subcellular compartments.
[0022] PI indicators of the invention are useful, inter alia, in high throughput assays for inhibitors of PI3K, including drug screening assays, and for diagnostic analysis of potential cancerous tissues. Furthermore, it may offer a generalizable design that relies on the binding of PIs to compete off a concatenated pseudoligand to generate the conformational change that leads to the RET change.
[0023] Components of PI Indicators
[0024] In some embodiments phosphoinositide indicators of the invention comprise a fusion protein comprising a pleckstrin homology (PH) domain of Akt (also known as protein kinase B) and a "pseudoligand" containing acidic amino acid residues, which is sandwiched between resonance energy transfer (RET) pairs, such as cyan and yellow mutants of GFP (a FRET pair). The pseudoligand portion of the indicator can be linked to either the N terminus or the C terminus the PH domain portion.
[0025] The pleckstrin homology (PH) domain of Akt binds specifically to two of the major PI3K products, PIPS and PI(3,4)P2 (18, 19), and can be used in PI indicators of the invention to detect PI3K activity by binding to these products. Other phosphoinositide binding domains, such as phox homology (PX), FYVE domains, or PH domains from other proteins can be used to detect other products, such as PI(4,5)P2, PI(3)P, etc. See (49).
[0026] Pseudoligand
[0027] The pseudoligand is a moiety (e.g., a peptide or small protein, or a compound) which binds to the same PH domain to which the respective PI binds; the binding sites on the PH domain to which the pseudoligand and the PI bind can be the same or different. The presence of the actual ligand (the PI) competes for binding with the pseudoligand or alter the binding with the pseudoligand. When the binding with the pseudoligand is changed, a conformational change is generated in the sensor containing the pseudoligand and the PH domain, which generates a RET response.
[0028] The amino acid sequence shown in SEQ ID NO:3 is a suitable pseudoligand for the PH domain of PKB/Akt that binds to PIP3 and PI(3,4)P2. Minor modifications of this amino acid sequence (e.g., VAEEDDDEEEDEDD; SEQ ID NO:17) can be made as long as the pseudoligand retains its ability to bind to the PH domain.
[0029] Subcellular Targeting Sequences
[0030] PI indicators of the invention can include a subcellular targeting sequence which can target an indicator to a subcellular domain such as a plasma membrane, a nuclear membrane, or other subcellular locations such as endoplasmic reticulum, Golgi apparatus, mitochondria, mitochondrial matrix, lysosomal lumen, or endosomal lumen. Many such targeting sequences are known in the art. Targeting sequences are known in the art. Examples include the plasma membrane targeting sequence shown in SEQ ID NO:4, the signal for geranylgeranylation (GerGer), a COOH-terminal CLLL from Rho; PalmPalm, an NH2-terminal MLCCMRRTKQ (SEQ ID NO:6) from Gap43; MyrPalm, an NH2-terminal MGCIKSKRKDNLNDDE (SEQ ID NO:7) from Lyn kinase; the nuclear localization signal sequence shown in SEQ ID NO:5, the mitochondrial localization sequence shown in SEQ ID NO:8, and the mitochondrial matrix targeting signal shown in SEQ ID NO:9.
[0031] Targeting sequences can be linked to PI indicators using, for example, a tetracysteine motif such as Cys Cys Xaa Xaa Cys Cys (SEQ ID NO:10). Targeting sequences can be linked at either the N- or C-terminus of a PI indicator (or a polypeptide portion of the PI indicator) or at intermediate points in the indicator.
[0032] Resonance Energy Transfer Pairs (Donor and Acceptor Moieties)
[0033] A resonance energy transfer pair (RET pair) contains a donor moiety and an acceptor moiety. As used here, a "donor moiety" is a fluorophore or a luminescent moiety. The absorption spectrum of the "acceptor moiety" overlaps the emission spectrum of the donor moiety. The acceptor moiety does not need to be fluorescent and can be a fluorophore, chromophore, or quencher. In some embodiments both the donor and acceptor moieties are fluorescent proteins. In other embodiments both the donor and acceptor moieties are luminescent moieties. In yet other embodiments, either one of the donor or acceptor moieties can be a fluorescent protein while the other moiety is a luminescent moiety. In other embodiments, the acceptor moiety is a "quencher moiety."
[0034] When both the donor and acceptor moieties are fluorophores, resonance energy transfer is detected as "fluorescence resonance energy transfer" (FRET). If a luminescent moiety is involved, resonance energy transfer is detected as "luminescent resonance energy transfer "(LRET). LRET includes "bioluminescent resonance energy transfer" (BRET; Boute et al., Trends Pharmacol. Sci. 23, 351-54, 2002; Ayoub et al., J. Biol. Chem. 277, 21522-28, 2002). Because excitation of the donor moiety does not require exogenous illumination in an LRET method, such methods are particularly useful in live tissue and animal imaging, because penetration of the excitation light is no longer a concern. LRET methods have a high contrast and high signal-to-noise ratio; 2) no photobleaching occurs; and 3) quantification is simplified because the acceptor moiety is not directly excited.
[0035] Suitable acceptor moieties include, for example, a coumarin, a xanthene, a fluorescein, a fluorescent protein, a circularly permuted fluorescent protein, a rhodol, a rhodamine, a resorufin, a cyanine, a difluoroboradiazaindacene, a phthalocyanine, an indigo, a benzoquinone, an anthraquinone, an azo compound, a nitro compound, an indoaniline, a diphenylmethane, a triphenylmethane, and a zwitterionic azopyridinium compound.
[0036] Suitable donor moieties include, but are not limited to, a coumarin, a xanthene, a rhodol, a rhodamine, a resorufin, a cyanine dye, a bimane, an acridine, an isoindole, a dansyl dye, an aminophthalic hydrazide, an aminophthalimide, an aminonaphthalimide, an aminobenzofuran, an aminoquinoline, a dicyanohydroquinone, a semiconductor fluorescent nanocrystal, a fluorescent protein, a circularly permuted fluorescent protein, and fluorescent lanthanide chelate.
[0037] Fluorescent Proteins
[0038] In some preferred embodiments either or both of the donor and acceptor moieties is a fluorescent protein. Suitable fluorescent proteins include green fluorescent proteins (GFP), red fluorescent proteins (RFP), yellow fluorescent proteins (YFP), and cyan fluorescent proteins (CFP). Useful fluorescent proteins also include mutants and spectral variants of these proteins which retain the ability to fluoresce.
[0039] RFPs include Discosoma RFPs, such Discosoma DsRed (SEQ ID NO:11) or a mutant thereof which includes an Ile125Arg mutation, or a non-oligomerizing tandem DsRed containing, for example, two RFP monomers linked by a peptide linker For example, a non-oligomerizing tandem RFP can contain two DsRed monomers or two mutant DsRed-I125R monomers linked by a peptide (having, for example, the amino acid sequence shown in SEQ ID NO:16).
[0040] Useful GFPs include an Aequorea GFP (e.g., SEQ ID NO:12), a Renilla GFP, a Phialidium GFP, and related fluorescent proteins for example, a cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), or a spectral variant of the CFP or YFP. CFP (cyan) and YFP (yellow) are color variants of GFP. CFP and YFP contain 6 and 4 mutations, respectively. They are Tyr66Try, Phe66Leu, Ser65Thr, Asn145Ile, Met153Thr, and Val163Ala in CFP and Ser65Gly, Val168Leu, Ser72Ala, and Thr203Tyr. Spectral variants include an enhanced GFP (EGFP; SEQ ID NO:13), an enhanced CFP (ECFP; SEQ ID NO:14), an enhanced YFP (EYFP; SEQ ID NO:15), and an EYFP with V68L and Q69K mutations. Other examples of fluorescent proteins comprising mutations are Aequorea GFP with one or more mutations at amino acid residues A206, L221 or F223 of SEQ ID NO:12 (e.g., mutations A206K, L221K, F223R, Q80R); mutations L221K and F223R of ECFP (SEQ ID NO:13), and EYFP-V68L/Q69K of SEQ ID NO:12. See also US 2004/0180378; U.S. Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079; Chalfie et al., Science 263:802-805, 1994. The truncated ECFP shown in SEQ ID NO:18 (which can be encoded by the nucleotide sequence shown in SEQ ID NO:19) is especially useful.
[0041] Other useful GFP-related fluorescent proteins include those having one or more folding mutations, and fragments of the proteins that are fluorescent, for example, an A. victoria GFP from which the two N-terminal amino acid residues have been removed. Several of these fluorescent proteins contain different aromatic amino acids within the central chromophore and fluoresce at a distinctly shorter wavelength than the wild type GFP species. For example, the engineered GFP proteins designated P4 and P4-3 contain, in addition to other mutations, the substitution Y66H; and the engineered GFP proteins designated W2 and W7 contain, in addition to other mutations, Y66W.
[0042] Folding mutations in Aequorea GFP-related fluorescent proteins improve the ability of the fluorescent proteins to fold at higher temperatures and to be more fluorescent when expressed in mammalian cells, but have little or no effect on the peak wavelengths of excitation and emission. If desired, these mutations can be combined with additional mutations that influence the spectral properties of GFP to produce proteins with altered spectral and folding properties, and, particularly, with mutations that reduce or eliminate the propensity of the fluorescent proteins to oligomerize. Folding mutations, with respect to SEQ ID NO:12, include the substitutions F64L, V68L, S72A, T44A, F99S, Y145F, N1461, M153T, M153A, V163A, I167T, S175G, 5205T, and N212K.
[0043] Luminescent Moieties
[0044] Luminescent moieties useful in a PI indicator include lanthanides, which can be in the form of a chelate, including a lanthanide complex containing the chelate (e.g, β-diketone chelates of cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, or ytterbium). Lanthanide chelates are well known in the art. See Soini and Kojola, Clin. Chem. 29, 65, 1983; Hemmila et al., Anal. Biochem. 137, 335 1984; Lovgren et al., In: Collins & Hoh, eds., Alternative Immunoassays, Wiley, Chichester, U.K., p. 203, 1985; Hemmila, Scand. J. Clin. Lab. Invest. 48, 389, 1988; Mikola et al., Bioconjugate Chem. 6, 235, 1995; Peruski et al., J. Immunol. Methods 263, 35-41, 2002; U.S. Patent 4,374,120; and U.S. Pat. No. 6,037,185. Suitable β-diketones are, for example, 2-naphthoyltrifluoroacetone (2-NTA), 1-naphthoyltrifluoroacetone (1-NTA), p-methoxybenzoyltrifluoroacetone (MO-BTA), p-fluorobenzoyltrifluoroacetone (F-BTA), benzoyltrifluoroacetone (BTA), furoyltrifluoroacetone (FTA), naphthoylfuroylmethane (NFM), dithenoylmethane (DTM), and dibenzoylmethane (DBM). See also US 20040146895.
[0045] Luminescent proteins include, but are not limited to, lux proteins (e.g., 1uxCDABE from Vibrio fischerii), luciferase proteins (e.g., firefly luciferase, Gaussia luciferase, Pleuromamma luciferase, and luciferase proteins of other beetles, Dinoflagellates (Gonylaulax; Pyrocystis), Annelids (Dipocardia), Molluscs (Lativa), and Crustacea (Vargula; Cypridina), and green fluorescent proteins of bioluminescent coelenterates (e.g., Aequorea Victoria, Renilla mullerei, Renilla reniformis; see Prendergast et al., Biochemistry 17, 3448-53, 1978; Ward et al., Photochem. Photobiol. 27, 389-96, 1978; Ward et al., J. Biol. Chem. 254, 781-88, 1979; Ward et al., Photochem. Photobiol. Rev 4, 1-57, 1979; Ward et al., Biochemistry 21, 4535-40, 1982). Many of these proteins are commercially available. Firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind. Recombinantly produced firefly luciferase is available from Promega Corporation, Madison, Wis. Jellyfish aequorin and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.
[0046] The DNA sequences of the aequorin and other luciferases employed for preparation of some PI indicators of the invention can be derived from a variety of sources. For example, cDNA can be prepared from mRNA isolated from the species disclosed above. See Faust, et al., Biochem. 18, 1106-19, 1979; De Wet et al., Proc. Natl. Acad. Sci. USA 82, 7870-73, 1985.
[0047] Luciferase substrates (luciferins) are well known and include coelenterazine (available from Molecular Probes, Eugene, Oreg.) and ENDUREN®. These cell-permeable reagents can be directly administered to cells, as is known in the art. Luciferin compounds can be prepared according to the methods disclosed by Hori et al., Biochemistry 14, 2371-76, 1975; Hori et al., Proc. Natl. Acad. Sci. USA 74, 4285-87, 1977).
[0048] Dark Quenchers
[0049] In some embodiments the acceptor moiety is a quencher moiety, preferably a "dark quencher" (or "black hole quencher") as is known in the art. In this case, the change in conformation which occurs upon PI binding eliminates quenching, resulting in an increase in energy emission from the donor moiety. "Dark quenchers" themselves do not emit photons. Use of a "dark quencher" reduces or eliminates background fluorescence or luminescence which would otherwise occur as a result of energy transfer from the donor moiety. Suitable quencher moieties include dabcyl (4-(4'-dimethylaminophenylazo)-benzoic acid), QSY®-7 carboxylic acid, succinimidyl ester (N,N'-dimethyl-N,N'-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbo- n yl) piperidinylsulfone-rhodamine (a diarylrhodamine derivative from Molecular Probes, Eugene, Oreg.). Suitable quencher moieties are disclosed, for example, in US 2005/0118619; US 20050112673; and US 20040146959.
[0050] Any suitable fluorophore may be used as the donor moiety provided its spectral properties are favorable for use with the chosen dark quencher. The donor moiety can be, for example, a Cy-dye, Texas Red, a Bodipy dye, or an Alexa dye. Typically, the fluorophore is an aromatic or heteroaromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, coumarin, a fluorescein (e.g., fluorescein, tetrachlorofluorescein, hexachlorofluorescein), rhodamine, tetramethyl-rhodamine, or other like compound. Suitable fluorescent moieties for use with dark quenchers include xanthene dyes, such as fluorescein or rhodamine dyes, including 6-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX). Suitable fluorescent reporters also include the naphthylamine dyes that have an amino group in the alpha or beta position. For example, naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).
[0051] Other suitable fluorescent moieties include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridin-e and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-1-carboxy-pentyl)-3'-ethyl-5,5'-dimethy-loxacarbocyanine (CyA); 1H,5H,1H,15H-Xantheno[2,3,4-ij:5,6,7-i'j']diquinol-izin-18-ium, 9-[2(or 4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or 2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahyd-ro-inner salt (TR or Texas Red); BODIPY® dyes; benzoxaazoles; stilbenes; pyrenes; and the like.
[0052] Nucleic Acid Molecules Encoding PI Indicators
[0053] In some embodiments, PI indicators contain only protein components. Such fusion proteins can be expressed recombinantly, and the invention provides nucleic acid molecules for this purpose. A nucleic acid molecule encoding a PI indicator can comprise any nucleotide sequence which encodes the amino acid sequence of the indicator. Nucleic acid molecules of the invention include single- and double-stranded DNA (including cDNA) and mRNA. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
[0054] In some embodiments the nucleic acid molecules are expression constructs which contain the necessary elements for the transcription and translation of an inserted coding sequence encoding a PI indicator. Expression constructs can be used as vectors for introducing PI indicators into cells. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding PI indicators and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.
[0055] Expression vectors of the invention can be expressed in a variety of host cells. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems, particularly mammalian systems, including human systems. See WO 01/98340, which is incorporated herein by reference in its entirety. The choice of vector components and appropriate host cells is well within the capabilities of those skilled in the art.
[0056] Alternatively, protein or non-protein donor and/or acceptor moieties can be linked to the polypeptide by covalent attachment. There are a variety of methods known in the art which are useful for this purpose. For example, the attachment can be direct, via a functional group on the polypeptide (e.g., amino, carboxyl and sulfhydryl groups) and a reactive group on the fluorophore. Free amino groups in the polypeptide can be reacted with fluorophores derivatized with isothiocyanate, maleic anhydride, N-hydroxysuccinimide, tetrafluorylphenyl and pentafluoryl esters. Free carboxyl groups in the polypeptide can be reacted with carbodiimides such as 1-ethyl-3-[dimethylaminopropyl]carbodiimide hydrochloride to create a reactive moiety that will react with an amine moiety on the donor or acceptor moiety. Sulfhydryl groups can be attached to donor or acceptor moieties modified with maleimide and iodoacetyl groups, although such linkages are more susceptible to reduction than linkages involving free amino groups. The polypeptide can also be linked indirectly via an intermediate linker or spacer group, using chemical groups such as those listed above.
[0057] It is also possible to produce PI indicators of the invention using chemical methods to synthesize the amino acid sequence of the polypeptide and, optionally, one or more fluorescent or luminescent proteins. Methods include direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of polypeptide portions of PI indicators can be separately synthesized and combined using chemical methods to produce a full-length indicator molecule. See WO 01/98340.
[0058] Delivery of PI Indicators to Cells
[0059] PI indicators of the invention can be introduced into cells in vitro using reversible permeabilization techniques. See U.S. Pat. No. 6,127,177; U.S. Pat. No. 6,902,931; Russo et al., Nature Biotechnology 15, 278-82, March 1997; Santangelo et al., Nucleic Acids Res. 32, 1-9, Apr. 14, 2004.
[0060] If the PI indicator is a fusion protein, expression vectors comprising a PI indicator-encoding nucleotide sequence can be transfected into any cell in vitro in which it is desired to monitor PI3K activity or PI distribution. Any transfection method known in the art can be used, including, for example, including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.
[0061] Useful vectors and methods of delivering the vectors to cells in vivo are disclosed, for example, in U.S. Pat. No. 6,825,012; U.S. Pat. No. 6,878,549; U.S. Pat. No. 6,645,942; U.S. Pat. No. 6,692,737; U.S. Pat. No. 6,689,758; U.S. Pat. No. 6,669,935; and U.S. Pat. No. 6,821,957.
[0062] Methods of Detecting PI3K Activity
[0063] The invention provides various methods for detecting PI3K activity or PI distribution by detecting conformational changes in a PI indicator. Broadly, the methods involve detecting a change in resonance energy transfer of a PI indicator of the invention when the indicator binds to a PI. PI binding to the indicator induces a conformational change that changes resonance energy transfer from the donor moiety to the acceptor moiety.
[0064] A change in resonance energy transfer can readily be detected using methods well known in the art. See, e.g., US 2005/0118619; US 2002/0137115; US 2003/0165920; US 2003/0186229; US 2004/0137479; US 2005/0026234; US 2005/0054573; US 2005/0118619; U.S. Pat. No. 6,773,885; U.S. Pat. No. 6,803,201; U.S. Pat. No. 6,818,420; Ayoub et al., 2002; Boute et al., 2002; Domin et al., Prog. Biomed. Optics and Imaging, Proc. SPIE, vol 5139, 2003, pp 238-242; Evellin et al., Methods Mol. biol. 284, 259-70, 2004; Honda et al., Proc. Natl. Acad. Sci. USA 98, 437-42, Feb. 27, 2001; Honda et al., Methods Mol. Biol. 3, 27-44, 1005; Mongillo et al., Cir. Res. 95, 67-75, Jul. 9, 2004; Mongillo et al., Methods Mol. Biol. 307, 1-14, 2005; Nagai et al., Proc. Natl. Acad. Sci. USA 101, 10554-59, July 20, 2004; Nikolaev et al., J. Biol. Chem. 279, 37215-18, 2004; Polit et al., Eur. J. Biochem. 270, 1413-23, 2003; Ponsioen et al., EMBO Rep. 5, 1176-80, 2004; Santangelo et al., Nucl. Acids Res. 32, 1-9, e-published Apr. 14, 2004; and Warrier et al., Am. J. Physiol. Cell Phiol. 289, C455-61, August 2005. Properties which can be detected as resonance energy transfer (RET) measurements include a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, anisotropy, a polarization of emitted light, resonance energy transfer, and a quenching of emission at a wavelength.
[0065] PI indicators of the invention can be used in cell-free systems, in isolated cells (for example, in primary cell culture or a cell line) or in cells in situ (e.g., in an isolated tissue sample, an isolated whole organ, or in a mammal). Subcellular distribution of PIs or changes in PI3K activity can be detected, for example, as described in the specific examples, below. Absolute PI levels can be detected by obtaining a RET measurement in the assay system and comparing it to a standard curve obtained in vitro.
[0066] Test compounds can be tested, for example, for the ability to inhibit PI3K activity (e.g., in drug-screening methods). Test compounds can be pharmacologic agents already known in the art to affect PI3K activity or can be compounds previously unknown to have such an activity. Compounds known to affect PI3K levels include, for example, wortmannin, LY294002, insulin, insulin-like growth factor 1 (IGF-1), PDGF, and PTEN inhibitors (e.g., bisperoxovanadium compounds and compounds disclosed in WO/2005/097119).
[0067] Test compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection.
[0068] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which describe the construction and testing of a PI indicator termed "InPAkt," which is a genetically targetable indicator for PIPS and PI(3,4)P2. An amino acid sequence for InPAkt is shown in SEQ ID NO:20; this amino acid sequence can be encoded by the nucleotide sequence shown in SEQ ID NO:21. SEQ ID NO:20 does not include a subcellular targeting peptide sequence which, as disclosed above, can be attached at a variety of positions in the indicator. These examples are provided for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1
[0069] Methods
[0070] Reporter construction. The Akt/PKB PH domain (1-164) was created by PCR using full length human Akt (SEQ ID NO:1) as the template. The pseudoligand peptide sequence VAEEEDDEEEDEDD (SEQ ID NO:3) was inserted between the C-terminus of PH domain and N-terminus of improved versions of YFP, Citrine (16) or Venus (17). Double mutation R23A/R25A was incorporated by the QUICKCHANGE® method (Stratagene) (FIG. 1). The constructs were first generated in pRSET B (Invitrogen) and subcloned into modified pcDNA3 (Invitrogen) behind a Kozak sequence for mammalian expression. For plasma membrane targeting of InPAkt, the sequence KKKKKSKTKCVIM (SEQ ID NO:4) was used. For nuclear targeting, the nuclear localization signal (NLS) PKKKRKVEDA (SEQ ID NO:5) was attached to the C terminus of the Venus-containing construct.
[0071] Cell Culture. HEK293 and NIH3T3 cells were plated onto sterilized glass coverslips in 35-mm dishes and grown to 50-90% confluency in DMEM (10% FBS) at 37° C. with 5% CO2. Cells were transfected with FUGENE®-6 (Roche), then serum starved for 24-36 h before imaging. Colocalization studies were performed by incubating transfected cells with Hoechst 33342 cell-permeable dyes (Molecular Probes) for staining nucleic acids.
[0072] Imaging. Cells were washed twice with Hanks' balanced salt solution buffer and maintained in the dark at room temperature with the addition of 50 ng/ml rat PDGF (Sigma), 100 ng/ml bovine insulin (Calbiochem), 50 nM IGF-1 (Sigma), or 20 μM LY294002 (Sigma) as described. Cells were imaged on a Zeiss Axiovert 200M microscope with a cooled CCD camera MicroMAX BFT512 (Roper Scientific, Trenton, N.J.) controlled by METAFLUOR 6.2 software (Universal Imaging, Downingtown, Pa.). Dual emission ratio imaging used a 420DF20 excitation filter, a 450DRLP dichroic mirror, and two emission filters (475DF40 for CFP and 535DF25 for YFP) alternated by a filter-changer Lambda 10-2 (Sutter Instruments, Novato, Calif.). Exposure time was 50-500 ms, and images were taken every 15-30 s. Fluorescence images were background-corrected by subtracting autofluorescence intensity of untransfected cells (or background with no cells) from the emission intensities of cells expressing fluorescent reporters. The ratios of yellow-to-cyan emissions were then calculated at different time points.
EXAMPLE 2
[0073] Development of a FRET Based Phosphoinositide Indicator
[0074] Design of the reporter. To measure the spatiotemporal dynamics of PIs in living cells, we employed a general molecular design in which binding of PIs in the "sensing unit" of the indicator can be translated into a change in the "reporting unit." A pair of fluorescent proteins that can undergo fluorescence resonance energy transfer (FRET), enhanced cyan fluorescent protein (ECFP) and improved versions of yellow fluorescent proteins (YFP) namely Citrine (16) or Venus (17), was employed as the reporting unit (FIGS. 1A, 1B). These two fluorophores can be genetically fused to a conformationally responsive element, the conformational change of which alters the relative distance and/or orientation of the two fluorophores and generates a change in the emission ratio. Recently many such reporters have been developed for measuring signaling molecules like Ca2+, cGMP, cAMP, or signaling events such as protein phosphorylation (14, 15).
[0075] For the sensing component, we chose the pleckstrin homology (PH) domain of Akt that binds specifically to two of the major PI3K products, namely PIPS and PI(3,4)P2 (18, 19). Crystal structure of this PH domain complexed to soluble inositol (1,3,4,5) tetrakisphosphate (IP4) (20) shows that this motif forms a bowl like structure lined with basic residues into which the highly negatively charged headgroup is accommodated (FIG. 1A).
[0076] To convert the PI binding to a conformational change, we engineered a "pseudoligand" sequence to associate with the basic patch of amino acids responsible for PI binding. The pseudoligand is a series of acidic residues taken from nucelolin 1 which was shown to bind to PH domain of insulin receptor substrate 1 (21). In the absence of PIs, the pseudoligand is expected to interact with the basic residues in the PH domain. Once the natural ligand is accumulated, the pseudoligand is competed off, unblocking the PH domain and generating a conformational change (FIG. 1A). This conformational change is relayed to the FRET pairs, thus yielding a change in FRET as readout to monitor PI dynamics.
[0077] Cellular response. When this construct was expressed in NIH3T3 cells, the fluorescence was uniformly distributed throughout the cell (FIG. 1D, first image). A similar expression pattern was seen in HEK293 cells. To verify that our reporter was expressed in full length, lysates of HEK293 cells expressing this chimera were separated on SDS-PAGE and probed with anti-GFP antibody. This chimeric protein was of the expected molecular weight showing no proteolysis (FIG. 1C).
[0078] We next checked the ability of this chimera to detect changes in 3' PIs in response to agonist stimulation. NIH3T3 cells expressing this construct were serum-starved then stimulated with 50 ng/ml platelet-derived growth factor (PDGF). Stimulation of endogenous PDGF receptor (PDGFR) generated a FRET increase, resulting in an increase in the ratio of yellow-to-cyan emissions (FIGS. 1D, 1E), which was detectable within several seconds and reached a plateau of 25.4±3.7% [average±std] (n=9) within minutes (t1/2=3.45±0.65 min). The FRET change occurred in the plasma membrane and was accompanied by the translocation of the reporter (FIG. 1D), reiterating that 3' PIs are mainly produced at the plasma membrane (22). To verify that the FRET response is caused by binding of PIP3 and PI(3,4)P2, we generated a variant of the reporter in which two critical residues in the PH domain, Arg23 and Arg25, which mainly contribute to D-3 phosphate recognition (20), are mutated. As shown in FIG. 1E, incorporating the mutations R23A/R25A into the chimeric protein completely abolished the FRET change.
[0079] To determine whether production of 3' PIs via PI3K activation is responsible for the FRET change, we pretreated cells with PI3K inhibitor LY294002 (20 μM). In the presence of the inhibitor, no emission ratio change was observed upon PDGF stimulation, but removal of the inhibitor recovered the response (FIG. 1F). In addition, this construct was shown to respond to microinjected dioctanoyl PIP3 and its soluble headgroup IP4 (FIG. 5), suggesting the FRET change is caused by ligand binding.
[0080] We next tested whether the FRET response was reversible. Addition of LY294002 resulted in a decrease in emission ratios (FIG. 1G), presumably due to the degradation of PIP3 and PI(3,4)P2 by lipid phosphatases. Similarly, another PI3K inhibitor, wortmannin, when added at 500 nM, could reverse the PDGF-stimulated response. Thus this reporter is useful in monitoring not only the production of PI3K products but also their degradation, and is designated as the indicator for Phosphoinositides based on Akt (InPAkt).
EXAMPLE 3
[0081] Differential Dynamics Stimulated by Different Growth Factors
[0082] Although PI3K is a crucial regulatory component shared by various growth factor pathways, their differential coupling to PI3K isoforms (23), and distinct modes of negative regulation (24), could lead to significant difference in PI dynamics, thereby differentiating downstream signaling. To compare the accumulation of PIP3 and PI(3,4)P2 induced by the activation of different tyrosine kinase receptor pathways, we applied three different ligands, namely insulin, insulin-like growth factor 1 (IGF-1) and PDGF, to serum-starved NIH3T3 cells expressing InPAkt. As shown above, PDGF stimulation produced an acute response that reached a plateau of 25.4±3.7% ratio increase in 5-9 min (t1/2=3.45±0.65 min) (FIG. 2A). In contrast, addition of 50 nM IGF-1 to activate insulin like growth factor receptor (IGF-IR) produced a more gradual response of 6.5±0.8% (n=5) in 10-12 min (1112=5.4±0.4 min) (FIG. 2A). Finally, stimulation with 100 ng/ml insulin did not generate any discernable response possibly due to low copies of insulin receptor (IR) in NIH3T3 cells (25). Thus, the magnitude of the responses differed significantly in the increasing order of insulin<<IGF-1<PDGF.
[0083] To determine whether the mechanisms that account for the suboptimal responses stimulated by insulin or IGF-1 could affect the PDGF-stimulated responses, we applied three agonists sequentially with sufficient time intervals in between. First, the addition of insulin did not generate any appreciable change in the emission ratio, as shown above. When IGF-1 was added to activate IGF-IR, the emission ratios increased by 6.8±0.9% in 10-12 min (t1/2=5.2±0.1 min) (n=3) and reached a plateau, indicating the production and degradation of PIP3 and PI(3,4)P2 reached an equilibrium. Lastly, when these fibroblasts were stimulated with PDGF, the emission ratio further increased by 14.6±1.4% in 6-7 min (n=4) (t1/2=3.2±0.2 min) (FIGS. 2B, 2C), indicating the balance between PI3K and phosphatases shifted more toward production of PIs and subsequently reached a new equilibrium. Conversely, when we reversed the order of the ligand addition, with PDGF addition first, followed by IGF-1, PDGF stimulated a full response of 24.2±1.5% in 7-9 min (n=2) (t1/2=3.9±0.2 min), which was not enhanced by the addition of IGF-1.
EXAMPLE 4
[0084] Phosphoinositide Dynamics within Subcellular Compartments
[0085] Plasma membrane. Taking advantage of the targetability of InPAkt, we constructed several fusions of InPAkt with various specific targeting motifs (FIG. 3A) to monitor PIP3 dynamics at different subcellular locations inside cells. Attaching the plasma membrane-targeting sequence of small guanosine trisphosphatase K-ras4B to the C terminus of the InPAkt, localized the reporter to the plasma membrane (FIG. 3B). Upon PDGF stimulation, we observed a ratio change of 9.25±0.4% (n=4) in 6-8 min (t1/2=3.9±0.7 min) (FIG. 3C). Compared to the responses from the untargeted reporter, (FIGS. 3C, 1G), the change in emission ratio was lesser for the membrane targeted InPAkt. Furthermore, upon addition of LY294002, we observed that the emission ratio decreased dramatically and reached below the initial ratio in the resting state. This data suggests the existence of basal levels of PIP3 and PI(3,4)P2 at the plasma membrane maintained by a critical balance between lipid kinase and phosphatase activities. Indeed, inhibition of PI3K led to a decrease in emission ratio in non-stimulated cells (FIG. 6).
[0086] Nucleus. Although PI signaling research has been mainly focused on events at the plasma membrane, recent experimental evidence suggested presence of PIP3 in the nucleus. For instance, it has been reported that PI3K translocates from cytoplasm to the nucleus (26), and presence of estrogen in cells containing a transmembrane intracellular estrogen receptor resulted in accumulation of PIP3-binding PH domain in the nucleus (27). To directly visualize the presence and dynamics of PIP3 and PI(3,4)P2 in the nucleus, we targeted InPAkt to the nucleus by attaching a nuclear localization signal (NLS) (FIG. 3A), shown by colocalization with the nuclear staining with Hoechst dye (FIG. 3B). Upon stimulation with PDGF, the nuclear targeted InPAkt did not elicit any response in NIH3T3 cells (FIG. 3D). Furthermore, treatment with LY294002 did not produce any detectable change in FRET. On the other hand, microinjection of dioctanoyl PIP3 and IP4 into the nuclei of cells expressing the same construct generated emission ratio increases of 5.4±2.2% (n=3), and 9.1±0.42% (n=2), respectively (FIG. 7), indicating the nuclear localized InPAkt was functional. Furthermore, surface plasmon resonance (SPR) analysis showed attaching NLS did not significantly alter the binding affinity (FIG. 8). Hence, results from our reporter indicate that there may not be any appreciable amounts of accessible PIP3 generated in the nucleus upon PDGF stimulation, in other words, these 3' PIs, if produced, may be accumulated in specific subnuclear compartments or complexed with other cellular components.
EXAMPLE 5
[0087] Simultaneous Imaging of Phosphoinositide Dynamics and Akt Phosphorylation
[0088] Increasing evidence has suggested that Akt, an immediate downstream effector of 3' PIs, is active within the nucleus (8). In the absence of accessible PIP3 and PI(3,4)P2 in the nucleus, how is nuclear Akt activity generated and maintained? It has been suggested that Akt gets activated at the plasma membrane by upregulated PIP3 and subsequently translocates to the cytosol and at a later time point, to the nucleus (28). To directly assess the signal propagation from the plasma membrane to the nucleus, we used two different FRET reporters: InPAkt to monitor the production and degradation of PIP3 and PI(3,4)P2 at the membrane; a nuclear targeted B kinase activity reporter (BKAR) to monitor Akt activity in the nucleus.
[0089] Briefly, BKAR is a genetically encoded fluorescent reporter (29) that monitors Akt activity. BKAR is comprised of ECFP and Citrine connected by a phosphoamino acid-binding domain (PBD) and an Akt specific substrate sequence, where phosphorylation of the substrate and its subsequent binding to PBD lead to a change in FRET, resulting in an increase in the ratio of cyan to yellow emissions. We previously reported that 3' PI production precedes Akt-mediated phosphorylation in the cytosol (29). To further examine the correlation between the PI dynamics at the membrane and Akt action in the nucleus with higher spatiotemporal resolution, we fused a NLS sequence to the C-terminus of BKAR (FIG. 4A) to target it to the nucleus (FIG. 4B), thereby achieving specific visualization of nuclear Akt activity. In addition, we used a plasma membrane targeted InPAkt to eliminate the possibility of probe translocation being a limiting step in kinetic measurement. When these two reporters were co-expressed, the spatial separation of the fluorescent signals allowed for simultaneous imaging of two closely coupled signaling events.
[0090] Following the aforementioned plan, we expressed both reporters in HEK293 cells (FIG. 4C). Upon IGF-1 treatment, we observed an immediate InPAkt response (t1/2=3.4±0.7 min) (n=4) at the plasma membrane. However, in the same time frame (phase I in FIG. 4D), the emission ratio of BKAR in the nucleus remained unchanged, suggesting the signal propagation from the membrane to nucleus does not occur instantaneously. After the response of InPAkt reached a peak, the emission ratio began to decrease gradually, depicting the ongoing turnover of PIP3 and PI(3,4)P2. Notably, within this time frame (phase II), the cyan-to-yellow emission ratio of BKAR in the nucleus began to increase gradually. The delay of 5-8 minutes can be correlated with the time frame of the departure of Akt from the plasma membrane, its translocation into the nucleus (30) and subsequent phosphorylation of BKAR. The nuclear Akt activity continued to increase even after the level of PIP3 and PI(3,4)P2 was below the starting point (phase III), indicating continuing accumulation of active Akt in the nucleus, and reached a plateau in 35-40 minutes. This time course is similar to those recorded from cells expressing nuclear targeted BKAR alone, indicating expression of InPAkt does not sequester 3' PIs or affect endogenous Akt activity. Similar results were obtained in NIH3T3 cells stimulated by PDGF.
EXAMPLE 6
[0091] Responses of InPAkt at the Plasma Membrane
[0092] The responses from the plasma membrane-targeted InPAkt upon activation or inhibition of PI3K indicated the presence of basal levels of PIP3 and PI(3,4)P2 at the plasma membrane. Thus, some of the reporter molecules were pre-saturated with PIs and activation of PI3K generated a moderate increase, yet subsequent PI3K inhibition allowed phosphatases to act on both stimulated and basal PIs, leading to a larger decrease. The basal levels of these 3' PIs may be involved in constitutively activating the plasma membrane-targeted Akt (m/pAkt). In fact, it was shown that m/pAkt was still subject to inhibition by PI3K inhibitors (28), supporting our finding that the basal levels of PIPS and PI(3,4)P2 are maintained by balanced activities of PI3K and phosphatases.
[0093] We observed distinct patterns of PI dynamics in response to activation of three different receptor tyrosine kinases (RTK), namely IR, IGF-IR and PDGFR in NIH3T3 cells, where PDGFR activation produces the largest response with fastest kinetics, consistent with data obtained using radioactive labeling (36). On the other hand, insulin induced a larger and more sustained response from PI3K activation than PDGF in 3T3 Ll adipocytes (37), one of the most insulin responsive cells expressing high levels of insulin receptors. However, the observed difference in responses stimulated by various growth factors cannot be accounted for solely by the difference in the total receptor numbers, as the numbers of PDGFR and IGF-IR per cell were similar [1-2×105] (25, 38). Instead, the different dynamics may result from differential coupling of the activated receptors to PI3K, involving, for instance, PI3K isoform specific activation by different receptor signaling pathways (23), or their regulation of lipid phosphatases (39, 40). Furthermore, as a distinct mode of negative regulation, the p85 regulatory subunit of PI3K was shown to translocate to discrete foci after the initial production of 3' PI, in response to IGF-1, but not to PDGF signaling (24). Our results from sequential activation of receptors suggested the mechanisms responsible for the suboptimal IGF-1 response do not affect the subsequent PDGF signaling, at least at the PI level. Thus differential regulation by shared negative regulators such as lipid phosphatases either does not account for the suboptimal IGF-1 response or can be counteracted by PDGF stimulation.
EXAMPLE 7
[0094] Signal Propagation from the Plasma Membrane to Nucleus
[0095] InPAkt targeted to the nucleus did not show any detectable response in NIH3T3 cells stimulated by PDGF or HEK293 cells stimulated by IGF-1, suggesting no substantial amounts of accessible PIP3 and PI(3,4)P2 are accumulated in the nucleus under these conditions. Tanaka at al, using radiolabeling and cell fractionation, showed that nuclear fraction of PDGF-treated NIH3T3 cells contain only a small amount of PIP3 (41). While it is possible that small amounts of PIP3 and PI(3,4)P2 are present yet below the detection limit of InPAkt, it is also plausible that these 3' PI are accumulated in subnuclear compartment or form complexes with other components, in that InPAkt detects free accessible PIP3 and PI(3,4)P2 compared to total PIP3 measured by Tanaka et al. Further experiments are needed to examine specific subnuclear compartments.
[0096] Upon growth factor stimulation, Akt gets activated at the plasma membrane and appears to maintain its activity during migration into the nucleus, rather than being activated by nuclear PI in situ. Using the plasma membrane-targeted PI indicator (InPAkt) and nuclear localized Akt activity reporter (BKAR), we show that Akt activity is gradually accumulated in the nucleus and sustained over 30-50 min, despite an immediate and transient production of PIP3 and PI(3,4)P2 at the plasma membrane. This observation is consistent with IGF-1 induced nuclear translocation of Aktl over a 30 min period (28) and increased nuclear Akt activity detected by in vitro kinase assay (30, 42). The fact that this accumulation occurs in the presence of high levels of PIP3 and PI(3,4)P2 at the plasma membrane (phase II in FIG. 4d) suggests that a pool of activated Akt is able to detach from the plasma membrane and translocate into nucleus. It is speculated that the membrane associated Akt may undergo a conformational change following activation, reducing its membrane affinity and facilitating its dissociation from the membrane. Supporting this theory, it was shown that the association between PH domain of Akt and the plasma membrane was more sustained than the full length Akt (30). Furthermore, Akt remains active during nuclear translocation and thereafter, presumably by maintaining its phosphorylation at T308 and 5473. Taken together, although lipid phosphatases are involved in maintaining the basal levels of 3' PIs and in shaping the stimulated transient response of the lipid second messenger, protein phosphatase activities are kept low to allow Akt mediated phosphorylation to sustain in the nucleus for 30-50 min or longer.
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Sequence CWU
1
251438DNAHomo sapiens 1atgcatatga gcgacgtagc cattgtgaag gagggctggc
tgcacaaacg aggggaatat 60attaaaacct ggcggccacg ctacttcctc ctcaagaacg
atggcacctt tattggctac 120aaggaacggc ctcaggatgt ggatcagcga gagtccccac
tcaacaactt ctcagtggca 180ctatgccagc tgatgaagac agagcggcca aggcccaaca
cctttatcat ccgctgcctg 240cagtggacca cagtcattga gcgcaccttc catgtggaaa
cgcctgagga gcgggaagaa 300tgggccaccg ccattcagac tgtggccgat ggactcaaga
ggcaggaaga agagacgatg 360gacttccgat caggctcacc cagtgacaac tcaggggctg
aagagatgga ggtgtccctg 420gccaagccca agcaccgt
4382146PRTHomo sapiens 2Met His Met Ser Asp Val
Ala Ile Val Lys Glu Gly Trp Leu His Lys1 5
10 15Arg Gly Glu Tyr Ile Lys Thr Trp Arg Pro Arg Tyr
Phe Leu Leu Lys 20 25 30Asn
Asp Gly Thr Phe Ile Gly Tyr Lys Glu Arg Pro Gln Asp Val Asp 35
40 45Gln Arg Glu Ser Pro Leu Asn Asn Phe
Ser Val Ala Leu Cys Gln Leu 50 55
60Met Lys Thr Glu Arg Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu65
70 75 80Gln Trp Thr Thr Val
Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu 85
90 95Glu Arg Glu Glu Trp Ala Thr Ala Ile Gln Thr
Val Ala Asp Gly Leu 100 105
110Lys Arg Gln Glu Glu Glu Thr Met Asp Phe Arg Ser Gly Ser Pro Ser
115 120 125Asp Asn Ser Gly Ala Glu Glu
Met Glu Val Ser Leu Ala Lys Pro Lys 130 135
140His Arg145314PRTArtificial Sequencepseudoligand 3Val Ala Glu Glu
Glu Asp Asp Glu Glu Glu Asp Glu Asp Asp1 5
10413PRTHomo sapiens 4Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile
Met1 5 10510PRTHomo sapiens 5Pro Lys Lys
Lys Arg Lys Val Glu Asp Ala1 5
10610PRTHomo sapiens 6Met Leu Cys Cys Met Arg Arg Thr Lys Gln1
5 10716PRTHomo sapiens 7Met Gly Cys Ile Lys Ser Lys
Arg Lys Asp Asn Leu Asn Asp Asp Glu1 5 10
15830PRTHomo sapiens 8Met Ala Ile Gln Leu Arg Ser Leu
Phe Pro Leu Ala Leu Pro Gly Met1 5 10
15Leu Ala Leu Leu Gly Trp Trp Trp Phe Phe Ser Arg Lys Lys
20 25 30916PRTHomo sapiens 9Met
Leu Ser Leu Arg Gly Ser Ile Arg Phe Phe Lys Arg Ser Gly Ile1
5 10 15106PRTArtificial
Sequencetetracysteine motif 10Cys Cys Xaa Xaa Cys Cys1
511225PRTDiscosoma sp. 11Met Arg Ser Ser Lys Asn Val Ile Lys Glu Phe Met
Arg Phe Lys Val1 5 10
15Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu
20 25 30Gly Glu Gly Arg Pro Tyr Glu
Gly His Asn Thr Val Lys Leu Lys Val 35 40
45Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro
Gln 50 55 60Phe Gln Tyr Gly Ser Lys
Val Tyr Val Lys His Pro Ala Asp Ile Pro65 70
75 80Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe
Lys Trp Glu Arg Val 85 90
95Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser
100 105 110Leu Gln Asp Gly Cys Phe
Ile Tyr Lys Val Lys Phe Ile Gly Val Asn 115 120
125Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly
Trp Glu 130 135 140Ala Ser Thr Glu Arg
Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu145 150
155 160Ile His Lys Ala Leu Lys Leu Lys Asp Gly
Gly His Tyr Leu Val Glu 165 170
175Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr
180 185 190Tyr Tyr Val Asp Ser
Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr 195
200 205Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg
His His Leu Phe 210 215
220Leu22512238PRTAequoria victoria 12Met Ser Lys Gly Glu Glu Leu Phe Thr
Gly Val Val Pro Ile Leu Val1 5 10
15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
Glu 20 25 30Gly Glu Gly Asp
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35
40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
Val Thr Thr Phe 50 55 60Ser Tyr Gly
Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln65 70
75 80His Asp Phe Phe Lys Ser Ala Met
Pro Glu Gly Tyr Val Gln Glu Arg 85 90
95Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
Glu Val 100 105 110Lys Phe Glu
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
Lys Leu Glu Tyr Asn 130 135 140Tyr Asn
Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly145
150 155 160Ile Lys Val Asn Phe Lys Ile
Arg His Asn Ile Glu Asp Gly Ser Val 165
170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly Pro 180 185 190Val
Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195
200 205Lys Asp Pro Asn Glu Lys Arg Asp His
Met Val Leu Leu Glu Phe Val 210 215
220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225
230 23513239PRTAequoria victoria 13Met Val Ser Lys
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1 5
10 15Val Glu Leu Asp Gly Asp Val Asn Gly His
Lys Phe Ser Val Ser Gly 20 25
30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45Cys Thr Thr Gly Lys Leu Pro Val
Pro Trp Pro Thr Leu Val Thr Thr 50 55
60Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65
70 75 80Gln His Asp Phe Phe
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85
90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu 100 105
110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125Ile Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135
140Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
Asn145 150 155 160Gly Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175Val Gln Leu Ala Asp His Tyr
Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185
190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
Ala Leu 195 200 205Ser Lys Asp Pro
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210
215 220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
Leu Tyr Lys225 230 23514239PRTAequoria
victoria 14Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
Leu1 5 10 15Val Glu Leu
Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly 20
25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
Leu Thr Leu Lys Phe Ile 35 40
45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50
55 60Leu Thr Trp Gly Val Gln Cys Phe Ser
Arg Tyr Pro Asp His Met Lys65 70 75
80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
Gln Glu 85 90 95Arg Thr
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100
105 110Val Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly 115 120
125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140Asn Tyr Ile Ser His Asn Val
Tyr Ile Thr Ala Asp Lys Gln Lys Asn145 150
155 160Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile
Glu Asp Gly Ser 165 170
175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190Pro Val Leu Leu Pro Asp
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200
205Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
Glu Phe 210 215 220Val Thr Ala Ala Gly
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys225 230
23515239PRTAequoria victoria 15Met Val Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu1 5 10
15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
Gly 20 25 30Glu Gly Glu Gly
Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35
40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
Leu Val Thr Thr 50 55 60Phe Gly Tyr
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys65 70
75 80Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90
95Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
Ala Glu 100 105 110Val Lys Phe
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115
120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
His Lys Leu Glu Tyr 130 135 140Asn Tyr
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145
150 155 160Gly Ile Lys Val Asn Phe Lys
Ile Arg His Asn Ile Glu Asp Gly Ser 165
170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
Ile Gly Asp Gly 180 185 190Pro
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 195
200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215
220Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys225
230 2351618PRTArtificial Sequencepeptide linker
16Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Gln Gly Ser Thr1
5 10 15Lys Gly1714PRTArtificial
Sequencepseudoligand 17Val Ala Glu Glu Asp Asp Asp Glu Glu Glu Asp Glu
Asp Asp1 5 1018228PRTAequoria victoria
18Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu1
5 10 15Val Glu Leu Asp Gly Asp
Val Asn Gly His Arg Phe Ser Val Ser Gly 20 25
30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
Lys Phe Ile 35 40 45Cys Thr Thr
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50
55 60Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro
Asp His Met Lys65 70 75
80Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95Arg Thr Ile Phe Phe Lys
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100
105 110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
Glu Leu Lys Gly 115 120 125Ile Asp
Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130
135 140Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala
Asp Lys Gln Lys Asn145 150 155
160Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175Val Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180
185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205Ser
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210
215 220Val Thr Ala Ala22519684DNAAequoria
victoria 19atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
cgagctggac 60ggcgacgtaa acggccacag gttcagcgtg tccggcgagg gcgagggcga
tgccacctac 120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180ctcgtgacca ccctgacctg gggcgtgcag tgcttcagcc gctaccccga
ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
taccatcttc 300ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
cgacaccctg 360gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
cctggggcac 420aagctggagt acaactacat cagccacaac gtctatatca ccgccgacaa
gcagaagaac 480ggcatcaagg cccacttcaa gatccgccac aacatcgagg acggcagcgt
gcagctcgcc 540gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
cgacaaccac 600tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
tcacatggtc 660ctgctggagt tcgtgaccgc cgcc
68420691PRTArtificial Sequencephosphoinositide indicator
20Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr1
5 10 15Gly Gly Gln Gln Met Gly
Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25
30Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
Val Pro Ile 35 40 45Leu Val Glu
Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser 50
55 60Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
Thr Leu Lys Phe65 70 75
80Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
85 90 95Thr Leu Thr Trp Gly Val
Gln Cys Phe Ser Arg Tyr Pro Asp His Ile 100
105 110Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
Gly Tyr Val Gln 115 120 125Glu Arg
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 130
135 140Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys145 150 155
160Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
165 170 175Tyr Asn Tyr Ile
Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 180
185 190Asn Gly Ile Lys Ala His Phe Lys Ile Arg His
Asn Ile Glu Asp Gly 195 200 205Ser
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 210
215 220Gly Pro Val Leu Leu Pro Asp Asn His Tyr
Leu Ser Thr Gln Ser Ala225 230 235
240Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
Glu 245 250 255Phe Val Thr
Ala Ala Arg Met His Met Ser Asp Val Ala Ile Val Lys 260
265 270Glu Gly Trp Leu His Lys Arg Gly Glu Tyr
Ile Lys Thr Trp Arg Pro 275 280
285Arg Tyr Phe Leu Leu Lys Asn Asp Gly Thr Phe Ile Gly Tyr Lys Glu 290
295 300Arg Pro Gln Asp Val Asp Gln Arg
Glu Ala Pro Leu Asn Asn Phe Ser305 310
315 320Val Ala Gln Cys Gln Leu Met Lys Thr Glu Arg Pro
Arg Pro Asn Thr 325 330
335Phe Ile Ile Arg Cys Leu Gln Trp Thr Thr Val Ile Glu Arg Thr Phe
340 345 350His Val Glu Thr Pro Glu
Glu Arg Glu Glu Trp Thr Thr Ala Ile Gln 355 360
365Thr Val Ala Asp Gly Leu Lys Lys Gln Glu Glu Glu Glu Met
Asp Phe 370 375 380Arg Ser Gly Ser Pro
Ser Asp Asn Ser Gly Ala Glu Glu Met Glu Val385 390
395 400Ser Leu Ala Lys Pro Lys His Arg Val Thr
Met Asn Glu Phe Glu Tyr 405 410
415Leu Lys Leu Leu Gly Lys Gly Thr Phe Gly Lys Val Ser Ala Gly Gly
420 425 430Ser Val Ala Glu Glu
Glu Asp Asp Glu Glu Glu Asp Glu Asp Asp Gly 435
440 445Gly Ser Glu Leu Met Val Ser Lys Gly Glu Glu Leu
Phe Thr Gly Val 450 455 460Val Pro Ile
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe465
470 475 480Ser Val Ser Gly Glu Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr 485
490 495Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val
Pro Trp Pro Thr 500 505 510Leu
Val Thr Thr Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro 515
520 525Asp His Met Lys Gln His Asp Phe Phe
Lys Ser Ala Met Pro Glu Gly 530 535
540Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys545
550 555 560Thr Arg Ala Glu
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 565
570 575Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
Gly Asn Ile Leu Gly His 580 585
590Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp
595 600 605Lys Gln Lys Asn Gly Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile 610 615
620Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
Pro625 630 635 640Ile Gly
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser His
645 650 655Gln Ser Ala Leu Ser Lys Asp
Pro Asn Glu Lys Arg Asp His Met Val 660 665
670Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
Asp Glu 675 680 685Leu Tyr Lys
690212082DNAArtificial Sequencecoding sequence for IP Indicator
21atgcggggtt ctcatcatca tcatcatcat ggtatggcta gcatgactgg tggacagcaa
60atgggtcggg atctgtacga cgatgacgat aaggatccca tggtgagcaa gggcgaggag
120ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacagg
180ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
240atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg
300ggcgtgcagt gcttcagccg ctaccccgac cacatcaagc agcacgactt cttcaagtcc
360gccatgcccg aaggctacgt ccaggagcgt accatcttct tcaaggacga cggcaactac
420aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
480ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacatc
540agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc ccacttcaag
600atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
660cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
720ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
780gcccgcatgc atatgagcga cgtggctatt gtgaaggagg gttggctgca caaacgaggg
840gagtacatca agacctggcg gccacgctac ttcctcctca agaatgatgg caccttcatt
900ggctacaagg agcggccgca ggatgtggac caacgtgagg ctcccctcaa caacttctct
960gtggcgcagt gccagctgat gaagacggag cggccccggc ccaacacctt catcatccgc
1020tgcctgcagt ggaccactgt catcgaacgc accttccatg tggagactcc tgaggagcgg
1080gaggagtgga caaccgccat ccagactgtg gctgacggcc tcaagaagca ggaggaggag
1140gagatggact tccggtcggg ctcacccagt gacaactcag gggctgaaga gatggaggtg
1200tccctggcca agcccaagca ccgcgtgacc atgaacgagt ttgagtacct gaagctgctg
1260ggcaagggca ctttcggcaa ggtgtctgca ggcggtagcg tggctgagga agaggatgac
1320gaggaggaag acgaggacga tggcggcagc gagctcatgg tgagcaaggg cgaggagctg
1380ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
1440agcgtgtccg gcgagggcga gggcgatgcc acttacggca agctgaccct gaagttcatc
1500tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
1560ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
1620atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
1680acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
1740atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
1800cacaatgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
1860cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
1920atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagccacca gtccgccctg
1980agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
2040gggatcactc tcggcatgga cgagctgtac aagtaagaat tc
20822215DNAArtificial Sequencecoding sequence for linker 22tctgcaggcg
gtagc
15235PRTArtificial Sequencelinker 23Ser Ala Gly Gly Ser1
52415DNAArtificial Sequencecoding sequence for linker 24ggcggcagcg agctc
15255PRTArtificial
Sequencelinker 25Gly Gly Ser Glu Leu1 5
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