CN1898564A - Methods of treating asthma - Google Patents

Abstract

Methods for agents useful for treating asthma are disclosed. The methods include screening for agents that inhibit the production of a PKC-theta protein, as well as for agents that inhibit the kinase activity of a PKC-theta protein, or a functional fragment thereof, wherein such agents are useful for treating asthma. The methods also include screening for agents that inhibit the production of a reporter gene product encoded by a nucleic acid sequence operably linked to a PKC-theta promoter. Also disclosed are methods of treating asthma that include administering an agent that inhibits the production of a functional PKC-theta protein or the kinase activity of a PKC-theta protein or a functional fragment thereof. An isolated mast cell lacking expression of endogenous PKC-theta is also disclosed.

Description

The method of treatment asthma

The cross reference of related application

[0001] the application requires the U.S. Provisional Application sequence number no.60/532 of submission on Dec 24th, 2003, the U.S. Provisional Application sequence number no.60/589 that on July 20th, 525 and 2004 submitted to, 415 rights and interests are introduced the complete content of each application here by reference.

Background of invention

[0002] the present invention relates to biology and field of immunology.Especially, invention relates to the method for asthma and treatment asthma.

[0003] asthma is chronic respiratory tract inflammatory disease, is characterised in that the outbreak repeatedly of reversible respiratory tract obst ruction and respiratory tract anaphylaxis (AHR).The frequent irritated and inflammation of respiratory tract of suffering from the patient of asthma.When asthmatic patient and anaphylactogen or when stimulating the material of its respiratory tract to contact, respiratory tract makes the patient be difficult to breathe with regard to shrinking (be around the respiratory tract wall muscle tighten).The respiratory tract inwall begins inflammation, causes the generation of other clinical manifestation of sticking phlegm and allergy.Other clinical manifestation of asthma comprise breathe hard, pant, cough and chest be tight, may become that life threatens or be fatal in some cases.

[0004],, people reinvent the effect in quickening the deterioration of asthmatic patient lung but having recognized long-term respiratory tract gradually although existing to focus on reduces symptomatic bronchial spasm and pulmonary inflammatory methods of treatment.Respiratory tract is reinvented and is meant many pathological characters, comprises that epithelium smooth muscle and myofibroblast hyperplasia and/or change are living, goes up subcutaneous fiberization and apposition.These processes cause being up under the fatal asthma situation about 300% respiratory tract jointly and thicken.Although obtained sizable progress aspect the asthma pathologic, physiologic illustrating, yet the popularity degree of this disease, M ﹠ M have increased between in the past twenty years.The data of up-to-date acquisition show in the U.S. has about 2,000 ten thousand people, the whole world to have 1.5 hundred million people of surpassing to suffer from asthma.In this early stage in 10 years, on annual basis,, only just have that nearly 1,900,000 people's emergency treatments are gone to a doctor, 454,000 people are in hospital and surpass 4000 people's death in the U.S. directly owing to asthma.

[0005] asthma is by unsuitable inflammatory reaction causes that this is generally accepted to the anaphylactogen in the air borne.The lung of asthmatic patient has confirmed the strong infiltration of lymphocyte, mast cell, particularly acidophic cell.

[0006] causes the cell and the interaction of molecules of the complexity of observed inflammation in asthma although current research has disclosed some, but still have great Knowledge Gap.

[0007] as the result of research asthma reason, can obtain large-scale various medicine and treat SOA.Yet many medicines have various shortcomings, and it is unsatisfactory to make that they are used for the treatment of asthma.For example, many medicines such as adrenaline and isoproterenol only are to alleviate SOA in very short a period of time.Other treatment had just been lost efficacy after having used a period of time.In addition, some medicine elephant hide matter steroids have serious side effects, have limited their long-term use.Clearly, both needed understanding, also needed more useful method of treatment for asthma the more molecular level of asthma.The present invention solves these demands.

Summary of the invention

[0008] the present invention is based on the inventor to small part and has found the effect of protein kinase C theta (PKC-θ) in comprising the breathing problem state of asthma.Therefore, the invention provides the method that is used to differentiate for treatment asthma useful reagent, be used for the treatment of the patient's who suffers from asthma or asthma sample symptom method, and the mast cell of not expressing the separation of endogenous PKC-θ albumen.

[0009] therefore, in first aspect, the invention provides the method for the correctives that is used to differentiate PKC-θ albumen.This method comprises PKC-θ albumen or its functional fragment is contacted with test agent; And whether definite test agent regulate the kinase activity of PKC-θ albumen or its functional fragment, and wherein when having test agent, the variation of PKC-θ albumen or its functional fragment kinase activity shows it is the correctives of PKC-θ albumen.In certain embodiments, determining step comprises compare test reagent with respect to the kinase activity under the condition that does not have test agent.

[0010] in some embodiments, the correctives of the PKC-θ albumen of reduction kinase activity is the inhibitor of PKC-θ albumen or its functional fragment.In some embodiments, the correctives of the PKC-θ albumen of increase kinase activity is the activator of PKC-θ albumen or its functional fragment.In some embodiments, the correctives of PKC-θ albumen makes the kinase activity of PKC-θ albumen or its functional fragment reduce twice at least.

[0011] in certain embodiments, PKC-θ albumen is total length PKC-θ albumen.In some embodiments, PKC-θ albumen is the functional variant of total length PKC-θ albumen.In specific embodiment, functional fragment is a PKC-θ kinase domain.

[0012] in some embodiments, contact procedure is to realize by the reaction mixture that PKC-θ albumen or its functional fragment and test agent are provided.In certain embodiments, reaction mixture is in the damping fluid of the NaCl that contains the concentration that is selected from 50mM-100mM, 100-150mM, 150-200mM and 200-250mM and 250-300mM.In specific embodiment, NaCl concentration is 250mM.

[0013] in some embodiments, the correctives of PKC-θ albumen is useful for the treatment mammal such as people's asthma.In some embodiments, asthma is the asthma of IgE-mediation.In specific embodiment, this method further is included in the external or body effectiveness of assessment test agent in the asthmatic model, wherein demonstrates in the asthmatic model to render a service the test agent that increases with respect to contrast agents and just differentiated to being useful for treatment asthma in external or body.

[0014] in some embodiments, PKC-θ albumen or its fragment obtain from prokaryotic such as bacterial cell (as Escherichia coli).

[0015] in some embodiments, contact procedure realizes in cell.

[0016] in certain embodiments, the kinase activity of PKC-θ albumen or its functional fragment is the autophosphorylation of PKC-θ albumen or its functional fragment.In some embodiments, the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that SEQ ID NO:1 is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues.In specific embodiment, autophosphorylation occurs on 538 threonine residues of SEQ ID NO:1.

[0017] in some embodiments, this method comprises PKC-θ albumen or its functional fragment is contacted with test agent and PKC-θ substrate.In certain embodiments, the kinase activity of PKC-θ albumen or its functional fragment is the phosphorylation of PKC-θ substrate.In some embodiments, PKC-θ substrate contains R-X-X-S motif or R-X-X-T motif, and wherein R is an arginine, and X can be unknown amino acid or can be any amino acid that S is a serine, and T is a threonine.For example, PKC-θ substrate can have and is selected from following amino acid sequence (based on general single-letter amino acid code): KKRFSFKKSFK (SEQ ID NO:5), FARKGSLRQKN (SEQ ID NO:6), FARKGSLRQ (SEQ ID NO:15), KKRFSFKKSFK (SEQ ID NO:16), QKRPSQRSKYL (SEQ IDNO:17), KIQASFRGHMA (SEQ ID NO:18), LSRTLSVAAKK (SEQID NO:19), AKIQASFRGHM (SEQ ID NO:20), VAKRESRGLKS (SEQ ID NO:21), KAFRDTFRLLL (SEQ ID NO:22), PKRPGSVHRTP (SEQ ID NO:23), ATFKKTFKHLL (SEQ IDNO:24), SPLRHSFQKQQ (SEQ ID NO:25), KFRTPSFLKKS (SEQID NO:26), IYRASYYRKGG (SEQ ID NO:27), KTRRLSAFQQG (SEQ ID NO:28), RGRSRSAPPNL (SEQ ID NO:29), MYRRSYVFQT (SEQ ID NO:30), QAWSKTTPRRI (SEQ ID NO:31), RGFLRSASLGR (SEQ ID NO:32), ETKKQSFKQTG (SEQ IDNO:33), DIKRLTPRFTL (SEQ ID NO:34), APKRGSILSKP (SEQID NO:35), MYHNSSQKRH (SEQ ID NO:36), MRRSKSPADSA (SEQ ID NO:37), TRSKGTLRYMS (SEQ ID NO:38), LMRRNSVTPLA (SEQ ID NO:39), ITRKRSGEAAV (SEQ IDNO:40), EEPVLTLVDEA (SEQ ID NO:41), SQKRPSQRHGS (SEQID NO:42), KPFKLSGLSFK (SEQ ID NO:43), AFRRTSLAGGG (SEQ ID NO:44), ALGKRTAKYRW (SEQ ID NO:45), VVRTDSLKGRR (SEQ ID NO:46), KRRQISIRGIV (SEQ ID NO:47), WPWQVSLRTRF (SEQ ID NO:48), GTFRSSIRRLS (SEQ IDNO:49), RVVGGSLRGAQ (SEQ ID NO:50), LRQLRSPRRTQ (SEQID NO:51), KTRKISQSAQT (SEQ ID NO:52), NKRRATLPHPG (SEQ ID NO:53), SYTRFSLARQV (SEQ ID NO:54), NSRRPSRATWL (SEQ ID NO:55), RLRRLTAREAA (SEQ IDNO:56), NKRRGSVPILR (SEQ ID NO:57), GKRRPSRLVAL (SEQID NO:58), QKKRVSMILQS (SEQ ID NO:59), and RLRRLTAREAA (SEQ ID NO:60).

[0018] in some embodiments, PKC-θ albumen or its functional fragment are in cell, in mast cell or CD4+T cell.

[0019] further, the invention provides the method for the correctives that is used to differentiate PKC-θ albumen, comprise that the cell that will express PKC-θ albumen or its functional fragment contacts with test agent and determine whether test agent reduces the amount of the functional PKC-θ albumen in the cell, the test agent that wherein reduces the amount of the functional PKC-θ albumen in the cell just is the correctives of PKC-θ albumen by discriminating.In some embodiments, the correctives of PKC-θ albumen is useful for the treatment mammal such as people's asthma.In some embodiments, asthma is the asthma of IgE-mediation.In specific embodiment, this method further is included in the external or body effectiveness of assessment test agent in the asthmatic model, wherein demonstrates in the asthmatic model to render a service the test agent that increases with respect to contrast agents and just differentiated to being useful for treatment asthma in external or body.

[0020] in some embodiments, reagent has reduced the expression of nucleic acid molecules in cell of encoding function PKC-θ albumen.In specific embodiments, asthma is the asthma of IgE-mediation.In some embodiments, mammal is the people.In certain embodiments, functional PKC-θ albumen is in cell, in mast cell or CD4+T cell (as the TH2T cell).

[0021] in certain embodiments, reagent has reduced the amount of the RNA of encoding function PKC-θ albumen.In some embodiments, reagent has suppressed the translation of the RNA of encoding function PKC-θ albumen.

[0022] further, the invention provides the method that is used to differentiate for treatment mammal asthma useful reagent, the nucleotide sequence that comprises the coding reporter product that will be operably connected to PKC-θ promoter contacts with test agent, and whether definite test agent reduced the generation of reporter product, wherein reduces reagent that the reporter product produces and just differentiated to for treatment asthma useful reagent.

[0023] in certain embodiments, be operably connected to the nucleotide sequence of coding reporter product of PKC-θ promoter in cell (as mast cell or CD4+T cell).In some embodiments, mast cell is not expressed endogenous PKC-θ albumen.In certain embodiments, the reporter product is luciferase, beta galactosidase, chloromycetin acyltransferase, β-glucuronidase, alkaline phosphatase or green fluorescent protein.

[0024] further, the invention provides the method for the correctives that is used to differentiate PKC-θ albumen.This method comprises that the cell of will express PKC-θ albumen or its functional fragment contacts with test agent; Determine whether test agent has reduced PKC-θ albumen or the autophosphorylation of its functional fragment in cell, the test agent that has wherein reduced PKC-θ albumen or its functional fragment autophosphorylation just is the correctives of PKC-θ albumen by discriminating.In some embodiments, determining step comprises compare test reagent with respect to the kinase activity under the condition that does not have test agent.

[0025] in some embodiments, the correctives of the PKC-θ albumen of reduction kinase activity is the inhibitor of PKC-θ albumen or its functional fragment.In some embodiments, the correctives of the PKC-θ albumen of increase kinase activity is the activator of PKC-θ albumen or its functional fragment.In some embodiments, the correctives of PKC-θ albumen makes the kinase activity of PKC-θ albumen or its functional fragment reduce twice at least.

[0026] in certain embodiments, PKC-θ albumen is total length PKC-θ albumen.In some embodiments, PKC-θ albumen is the functional variant of total length PKC-θ albumen.In some embodiments, functional fragment is a PKC-θ kinase domain.

[0027] in some embodiments, the correctives of PKC-θ albumen is useful for the treatment mammal such as people's asthma.In some embodiments, asthma is the asthma of IgE-mediation.In specific embodiments, this method further is included in the external or body effectiveness of assessment test agent in the asthmatic model, wherein demonstrates in the asthmatic model to render a service the test agent that increases with respect to contrast agents and just differentiated to being useful for treatment asthma in external or body.

[0028] in some embodiments, cell is a prokaryotic, such as bacterial cell (as Escherichia coli).

[0029] in some embodiments, the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that SEQ ID NO:1 is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues.

[0030] also has on the other hand, disclosure of the Invention be used for the treatment of the method for asthma, comprise to the administration of suffering from asthma or suffering from SOA and reduce the kinase activity of PKC-θ albumen or its functional fragment or reduce the reagent that functional PKC-θ albumen produces.In some embodiments, reagent is used with pharmaceutically acceptable carrier.In some embodiments, carrier is an aerosol form.

[0031] in some embodiment of inventive method, reagent by intravenous, oral, approach is used in skin and/or muscle.In specific embodiments, reagent is used through suction.In some embodiments, asthma is the asthma of IgE-mediation.In some embodiments, reagent is used altogether with medicine, and medicine can be beta-adrenergic reagent, theophylline compound, corticosteroid, anticholinergic, antihistaminic, calcium channel blocker, nasmil or their combination.In specific embodiments, reagent is the antibody of specificity in conjunction with PKC-θ albumen or its fragment.In some embodiments, antibody is polyclonal antibody.In some embodiments, antibody is monoclonal antibody.

[0032] in some embodiments, test agent is a nucleic acid molecules.In certain embodiments, nucleic acid molecules is a ribonucleic acid molecule.In some embodiments, ribonucleic acid molecule comprises the nucleotide sequence with the part complementation of the described nucleotide sequence of SEQ ID NO:3.

[0033] in certain embodiments, the kinase activity of PKC-θ albumen or its functional fragment is the autophosphorylation of PKC-θ albumen or its functional fragment.In some embodiments, the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that SEQ ID NO:1 is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues.

[0034] in certain embodiments, the kinase activity of PKC-θ albumen or its functional fragment is the phosphorylation of PKC-θ substrate.In some embodiments, PKC-θ substrate contains R-X-X-S motif or R-X-X-T motif, and wherein R is an arginine, and X is unknown or any known amino acid, and S is a serine, and T is a threonine.For example, PKC-θ substrate can have and is selected from following amino acid sequence (based on general single-letter amino acid code): KKRFSFKKSFK (SEQ ID NO:5), FARKGSLRQKN (SEQ ID NO:6), FARKGSLRQ (SEQ ID NO:15), KKRFSFKKSFK (SEQ ID NO:16), QKRPSQRSKYL (SEQ ID NO:17), KIQASFRGHMA (SEQ IDNO:18), LSRTLSVAAKK (SEQ ID NO:19), AKIQASFRGHM (SEQID NO:20), VAKRESRGLKS (SEQ ID NO:21), KAFRDTFRLLL (SEQ ID NO:22), PKRPGSVHRTP (SEQ ID NO:23), ATFKKTFKHLL (SEQ ID NO:24), SPLRHSFQKQQ (SEQ IDNO:25), KFRTPSFLKKS (SEQ ID NO:26), IYRASYYRKGG (SEQID NO:27), KTRRLSAFQQG (SEQ ID NO:28), RGRSRSAPPNL (SEQ ID NO:29), MYRRSYVFQT (SEQ ID NO:30), QAWSKTTPRRI (SEQ ID NO:31), RGFLRSASLGR (SEQ ID NO:32), ETKKQSFKQTG (SEQ ID NO:33), DIKRLTPRFTL (SEQ IDNO:34), APKRGSILSKP (SEQ ID NO:35), MYHNSSQKRH (SEQID NO:36), MRRSKSPADSA (SEQ ID NO:37), TRSKGTLRYMS (SEQ ID NO:38), LMRRNSVTPLA (SEQ ID NO:39), ITRKRSGEAAV (SEQ ID NO:40), EEPVLTLVDEA (SEQ IDNO:41), SQKRPSQRHGS (SEQ ID NO:42), KPFKLSGLSFK (SEQID NO:43), AFRRTSLAGGG (SEQ ID NO:44), ALGKRTAKYRW (SEQ ID NO:45), VVRTDSLKGRR (SEQ ID NO:46), KRRQISIRGIV (SEQ ID NO:47), WPWQVSLRTRF (SEQ ID NO:48), GTFRSSIRRLS (SEQ ID NO:49), RVVGGSLRGAQ (SEQ IDNO:50), LRQLRSPRRTQ (SEQ ID NO:51), KTRKISQSAQT (SEQID NO:52), NKRRATLPHPG (SEQ ID NO:53), SYTRFSLARQV (SEQ ID NO:54), NSRRPSRATWL (SEQ ID NO:55), RLRRLTAREAA (SEQ ID NO:56), NKRRGSVPILR (SEQ IDNO:57), GKRRPSRLVAL (SEQ ID NO:58), QKKRVSMILQS (SEQID NO:59), and RLRRLTAREAA (SEQ ID NO:60).

[0035] further, invention provides the mast cell of the separation of not expressing endogenous PKC-θ albumen.In some embodiments, cellular expression external source PKC-θ albumen or its fragment.

[0036] these and other aspect of the present invention, embodiment and advantage will be conspicuous from the description here.

The accompanying drawing summary

[0037] Figure 1A-1C is the photo figure of Western engram analysis, PKC-θ film transposition and derivable activation cycli phosphateization when illustrating TCR and stimulating the human T-cell altogether.

[0038] Fig. 2 A-2C is photo (Fig. 2 A and 2C) and picture (Fig. 2 B), and the autophosphorylation that has shown PKC-θ activation ring is that the kinase activity in the cell is necessary.

[0039] Fig. 3 A-3D is the synoptic diagram (Fig. 3 A) that shows PKC-θ kinase domain (PKC-θ KD) autophosphorylation feature, as determined by mass spectrophotometry, and peptide NFpSFMNPGMER (SEQ ID NO:64; Wherein pS represents that serine is by phosphorylation; Spanning position 693-703) product ion is composed at m/z 705.52 (Fig. 3 B), peptide ALINpSMDQNMFR (SEQ ID NO:65; Spanning position 681-692) at m/z 760.48 (Fig. 3 C), peptide TNTFCGTPDYIAPEILLGQK (SEQ ID NO:66; Spanning position 536-555) at m/z 1159.71 (Fig. 3 D).Attention is represented with # by the alkylating halfcystine of iodoacetamide in Fig. 3 D.

[0040] Fig. 4 A-4C is with anti--pT538PKC-θ Western blotting (Fig. 4 A) and anti-His Western blotting (Fig. 4 B), to shown in the Western engram analysis of Escherichia coli pyrolysis product of PKC-θ KD albumen and mutant confirming equivalents, and the chart (Fig. 4 C) of the external lytic activity of PKC-θ KD albumen shown in showing and mutant.

[0041] Fig. 5 A-5D is a series chart, has shown that intercept is to 1/[peptide 1 when 100mM NaCl] recast figure (Fig. 5 A); Slope is to 1/[peptide 1 when 100mM NaCl] recast figure (Fig. 5 B); Intercept is to 1/[peptide 1 when 625mM NaCl] recast figure (Fig. 5 C); Slope is to 1/[peptide 1 when 625mMNaCl] recast figure (Fig. 5 D).

[0042] Fig. 6 A-6C shows that the dynamics of PKC-θ KD shows the diagrammatic series of views of possible various mechanism.Fig. 6 A has shown order ordered mechanism, and ADP is the last product that discharges thus; Fig. 6 B has shown kinetic mechanism, and ADP is the last product that discharges thus; Fig. 6 C has shown chance mechanism.In Fig. 6 A-6C, " E " represents enzyme, and " A " represents substrate A, and " B " represents substrate B, and " P " represents product P, and " Q " represents product Q.

[0043] Fig. 7 A-7D has shown the k of solvent viscosity to PKC-θ KD _Cat(Fig. 7 A and 7C) and k _Cat/ K _mThe influence of (Fig. 7 B and 7D).Fig. 7 A has shown when ATP remains on 2.0mM influences k with different peptide 1 _CatFig. 7 B has shown when ATP remains on 0.125mM, the k of peptide 1 _Cat/ K _mFig. 7 C has shown when ATP remains on 2.0mM influences k with different peptide 3 _CatFig. 7 D has shown the k of peptide 3 when ATP remains on 2.0mM _Cat/ K _mOpen circles symbol (zero) is illustrated in the 100mM NaCl in the sucrose of increase; Hollow inverted triangle symbol () is illustrated in the 250mM NaCl in the sucrose of increase; Filled circles symbol (●) is illustrated in the 100mM NaCl among the Ficoll 400 of increase; Solid inverted triangle symbol () is illustrated in the 250mM NaCl among the Ficoll 400 of increase.Dotted line among Fig. 7 A-7D is represented slope 1.

[0044] Fig. 8 is the synoptic diagram that the show suppression substrate may disturb the different mechanisms of PKC-θ KD catalytic activity.In Fig. 8, " E " represents enzyme, and " A " represents substrate A, and " B " represents substrate B, and " P " represents product P, and " Q " represents product Q.

[0045] Fig. 9 A-9B illustrates the peptide array (Fig. 9 A) of several peptide substrates sequences that can differentiate PKC-θ and identifies by the peptide of PKC-θ phosphorylation (Fig. 9 B).

[0046] Figure 10 A-10B is the photo figure of Western engram analysis, shows when PKC-θ activates mast cell (BMMC) that ring is linked to bone marrow derived at the IgE acceptor and goes up by phosphorylation inductively.

[0047] Figure 11 A-11C is the photo figure of Western engram analysis of film fraction (Figure 11 A), the insoluble fraction of detergent (DI) (Figure 11 B) and the intact cell extract (WCE) (Figure 11 C) of the crosslinked BMMC of IgE acceptor, proves the PKC-θ film transposition in the BMMC of IgE receptor for stimulating.

[0048] Figure 12 A-12B is the photo figure of Western engram analysis, has confirmed when the IgE acceptor is linked on the BMMC, and the distribution of PKC-δ (Figure 12 A) and PKC-β (Figure 12 B) is not obviously changed.

[0049] Figure 13 A-13B is histology (Figure 13 A) and diagram (Figure 13 B) figure, illustration the BMMC of PKC-θ knock-out mice contain BMMC particle still less than wild-type mice.Figure 13 B has shown that cell average fluorescent strength (MFI) is as the function of time or as the function of DNP-BSA concentration.

[0050] Figure 14 A-14B is diagram figure, has confirmed that the peritonaeum mast cell of PKC-θ knock-out mice has than the lower level cell surface IgE of wild-type mice (Figure 14 A), but the cell surface ckit (Figure 14 B) with similar level.The p value is determined by the t-check.

[0051] Figure 15 A-15C is diagram figure, has confirmed that PKC-θ knock-out mice compares SERUM IgE (Figure 15 A) and the IgG1 (Figure 15 B) with reduction level with wild-type mice, but the IgA (Figure 15 C) with increase level.The p value is determined by the t-check.

[0052] Figure 16 A-16C is diagram figure, shows after the IgE acceptor is crosslinked, is derived from following production of cytokines deficiency: TNF-α (Figure 16 A), IL-13 (Figure 16 B) and IL-6 (Figure 16 C) among the BMMC of PKC-θ knock-out mice.

[0053] Figure 17 A-17B is diagram figure, tranquillization CD4+T cell, TH1 cell and the TH2 cell that demonstrates PKC-θ knock-out mice lack IL-2 and exist 0.5 μ g/ml anti--shown the IL-4 (Figure 17 A) and the IL-5 (Figure 17 B) of reduction level after cultivating under the condition of CD3.

[0054] Figure 18 is diagram figure, and proving in PKC-θ knock-out mice passive cutaneous anaphylaxis (PCA) model that the antagonism-IgE of embodiment 7 descriptions replys does not below have the swollen increase of ear.The swollen Δ that is expressed as from baseline of ear changes.Statistical study is to use the unpaired t check of Si Shi to determine.The P value that shows is compared wild type with PKC-θ knock-out animal.

[0055] Figure 19 is diagram figure, and proving does not have the swollen increase of ear in the passive cutaneous anaphylaxis that has external source IgE (PCA) model that PKC-θ knock-out mice is described below.The swollen Δ that is expressed as from baseline of ear changes.Statistical study is to use the unpaired t check of Si Shi to determine.The p value that shows is compared wild type with PKC-θ knock-out animal.

[0056] Figure 20 A-20D is a histogram, and the TH1 that demonstrates PKC-θ knock-out mice has shown that with the TH2T cell comparing antagonism-CD3 with the TH2T cell with the TH1 of PKC-θ wild-type mice stimulates the propagation of (0.5 μ g/ml) to reduce.TH0, the TH1 of PKC-θ wild-type mice (light gray bar) or PKC-θ knock-out mice (dark-grey) or TH2 cell in addition with anti--CD28 (Figure 20 A), anti--CD28 add IL-2 (Figure 20 B), need not resist-CD28 and without IL-2 (Figure 20 C) and not anti--during CD28 with IL-2 (Figure 20 D) stimulation.

[0057] Figure 21 A-21D is a histogram, and the TH1 that demonstrates PKC-θ knock-out mice has shown that with the TH2T cell comparing antagonism-CD3 with the TH2T cell with the TH1 of PKC-θ wild-type mice stimulates the propagation of (0.05 μ g/ml) to reduce.TH0, the TH1 of PKC-θ wild-type mice (light gray bar) or PKC-θ knock-out mice (dark-grey) or TH2 cell in addition with anti--CD28 (Figure 21 A), anti--CD28 add IL-2 (Figure 21 B), need not resist-CD28 and without IL-2 (Figure 21 C) and not anti--during CD28 with IL-2 (Figure 21 D) stimulation.

The description of preferred embodiment

[0058] invention is based on the reagent of amount of the reagent of regulating protein kinase C theta (PKC-θ) or regulatory function PKC-θ albumen for the useful discovery of treatment asthma.Here the new discovery of Ti Chuing has been supported to reduce PKC-θ catalytic activity or has been reduced the purposes of the mast cell of the reagent of amount of functional PKC-θ albumen and asthma irritated as target.

[0059] in order to promote understanding, will mention preferred embodiment now, and will use concrete syntax to describe them inventive principle.But should be appreciated that, therefore plan limits invention scope, these of invention change and further revise and as these further application of the graphic inventive principle of institute here, will be contemplated to for the relevant those skilled in the art of invention is normal generation.

[0060] patent of quoting here and scientific literature have been established the obtainable knowledge of those skilled in the art.Application (U.S. and foreign country) of authorizing United States Patent (USP), the application of allow authorizing, announcement of quoting here and list of references comprise that the GenBank database sequence introduces by reference, and be all concrete and show the same degree that is introduced into by reference separately as every kind.

[0061] PKC-θ is the member who does not rely on the new classification of PKCs of Ca+2.It is high expressed in T cell and muscle.As described here, have been found that PKC-θ albumen works in such as asthma in breathing problem, and with for example induce symptom relevant and/or complication relevant with asthma, comprise the allergic asthma that for example comprises IgE-mediation asthma; Non-allergic asthma, occupational asthma and drug-induced asthma.Based on the discovery that proposes here, invention provides the compositions and methods of differentiating treatment asthma, and provides by treat the method for asthma to the reagent of adjusting (as by suppressing or enhancing) the PKC-θ generation of administration treatment effective dose and/or kinase activity.In addition, invention provides the mast cell of the separation that lacks endogenous PKC-θ protein expression.

[0062] on the one hand, invention provides the method for the correctives that is used to differentiate PKC-θ albumen.This method comprises PKC-θ albumen or its functional fragment is contacted with test agent; And whether definite test agent suppresses the kinase activity of PKC-θ albumen or its functional fragment.The test agent that reduces the kinase activity of PKC-θ albumen or its functional fragment just is the correctives of PKC-θ albumen by discriminating.

[0063] as used herein, " test agent " is chemicals (as organism or inorganics), micromolecular compound, nucleic acid molecules, peptide or protein, such as hormone, antibody, and/or their part.By---increase or the reduce---reagent of the kinase activity of PKC-θ albumen or its functional fragment that is to regulate of " correctives of PKC-θ albumen " expression, the reagent of amount (as by transcribing or translating) that perhaps can regulatory function PKC-θ albumen.In some embodiments, the correctives of the PKC-θ albumen of reduction kinase activity is the inhibitor of PKC-θ albumen or its functional fragment.In some embodiments, the correctives of the PKC-θ albumen of increase kinase activity is the activator of PKC-θ albumen or its functional fragment.

[0064] in a kind of form of invention, the method that is used to differentiate the correctives of PKC-θ albumen comprises PKC-θ albumen or its functional fragment is contacted with test agent, and the autophosphorylation that detects PKC-θ albumen or its functional fragment changes (as the variation of the phosphorylation of following residue among the SEQ ID NO:1: 695 serine, 685 serine, 538 threonine, 536 threonine).In alternative form, this method comprises PKC-θ albumen or its functional fragment contacted with test agent and PKC-θ substrate, and the phosphorylation that detects PKC-θ substrate changes.Test agent is a kind of reagent that is considered to the amount (for example passing through the RNA of change encoding function PKC-θ albumen or the amount of DNA) of the kinase activity or the functional PKC-θ albumen of effective adjusting (promptly suppressing or increase) PKC-θ albumen or its functional fragment.In certain embodiments, the correctives of PKC-θ albumen makes the kinase activity of PKC-θ albumen or its functional fragment reduce twice at least.In some embodiments, correctives makes the PKC-θ protein kinase activity of PKC-θ albumen or its functional fragment reduce at least four times or at least ten times.In some embodiments, correctives has been eliminated the kinase activity of PKC-θ albumen or its functional fragment.PKC-θ protein kinase activity can be by quantitatively, for example uses the standard technique of analyzing such as vitro kinase that describes below.

[0065] in the another kind of non-limiting embodiments of invention, the amount of functional PKC-θ albumen is reduced by the correctives of PKC-θ albumen.

[0066] as used herein, the meaning of " functional " is PKC-θ albumen or its fragment (for example having and the same kinase activity of wild type PKC-θ albumen) of proper function.Determine whether PKC-θ albumen or its fragment are functional, can be easy to carry out by common skilled biologist.Be used for determining whether described PKC-θ albumen or its fragment are that functional a kind of nonrestrictive method is, analyze (referring to for example Ausubel et al. at the standard protein kinases, eds., Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, Inc., NewYork, New York (1995, till 2003, added renewal subsequently)) middle more described PKC-θ albumen or its fragment and wild type PKC-θ albumen or wild type PKC-θ fragment, the kinases analysis is described in the following embodiments.

[0067] a kind of non-limitative example of PKC-θ protein functional fragment is a PKC-θ kinase domain.Described in following embodiment (particularly embodiment 3), the kinase domain of PKC-θ albumen (be also referred to as " PKC-θ kinase domain " or be called for short " PKC-θ KD ") surprisingly can autophosphorylation.This is surprising, because other enzyme in known this classification can autophosphorylation.As employed, term " PKC-θ kinase domain " is meant the kinase domain of PKC-θ albumen, and it comprises crosses over the protein portion of about amino acid residue 362 to about amino acid residue 706.In some embodiments, the amino acid sequence that provides among the SEQID NO:61 is provided the PKC-θ KD of invention.In some embodiments, the amino acid sequence that provides among the SEQ ID NO:62 (notice that two the-terminal amino acid residue methionines of SEQ ID NO:62 and glycocoll are the expression for the ease of PKC-θ KD fragment, but do not appear in the total length PKC-θ albumen) is provided the PKC-θ KD of invention.

[0068] in some embodiments, the PKC-θ kinase domain of invention is expressed in prokaryotic, such as bacterium, such as Escherichia coli.In some embodiments, PKC-θ kinase domain on following one or more amino acid residues by phosphorylation (for example passing through autophosphorylation): 695 the serine of SEQ ID NO:1,685 serine, 538 threonine and 536 s' threonine.

[0069] in certain embodiments, the correctives of PKC-θ albumen makes the amount of functional PKC-θ albumen reduce twice at least.In some embodiments, the correctives of PKC-θ albumen makes the amount of functional PKC-θ albumen reduce at least four times.In some embodiments, the correctives of PKC-θ albumen makes the amount of functional PKC-θ albumen reduce at least ten times.In some embodiments, the correctives of PKC-θ albumen has been eliminated the amount of functional PKC-θ albumen.The level of functional PKC-θ albumen can for example be used the standard technique such as the Western engram analysis that describes below by quantitatively.

[0070] further, invention provides the another kind of method that is used to differentiate the correctives of PKC-θ albumen, comprise that the cell that will contain functional PKC-θ albumen or its functional fragment contacts with test agent, determine whether test agent has reduced functional PKC-θ albumen or the amount of its functional fragment in cell, the test agent that wherein reduces functional PKC-θ albumen or the amount of its functional fragment in cell just is the correctives of PKC-θ albumen by discriminating.The correctives of these PKC-θ albumen can for example transcribed or translation skill works.

[0071] in certain embodiments, the correctives of PKC-θ albumen is useful for the treatment mammal such as people's respiratory disease.Respiratory disease includes but not limited to asthma (as allergic asthma or non-allergic asthma); Bronchitis (as chronic bronchitis); Chronic obstructive pulmonary disease (COPD) (as pulmonary emphysema); The situation that relates to respiratory inflammation, Eosinophilia, fiberization and excessive mucus generation, for example cystic fibrosis, pulmonary fibrosis and allergic rhinitis.

[0072] in some embodiments, the correctives of PKC-θ albumen is useful for the treatment atopic diseases." idiocrasy " is meant often have one group of disease that the allergic reaction genetic predisposition takes place.The non-limitative example of idiocrasy illness comprises that allergy, allergic rhinitis (hay fever, its symptom comprise itch, have a running nose, sneeze or have a stuffy nose and eye is itched), atopic dermatitis (are also referred to as eczema usually; Cutaneous chronic disease), asthma and hay fever.

[0073] in specific embodiments, the correctives of PKC-θ albumen is useful for the treatment mammal such as people's asthma.As " asthma " the used herein illness that to be meant with labored breathing continuation or paroxysmal be sign, be accompanied by pant, sensation and frequent cough or the outbreak of panting that chest is tight.These any or all symptoms are included as " SOA ".As used herein, " asthma " include but not limited to the combination, exercise-induced asthma (be also referred to as and mix asthma) of non-allergic asthma (being also referred to as the asthma of endogenous or ergotropy), allergic asthma (being also referred to as exogen or atopic asthma), nonallergic and allergic asthma, drug-induced asthma, occupational asthma and late period asthma.Asthma exogen or hypersensitive comprises by anaphylactogen for example, such as incidents that cause or associated such as pollen, spore, grass or weeds, pet soft flocks, dust, mites.Because anaphylactogen and other stimulus occur at the difference of the whole year, the incident of these types is also referred to as seasonal asthma.The extrinsic asthma class also comprises bronchial astehma and allergic bronchopulmonary aspergillosis.

[0074] asthma is the phenotype heterogeneous illness irritated relevant with reversible airflow obstruction with interrupted respiration tract disease symptom such as bronchus.Immuning tissue's pathological characteristics of asthma comprises that airway epithelial for example is exposed, the collagen deposition under the basilar memebrane; Oedema; Mast cells activation; And inflammatory cell infiltration (for example by neutrocyte, acidophic cell and lymphocyte).Respiratory inflammation may further be reinvented and other symptoms of respiratory disease owing to formation, the respiratory tract wall of respiratory tract anaphylaxis, flow limitation, acute bronchoconstriction, mucus plugging.

[0075] can comprise those that cause by infectious agent by this method treatment or the asthma that alleviates, described infectious agent such as virus (as flu and influenza virus, Respiratory Syncytial Virus(RSV) (RSV), paramyxovirus, rhinovirus and influenza virus.RSV, rhinovirus and influenza infection are very general in children, are a kind of main reasons of baby and children's breathing problem.The children of ill toxicity capillary bronchitis may develop into chronic panting and asthma, and it can use method of the present invention to treat.Also comprising may be by the asthma situation of taking exercise and/or cold air causes in some asthmatic patients.Method of the present invention is for expose (induce as cigarette or industrial smog) and industry and occupational exposure with cigarette, such as cigarette, ozone, poison gas, sulphuric dioxide, nitrous oxide, flue dust, comprise the isocyanate from coating, plastics, polyurethane, varnish etc., the asthma that wood, plant or other organic dust etc. are relevant is useful.This method also is useful for the asthma incident relevant with food additives, antiseptic or pharmacologic agent.Method of the present invention also is useful for treating, suppressing or alleviate the various asthma that are called as reticent asthma or CVA.

[0076] in addition, method of the present invention is useful for treatment with alleviating the asthma relevant with the gastroesophageal reflux that can stimulate bronchoconstriction (GERD).

[0077] in some embodiments, asthma is the asthma of IgE-mediation.In specific embodiments, this method further is included in the external or body effectiveness of assessment test agent in the asthmatic model, wherein demonstrates in the asthmatic model to render a service the test agent that increases with respect to contrast agents and just differentiated to being useful for treatment asthma in external or body.

[0078] various asthmatic models are that prior art is known.Soler et al. for example, J.Appl.Physiol.70 (2): 617-23 (1991) and Long et al., J.Appl.Physiol.69 (2): 584-590 (1990) has described the model that is used for the sheep bronchoconstriction.Sheep is natural sensitization to roundworm parasite ascaris suum (Ascaris suum).Suck with ascaris suum antigen attack after, animal stood early stage and late period bronchoconstriction react the reaction when being similar to asthmatic patient and being exposed to the sensitization anaphylactogen.Roundworm is attacked the respiratory tract anaphylaxis also induced sheep, and it is attacking the back as the increase of lung resistance and determined with the cholinergic agonist carbachol.The dosage of carbachol need cause the particular responses that roundworm reduced in attack in back 24 hours, and this is the sign of respiratory tract anaphylaxis.

[0079] Bischof et al. (Clin.Exp.Allergy 33 (3): the model that is used for the sheep allergic asthma has been described 367-75 (2003)), wherein subcutaneous immunity the sheep of dermatophagoides pteronyssinus extract of dissolving carry out the single bronchus with dermatophagoides pteronyssinus subsequently and attack.In this model, bronchoalveolar lavage (BAL) and the peripheral blood lymphocyte of collecting dermatophagoides pteronyssinus bronchus attack front and back are used for flow cytometry, get tissue samples in back 48 hours in attack and be used for histology and immunohistochemical analysis (Bischof et al. sees above).The test agent that is considered to the correctives of PKC-θ albumen, the test agent that particularly is considered to PKC-θ protein inhibitor can be administered to sheep, reduces the ability of the lymphocytic number of BAL by contrast with the lymphocytic number of BAL in the sheep of assessing the test agent of attacking the back and not using invention.

[0080] also having another known asthmatic model is that (referring to for example Gundel et al., Clin.Exp.Allergy 22 (1): 51-57 (1992)) for the non-human primates model of respiratory inflammation of roundworm-induce.Rhesus macaque is natural sensitization to roundworm parasite ascaris suum, and it is by inducing strong IgE and reply and working as anaphylactogen.When attacking in tracheae with antigen, animal has shown the respiratory inflammation of mainly being made up of acidophic cell.This can attack the back 24 hours lymphocytes in the counting inflow bronchoalveolar lavage fluid by lung section anaphylactogen and measure.

[0081] also have another kind of nonrestrictive asthmatic model be ovalbumin in mouse (OVA) respiratory tract anaphylaxis of inducing (referring to for example Kips et al., Eur.Respir.J.22 (2): 374-382 (2003); Taube et al., Int.Arch.Allergy Immunol.135 (2): 173-186 (2004); And Reader et al., Am.J.Pathol.162 (6): 2069-2078 (2003)).In this model, with ovalbumin (OVA) immune mouse in the adsorbed onto alum adjuvant, strengthen, give aerosol with OVA then and attack.When attacking, animal demonstrates the resistance of respiratory tract of increase, and lymphocytic infiltration is in bronchoalveolar lavage (BAL) liquid.In addition, the serum cytokines level increases, and lung tissue has shown that tissue inflammation and mucus produce.

[0082] other non-limiting asthmatic model well known in the prior art be included in ascaris suum antigen in dog and the monkey-induce asthmatic model (referring to for example Hirshman et al., J.Appl.Physiol.49:953-957 (1980); Mauser et al., Am.J.Respir.Crit.CareMed.152 (2): 467-472 (1995)).

[0083] external asthmatic model also is that common skilled biologist is known.For example, for the treatment of T cell-targeting, a kind of nonrestrictive example is the inhibition to the cell factor generation of TH2 cell.The T cell can be at the antibody of stimulated in vitro CD3 and CD28, with the activation of simulation TCR-mediation.This produces the inducing cell factor, and it can be analyzed in the supernatant after 48 hours.Key cytokines is IL-4 and IL-13.IL-13 is the main inducer (referring to for example Wills-Karp M., Immunol Rev.202:175-190 (2004)) of asthma morbidity in the animal model especially.

[0084] correctives that is used to assess PKC-θ albumen is the inhibition of T cell proliferation when replying anti--CD3 and anti--CD28 or inducing of nuclear factor NF-kB or NFAT to the another kind of non-limiting in-vitro method of the effect of asthma.T cell proliferation can for example be passed through ³(referring to method, for example at Ausubel et al., seeing above) analyzed in the picked-up of H-thymidine.In replying t cell activation, NFAT or NF-kB have experienced activation and consideration convey moves, and it can be analyzed by the Western trace of product of cell lysis.

[0085] the PKC-θ protein inhibitor TH2 that should also reduce in the ovalbumin mice immunized replys, and it can produce owing to the minimizing of ovalbumin specific IgG 1 or total IgE and analyze.The level of these antibody can be analyzed by the ELISA of mice serum.

[0086] as used herein, the PKC-θ albumen of invention can be from the people, and can have amino acid sequence shown in the SEQ ID NO:1 (GenBank Accession No:NM_006257).In another embodiment, the PKC-θ albumen of invention can be from mouse, and can have amino acid sequence shown in the SEQ ID NO:2 (GenBank Accession No:NM_008859).PKC-θ albumen useful in the invention also can be by nucleotide sequence coded shown in SEQ ID NO:3 (people) (GenBankAccession No:NM_006257) or the SEQ ID NO:4 (mouse) (GenBank AccessionNo:NM_008859).Encoding the other PKC-θ protein sequence and the nucleotide sequence of these albumen can be at GenBank Accession No:NM_178075 (Niino et al., J.Biol.Chem.276 (39): 36711-36717 (2001); (mouse)); GenBank Accession No.AF473820 (Nonneman and Rohrer, Anim.Genet.34 (1): 42-46 (2003); Pig)) obtain.

[0087] as used herein, nucleotide sequence plans to mean natural or the synthetic linear or continuous array of nucleotide and/or nucleosides, and their derivant.Term " coding (encoding) " and " coding (coding) " are meant such process, by transcribing and translating mechanism, nucleotide sequence offers cell with information by this process, and a series of amino acid can be assembled into the specific amino acids sequence thus to produce polypeptide.The process of coding specific amino acids sequence can relate to and has one or more sequence changes (promptly insert, lack, replace) and do not cause the dna sequence dna that amino acids coding changes, and perhaps it relates to and may change one or more amino acid but do not eliminate functional sequence change by the polypeptide of dna sequence encoding.

[0088] PKC-θ this discovery relevant with inducing of SOA and/or complication makes PKC-θ sequence useful in the compositions and methods of differentiating invention.These methods comprise the potential reagent of analysis for the ability of regulating (as suppressing or enhancing) PKC-θ kinase activity.Useful PKC-θ nucleic acid molecules (as PKC-θ promoter sequence) and albumen not only comprises gene disclosed herein and encoded protein in the analysis of invention, also comprises their variant that has basically same activity with wild type gene and polypeptide.As " variant " used herein, comprise the polynucleotide or the polypeptide that contain one or more disappearances, insertion or replacement, as long as variant keeps and wild type polynucleotide or the same basically activity of polypeptide.With regard to polypeptide, the expection of disappearance variant comprises the fragment that lacks for the unnecessary polypeptide portion of biologically active, inserts the variant expection and comprises that wild type peptide or its fragment are fused to the fused polypeptide of another polypeptide.

[0089] therefore, in certain embodiments, the PKC-θ albumen of invention is the functional variant of total length PKC-θ albumen.Therefore be understandable that PKC-θ albumen is not limited to by nucleotide sequence coded shown in SEQ IDNO:3 or the SEQ ID NO:4.For example, as discussed above, the nucleotides sequence of coding variant amino acid sequence is listed within the scope of nucleotide sequence of encoded pkc-θ.The sequence modification that " silence " that producing does not influence PKC-θ protein function characteristic basically changes such as sequence deletion, insertion or replacement, is specially expected here.For example, be understandable that reflection genetic code degeneracy or the nucleotide sequence change that causes chemical equivalence amino acid to produce at specific site are as expected.Therefore, the codon of hydrophobic amino acid ala can be encoded another more not the codon of hydrophobic residue replace such as glycocoll, perhaps replaced by more hydrophobic residue such as valine, leucine or isoleucine.Same, cause a negative charge residue to be substituted by another variation, such as replacing glutamic acid with aspartic acid, perhaps positive charge residue is substituted by another variation, such as replacing arginine, also can expect to produce biology PKC-θ of equal value albumen with lysine.

[0090] with regard to the use in the analysis described here, PKC-θ albumen can be bought from each suppliers is commercial, and (Madison WI), perhaps can produce by those of skill in the art known genetic engineering and method for purifying proteins such as Panvera.For example, the nucleotide sequence of encoding mammalian PKC-θ albumen can be introduced in the host cell of expectation, be cultivated, separate and purifying.Can at first these nucleotide sequences be inserted in the recombinant expression carrier suitable or other expectation.For example, the nucleotide sequence subclone of encoding mammalian PKC-θ albumen can be expressed in the pcDNA3 expression vector and in people's 293 cells, described in following embodiment.PKC-θ albumen or the expression of PKC-θ kinase domain in prokaryotic also are as expected.For example, described in following embodiment, can with PKC-θ albumen or PKC-θ kinase domain subclone to bacterial expression vector such as pET16b (for example commercial available from EMDBiosciences/Merck Biosciences.San Diego, CA) in, and in bacterial cell, express.As used herein and as be known in the art, " carrier " is meant and comprised the construct that designs the genetic stocks that instructs the target cell conversion.That carrier can contain multiple location and the directed genetic elements of order promptly operationally is connected with other element essential or expectation so that the nucleic acid in the nucleic acid box can be transcribed, and if expect, in cells transfected, translate.

[0091] recombinant expression carrier can make up by above-mentioned nucleotide sequence is inserted in the carrier, it carries out according to the known method of those of skill in the art, for example as Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Springs HarborLaboratory, 2 ^NdEd., described in the Cold Springs Harbor, New York (1989).Other list of references of describing molecular biology and recombinant DNA technology also is discussed in for example DNA Cloning 1:Core Techniques, (D.N.Giover et al., eds., IRL Press, 1995); DNA Cloning 2:Expression Systems, (B.D.Hameset al., eds., IRL Press, 1995); DNA Cloning 3:A Practical Approach, (D.N.Glover et al., eds., IRL Press, 1995); DNA Cloning 4:Mammalian Systems, (D.N.Glover et al., eds., IRL Press, 1995); Oligonucleotide Synthesis (M.J.Gait, ed., IRL Press, 1992); NucleicAcid Hybridization:A Practical Approach, (S.J.Higgins and B.D.Hames, eds., IRL Press, 1991); Transcription and Translation:APractical Approach, (S.J.Higgins ﹠amp; B.D.Hames, eds., IRL Press, 1996); R.I.Freshney, Culture of Animal Cells:A Manual of BasicTechnique, 4th Edition (Wiley-Liss, 1986); And B.Perbal, A PracticalGuide To Molecular Cloning, 2nd Edition, (John Wiley ﹠amp; Sons, 1988); With Current Protocols in Molecular Biology (Ausubel et al., eds., JohnWiley ﹠amp; Sons), they are regular and periodically update.

[0092] useful variety carrier is known in invention.Suitable carriers comprises plasmid vector, viral vectors, comprise that retroviral vector (for example referring to Miller et al., Methodsof Enzymology 217:581-599 (1993)), adenovirus vector are (for example referring to Erzurumet al.Nucleic Acids Res.21:1607-1612 (1993); Zabner et al., NatureGenetics 6:75-83 (1994); And Davidson et al., Nature Genetics 3:219-223 (1993)), adeno-associated virus vector is (for example referring to Flotte et al., Proc.Natl.Acad.Sci.USA 90:10613-10617 (1993)) and herpesvirus vector (for example referring to Anderson et al., Cell Mol.Neurobiol.13:503-515 (1993)).Carrier can comprise to nucleic acid in particular host cell effective expression other known genetic elements of essential or expectation, comprise regulating element.For example, carrier can comprise promoter and any essential and the collaborative enhancer sequence that realizes genetic transcription of promoter." enhancer " mean can irritation cell such as the promoter activity in the eukaryotic host cell nucleotide sequence elements.

[0093] as herein defined, when nucleotide sequence be placed in another nucleotide sequence functional relationship in the time, nucleotide sequence " is operably connected " to another nucleotide sequence.For example, if when coded sequence is operably connected to promoter sequence, this means that generally promoter can promote transcribing of coded sequence.Being operably connected means connected dna sequence dna adjacency typically, adds two protein-coding regions in case of necessity, its in abutting connection with and in reading frame.Yet because enhancer also can work when being separated by several thousand bases with promoter, intron sequences can be a variable-length, so some nucleotide sequences can be operably connected, but adjacency not.

[0094] for the nucleotide sequence of encoded pkc-θ albumen is introduced host cell, the known method of many prior aries is available, and described nucleotide sequence can be included in the recombinant expression carrier.These methods include but not limited to mechanical means, chemical method, lipophilicity method and electroporation.Representational mechanical means comprises the use of microinjection for example and particle gun, with golden particulate for example as the matrix that will introduce DNA.Representational chemical method comprises for example uses calcium phosphate or DEAE-Dextran.Representational lipophilicity method comprises uses liposome and other cation reagent to carry out fat mediation transfection.These methods are that prior art is known, many such methods are described in for example Gene Transfer Methods:Introducing DNAinto Living Cells and Organisms, (P.A.Norton and L.F.Steel, eds., Biotechniques Press, 2000); And Current Protocols in MolecularBiology (Ausubel et al., eds., John Wiley ﹠amp; Sons), they are regular and periodically update.

[0095] the various host cells that can utilize wide scope in the present invention are used for screening for example described herein and analyze to produce PKC-θ albumen or its functional fragment of the amount of expecting.These cells comprise eucaryon and prokaryotic, comprise mammal and bacterial cell that prior art is known.Many host cells can be commercial available from American type culture collection, Manassas, VA.

[0096] PKC-θ albumen or its functional fragment can separate and purifying by those of skill in the art's technique known, comprise chromatogram, electrophoresis and centrifugation technique.These methods are that prior art is known, can be at for example Current Protocols in Protein Science, J.Wiley andSons, New York, NY, Coligan et al. (Eds.) (2002); And Harris, E.L.V., and S.Angal in Protein Purification Applications:A PracticalApproach, Oxford University Press, New York, NY finds in (1990).

[0097] be the PKC-θ albumen that helps recombinant production or the purifying and the detection of its functional fragment, can be to PKC-θ albumen or its functional fragment through engineering approaches so that it be " marked ".In some embodiments below, PKC-θ albumen and PKC-θ kinase domain (non-limitative example of PKC-θ protein functional fragment) carry out mark with histidine mark.This protein combination that can make the his-mark to nickel-NTA, thereby be purified.Among other embodiment below, PKC-θ albumen carries out mark with hemagglutinin (HA) mark and expresses in 293 cells.Commerce that other is nonrestrictive, can be used for helping purifying and/or detect PKC-θ albumen (or its functional fragment) can obtain mark and include but not limited to that myc mark (being attached to anti--myc labelled antibody), GST mark (are attached to glutathione-Sepharose) and flu mark (being attached to anti--flu labelled antibody).

[0098] for determining whether test agent suppresses the kinase activity of PKC-θ albumen or its functional fragment, a kind of non-limiting analysis that can adopt is that PKC-θ albumen (or its functional fragment) is contacted the time period of enough suppressing PKC-θ protein kinase activity with test agent.This time period can change according to the character of selected inhibitor and PKC-θ albumen or its functional fragment.Such time can easily be determined without undo experimentation by those of skill in the art.Nonrestrictive invention test agent is to reduce the reagent of PKC-θ (or its functional fragment) kinase activity, although also be as expected by for example being attached to the test agent that PKC-θ substrate suppresses PKC-θ or other mechanism suppresses PKC-θ kinase activity by some.

[0099] describes as following, when the IgE acceptor is crosslinked, induced phosphorylation on the residue of PKC-θ below at least one in BMMC: 695 serines of SEQ ID NO:1,685 serines, 538 threonines or 536 serines.Therefore, in specific embodiments, test agent can be confirmed as suppressing the reagent (thereby be useful for treatment asthma) of PKC-θ kinase activity by the ability that it suppresses PKC-θ albumen autophosphorylation.In some embodiments, the autophosphorylation of the amino acid residue of PKC-θ albumen activation ring is suppressed.

[0100] can utilize many analyses to determine whether test agent suppresses the kinase activity of PKC-θ albumen.Because PKC-θ albumen is kinases, these analyses are included in when having a kind of phosphoric acid such as triphosphoric acid (ATP) adenosine or can be transferred to other phosphoric acid of PKC-θ substrate, measure the influence of test agent to PKC-θ ability of autophosphorylation it self on 538 threonine residues.Same, these analyses can be measured the influence of test agent to PKC-θ phosphorylation PKC-θ substrate ability when phosphoric acid exists.Can utilize based on radioactive analysis with based on inactive analysis, comprise analysis based on fluorescence.Based on radioactive assay determination for example [γ-32P]-ATP mix in the PKC-θ substrate and and measure by liquid scintillation counting (LSC).Adopt external substrate phosphorylation and based on other analysis of the colorimetric detection of antibody or other detection method from various sources easily commercial the acquisition, comprise Promega (Madison, WI; CatalogNos.V7470 and V5330), Calbiochem (San Diego, CA; Catalog Nos.539484,539490,539491), Panvera Discovery Screening (Madison, WI; Catalog Nos.P2747 and P2748; It is Invitrogen, Carlsbad, the subsidiary company of CA).The on-radiation analysis comprises that (it comprises the phosphorylation of the substrate with R-X-X-S/T consensus motif and the substrate that passes through the fluorescence polarization determination phosphorylation for Madison, WI) those that sold by Panvera.

[0101] in a limiting examples, available anti--the IgE receptor antibody stimulate be exposed to test agent and ³²The BMMC of P-ATP is with crosslinked IgE acceptor.After crosslinked 15 minutes, then can be with lysis.Next, the available commercial antibody mediated immunity that obtains precipitates endogenous PKC-θ (for example uses Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) anti--PKC-θ antibody of providing of commerce, it is described in the following embodiments), differentiate by SDS-PAGE.The PKC-θ of the BMMC that handles with the test agent that suppresses PKC-θ autophosphorylation will demonstrate the phosphorylation of comparing with the PKC-θ of untreated cell to be reduced (promptly ³²P-ATP mixes minimizing).

[0102] in the substituting of this example, do not having ³²During P-ATP BMMC is exposed to test agent.At anti--IgE acceptor after crosslinked 15 minutes, with lysis, the endogenous PKC-θ of immunoprecipitation also differentiates by SDS-PAGE.(commercial available from for example Zymed Laboratories Inc., San Francisco CA) carries out the Western engram analysis with anti--phosphothreonine antibody with the SDS-PAGE gel then.The PKC-θ of the BMMC that handles with the test agent that suppresses PKC-θ autophosphorylation will demonstrate the phosphorylation of comparing with the PKC-θ of untreated cell to be reduced (promptly ³²P-ATP mixes minimizing).

[0103] PKC-θ kinase activity also can be determined by the ability of its phosphorylated substrate.Therefore, can be with various oligopeptides and peptide substrate are used in the analysis to measure PKC-θ kinase activity widely.Useful peptide has total R-X-X-S/T motif (wherein R is an arginine, and X is unknown or any known amino acid in invention; S is that serine and T are threonines).Other protein substrate includes but not limited to the C-kinase substrate (MARCKS) that is rich in alanine (the amino acid sequence KKRFSFKKSFK (SEQ ID NO:5) of nutmeg acidylate; wherein underlined serine residue is by phosphorylation), PKC-α intends substrate (amino acid sequence FARKGSLRQKN (SEQ ID NO:6), wherein underlined serine is by phosphorylation.Use the peptide array technique, identified several potential PKC-θ substrates, they can contain the sequence of PKC-θ physiological substrate uniqueness, may be the therapeutic purposes (seeing Fig. 9 B and the following examples 4) that has same application with PKC-θ.Substrate can have various modifications, as long as substrate participates in the reaction by PKC-θ catalysis.

[0104] also having the method for the another kind of PKC-of mensuration θ kinase activity is to measure its autophosphorylation ability.Among the embodiment that is described below, when finding surprisingly that PKC-θ kinase domain is expressed in bacterial cell by phosphorylation.This phosphorylation is owing to autophosphorylation, because bacterial cell can phosphorylating protein.Therefore, invention also provides the method that is used for differentiating to the immune disorders useful reagent of treatment mammal, and this method is to contact with test agent by the cell that will express PKC-θ albumen (or its functional fragment); Determine whether test agent has reduced the autophosphorylation of PKC-θ albumen (or its functional fragment) in cell, wherein reduce the test agent of PKC-θ albumen (or its functional fragment) and just differentiated to treating the immune disorders useful reagent.In some embodiments, cell is bacterial cell (as Escherichia coli).In some embodiments, immune disorders is an asthma.

[0105], can make cellular expression PKC-θ albumen or its functional fragment by the nucleotide sequence of encoded pkc-θ albumen or its functional fragment is introduced in the cell according to invention.As mentioned above, nucleotide sequence is operably connected to the adjusting sequence (as promoter sequence and enhancer) that allows cellular expression PKC-θ albumen (or its functional fragment).Common those of skill in the art will understand that the type of the cell that the required adjusting sequence type of nucleotide sequence expression of realization encoded pkc-θ albumen (or its functional fragment) will be introduced into according to the nucleotide sequence of encoded pkc-θ albumen (or its functional fragment) changes.For example, if cell is a bacterial cell, so preferably use the adjusting sequence of bacterial cell.The adjusting sequence that is used for many dissimilar cells (as insect, mammal and bacterium) be commonly known in the art (referring to for example Ausubelet al., Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, New York, NY, it is regular and periodically updates).

[0106] also have on the other hand, invention provides the method that is used for differentiating to treatment mammal immune disorders such as asthma useful reagent, by functional PKC-θ albumen or PKC-θ kinase domain are contacted with test agent; And whether definite test agent reduces the autophosphorylation of functional PKC-θ albumen or PKC-θ kinase domain, wherein reduces the test agent of functional PKC-θ albumen or PKC-θ kinase domain autophosphorylation and is just differentiated to treating the immune disorders useful reagent.In some embodiments, contact is carried out external.

[0107] in some embodiments, functional PKC-θ or PKC-θ kinase domain carry out with contacting in damping fluid of test agent.In some embodiments, damping fluid has the high total ionic strength adjustment buffer degree with respect to the ionic strength of finding in the cell (approximately 100mM NaCl).For example, in some embodiments, damping fluid has the ionic strength of 100mM at least.In some embodiments, damping fluid has 200mM or the ionic strength of 250mM at least at least.

[0108] in certain embodiments, contain NaCl in functional PKC-θ albumen or PKC-θ kinase domain and the contacted damping fluid of test agent.For example, damping fluid can contain at least 50mM NaCl (noticing that other salt (promptly except that NaCl) can be present in the damping fluid).In some embodiments, damping fluid contains 100mM NaCl or 150mMNaCl or 200mM NaCl at least at least at least.In some embodiments, damping fluid contains 250mM NaCl at least.Certainly, common those of skill in the art will understand, be different from or the salt except NaCl can be used to obtain the damping fluid of high ionic strength.Some non-limitative examples of these salt comprise ammonium acetate, sodium acetate and potassium chloride.

[0109] according to invention, " immune disorders " means the undesired illness that works of immune system cell (as T cell, B cell, natural killer cell, mast cell, neutrophil and macrophage) wherein.In some embodiments, immune disorders is an asthma.Other immune disorders includes but not limited to autoimmune disease (such as type i diabetes and rheumatoid arthritis), graft rejection and breathing problem, and such as allergy, immunocyte works therein.

[0110] therefore, invention provides the method that is used to differentiate to treatment immune disorders such as asthma useful reagent, and it regulates the reagent of (as reducing) functional PKC-θ protein level or the reagent of adjusting (as reducing) PKC-θ kinase activity by differentiating.The reagent of regulating (as reducing) functional PKC-θ albumen generation or PKC-θ kinase activity includes but not limited to micromolecular compound, chemicals, nucleic acid molecules, peptide and protein, such as hormone and antibody.Reagent also comprises for example oligonucleotides or polynucleotide, such as antisense RNA and siRNA s (siRNA).Antisense base sequences and siRNAs typically comprise and the nucleotide sequence part complementation of target nucleotide sequences or that can hybridize with the part of target nucleotide sequences.In a kind of non-limitative example, antisense base sequences and/or siRNA hybridize to nucleotide sequence CAGAATATGTTCAGGAACTTTTCCTTCATGAACCCCG (SEQ IDNO:7), its encoding amino acid sequence QNMFRNFSFMNP (SEQ ID NO:8), corresponding to amino acid residue 688 to 699, contain serine residue at 695, this is that the T538 autophosphorylation is required.In another non-limitative example, antisense RNA and/or siRNA hybridize to nucleotide sequence GGAGATGCCAAGACGAATACCTTCTGTGGGACACCT (SEQ ID NO:9), its encoding amino acid sequence GDAKTNTFCGTP (SEQ IDNO:10), corresponding to amino acid residue 532 to 543, contain threonine at 536 and 538, at least required (referring to for example Fig. 2 B and the 2C) of first kinase activity.Antisense base sequences can have the length of about 20 nucleotide, but can perhaps can be the full-length gene target at about 20 length ranges to about 200 nucleotide.Those of skill in the art can select the suitable target and the antisensenucleic acids of suitable length by the known standard program of prior art, to have the curative effect of expectation, for example at Methods in Enzymology, AntisenseTechnology, Parts A and B (Volumes 313 and 314) (M.Phillips, ed., Academic Press, 1999) described in.The non-limitative example of useful antisense molecule is to be described in Bennett et al. among the present invention, U.S. Patent No. 6,190, and those in 869 (issue February 20 calendar year 2001) are incorporated into thus by reference.

[0111] RNA disturbs and to relate to gene silencing sequence-specific, after transcribing, be by with (Lee, N.S.etal., the Nature Biotech.19:500-505 (2002)) that caused by the little interference double-stranded RNA fragment of reticent gene target homology.These siRNA are target and remove natural mRNA molecule specifically.The method of utilizing siRNAs Profilin matter to produce is that prior art is known, is disclosed in for example PCT international application number WO 01/75164; WO00/63364; WO 01/92513; WO 00/44895; And WO 99/32619.

[0112] functional PKC-θ albumen produces or other reagent of adjusting (as reducing) PKC-θ kinase activity includes but not limited to block the reagent that PKC-θ transfers to surface of cell membrane to can be used for regulating (as reducing).Other reagent that can be utilized comprises those that find in the screening analysis of describing here.

[0113] other reagent, perhaps the inhibitor of PKC-θ or antagonist comprise that specificity for example is attached to the antibody and the micromolecule of PKC-θ albumen or PKC-θ protein part." specificity combination " means the antibody of invention with at least 10 ^-5Dissociation constant (the K of M _D) or at least 10 ^-6The K of M _D, or at least 10 ^-7The K of M _D, or at least 10 ^-8The K of M _D, or at least 10 ^-10The K of M _DIdentification also is attached to PKC-θ albumen (or its part).Be used for determining that the standard method of combination and binding affinity is known.Therefore, provide specificity to be attached to the antibody of PKC-θ albumen here.

[0114] specificity used herein is attached to the antibody of PKC-θ albumen and can is, but be not limited to, polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, genetically engineered antibody, bispecific antibody, antibody fragment (include but not limited to " Fv ", " F (ab ') ₂", " F (ab) " and " Dab ") and the strand of performance antibody activity part.The method that produces each above-mentioned antibody formation is that prior art is known.

[0115] for example, polyclonal antibody can inject the antibody that various animals and separation of serum produce by the acidic mammalian PKC-θ albumen with purifying and obtain, be described in for example Ausubel et al. more fully, Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, it is regular and periodically updates.Antibody can be monoclonal antibody, and its production method is that prior art is known.

[0116] specific monoclonal antibody can commercially obtain, perhaps in addition can be by Kohler and Milstein, and the technology of Eur.J.Immunol.6:511-519 (1976) and improvement thereof or change prepare.In brief, these methods comprise that preparation can produce the immortal cell line of expectation antibody.The generation of immortal cell line can be injected animal such as mouse by the antigen that will select, be merged to form hybridoma from the spleen results B cell of animal and with cell and myeloma cell.Can select single colony and test the ability that the high-affinity antibody of epi-position is expected in their secretions by conventional program of the prior art.

[0117] alternatively, can pass through the known the whole bag of tricks recombinant production antibody from expression library of prior art.For example, can be from separating from lymphocyte, preferable separate from bone-marrow-derived lymphocyte and more preferably separate from the RNA (ribonucleic acid) (RNA) of the animal of having injected expectation antigen and produce cDNA.CDNA such as the various immunoglobulin genes of coding can be amplified and be cloned in the suitable carrier, such as Vector for Phage Display by PCR (PCR).These carriers can be added in the bacterial suspension, preferably include in the colibacillary suspending liquid, can produce bacteriophage or phage particle, and it has showed the corresponding antibodies fragment that is connected to the phage particle surface.Can comprise that the phage particle of expecting antibody makes up Ya Wenku by screening, it passes through for example affinity purification technology of the known method of prior art, such as elutriation.Can utilize inferior library to come then from the cell type of expectation, such as separation antibody in bacterial cell, yeast cells or the mammalian cell.Method and improvement thereof as production recombinant antibodies described herein can be at for example Griffiths, W.G.et al., Ann.Rev.Immunol.12:433-455 (1994); Marks, J.D.et al., J.Mol.Biol.222:581-597 (1991); Winter, G.and Milstein, C., Nature 349:293-299 (1991); Hoogenboom, H.R.and Winter, G., J.Mol.Biol.227:381-388 finds in (1992).

[0118], before being used to produce antibody, at first can pass through those skilled in the art's technique known purifying PKC-θ albumen previously discussed here in order to be used for the present invention.

[0119] the further embodiment of invention provides the non-limiting way that reduces the amount of test agent by the prescreen test agent.For example have only those to have the test agent that is attached to PKC-θ albumen ability or instruct in the functional selection that the promoter of PKC-θ gene expression just can be used to invent.

[0120] in non-limiting instance, at first filler test reagent is attached to the ability of PKC-θ albumen, and the PKC-θ albumen of separable purifying also is used for filler test reagent.For example, can test agent be contacted with the immobilization PKC-θ albumen of purifying with the PKC-θ proteopexy of purifying at (for example on sepharose 4B or the plastics) on the solid phase surface.In an alternative embodiment, after PKC-θ is exposed to test agent, can adds at the antibody of PKC-θ albumen and be used for immunoprecipitation PKC-θ albumen, with determine test agent whether with PKC-θ albumen coimmunoprecipitation.Have only those test agent that can be attached to PKC-θ albumen just to be used to functional analysis at next step, with determine whether they can regulate (as reducing) PKC-θ kinase activity or adjusting (as reducing) cell, such as the amount of functional PKC-θ albumen in mast cell or the T cell (as TH1 or TH2 helper cell).

[0121] in limiting examples, at first filler test reagent is attached to the ability of PKC-θ promoter, can be with the immobilization of PKC-θ promoter sequence, as in the DNA microchip array.Can screen the ability that different test agent are attached to promoter then.Have only those test agent that can be attached to PKC-θ promoter just to be used in the functional analysis, with (as the reducing) cell of determining whether they can regulate, such as the amount of functional PKC-θ albumen in mast cell or the T cell (as TH1 or TH2 helper T lymphocyte).

[0122] further, invention provides the method that is used for differentiating to treatment mammal (as the people) asthma useful reagent, the nucleotide sequence that comprises the coding reporter product that will be operably connected to PKC-θ promoter contacts with test agent, determine whether test agent reduces the generation of reporter product, wherein reduce the test agent of reporter product generation and just differentiated to be for treatment asthma useful reagent.In certain embodiments, the nucleotide sequence that is operably connected to the coding reporter product of PKC-θ promoter is in cell (as mast cell or T cell, such as TH1 or TH2 helper T lymphocyte).

[0123] nucleotide sequence of PKC-θ promoter is determined by means commonly known in the art.The nucleotide sequence that a non-limitative example of these methods is to use PKC-θ screens the genomic library (as YAC people's gene group library) of interested promoter sequence as probe, then the nucleotide sequence 5 of separate probe institute combination ' nucleotide sequence.Another non-limitative example of determining the method for suitable promoter sequence is the Southern engram analysis that carries out the human gene group DNA, this is by detecting the human gene group DNA of resolution with probe (for example having comprised the nucleotide sequence of coding people PKC-θ albumen or the probe of its part) electrophoresis, determining the position of cDNA probe hybridization then.In case determine the band of probe hybridization, just band can be separated (as gel is cut away) and carries out sequential analysis.So just allowed the detection of the nucleotide fragments of nucleotide ATG (being the initial of transcription site) 5 '.This nucleotide fragments is the promoter of PKC-θ, can carry out sequential analysis.This nucleotide fragments can be in the length of about 500 to 1000 nucleotide.The present invention also comprises with these sequences having about at least 70%, about at least 80% or about at least 90% homogeneity and as for example the work nucleotide sequence of the gene expression of instructing coding PKC-θ albumen described herein of promoter.

[0124] various widely reporters can be operably connected on the above-mentioned PKC-θ promoter.These gene codifieds are luciferase, beta galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, alkaline phosphatase and green fluorescent protein or other known reporter product of prior art for example.

[0125] in a kind of form of invention, the nucleotide sequence that is operably connected to the coding reporter of PKC-θ promoter is introduced in the host cell.As mentioned above, in invention, can adopt many host cells.At first these nucleotide sequences can be inserted in the recombinant expression carrier suitable or expectation, as previously described here.

[0126] carrier of this form of invention can comprise that other is known to from PKC-θ promoter expression reporter genetic elements essential or expectation, comprises the regulating element in the mammalian cell.For example, carrier can comprise any essential enhancer sequence collaborative in vivo with promoter, for example realizes in vivo that reporter transcribes.The method of nucleotide sequence being introduced host cell is used to produce the same of PKC-θ albumen with previously described.

[0127] nucleotide sequence of coding reporter that will be operably connected to PKC-θ promoter determines with after test agent contacts whether test agent suppresses the generation of reporter product.This terminal point can be determined by the amount or the activity of quantitative reporter product.Quantivative approach will depend on employed reporter, but can relate to the use of the enzyme-linked immuno assay that the antibody with anti-reporter product carries out.In addition, analysis can measure chemiluminescence, fluorescence, radioactivity decay, or the like.If test agent suppresses the generation of reporter product, it just is classified as the reagent of treatment asthma.

[0128] is used for determining that the method active or amount of reporter product described herein is that prior art is known, is discussed in for example Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley ﹠amp; Sons), it is regular and periodically updates.Further describe and can for example find in the following publication what the reporter product of discussing was here analyzed: for luciferase, referring to Nguyen, V.T.et al., Anal.Biochem.171:404-408 (1988); For beta galactosidase, referring to for example Martin, C.S.et al., Bioluminescence and Chemiluminescence:MolecularReporting with Photons pp.525-528 (J.W.Hastings et al., eds., JohnWiley ﹠amp; Sons, 1997); Jain, V.K.and Magrath, I.T., Anal.Biochem.199:119-124 (1991); For beta galactosidase, β-glucuronidase and alkaline phosphatase, referring to for example Bronstein, I.et al.Bioluminescence andChemiluminescence:Fundamentals and Applied Aspects, pp.20-23, (A.K.Campbell et al., eds., John Wiley ﹠amp; Sons, 1994); For chloramphenicol acetyltransferase, referring to Cullen, B., Methods.Enzymol.152:684 (1987); Gorman, C.et al.Mol.Cell.Biol.2:1044 (1982); Miner, J.N.et al., J.Virol.62:297-304 (1988); Sleigh, M.J., Anal.Biochem.156:251-256 (1986); Hruby, D.E.and Wilson, E.M., Methods Enzymol.216:369-376 (1992).

[0129] micromolecule of selectivity inhibition PKC-θ activity also is the agent of treatment treatment of asthma.Selectivity can be restricted to by suppressing PKC-θ than other PKC-θ isoform and surpass about 20 times IC50.(IC50 is defined as producing the inhibitor concentration of 50% inhibitor target activity).

[0130] on the other hand in invention, invention provides the method for treatment asthma, comprise to suffer from asthma or suffer from SOA mammal (as the people) administering therapeutic effective dose reduction PKC-θ catalytic activity or reduce the reagent that functional PKC-θ albumen produces.In one embodiment, mammal is the people.In some embodiments, asthma is the asthma of IgE-mediation.

[0131] " treatment " used herein mean prevention, reduce or eliminate at least a SOA or complication.The amount of " treatment effective dose " expression reagent can suppress or reduce the generation of functional PKC-θ albumen or can suppress or reduce the kinase activity of PKC-θ albumen, and causes noticeable response clinically.Noticeable response includes but not limited to by the prevention of the improvement of the situation for the treatment of or situation clinically.The given dose of the reagent of using will be determined by the relevant particular case of case certainly according to the present invention, comprise the reagent used, by specific asthma and similar state in treating.Treatment of asthma can be by for example reducing respiratory tract anaphylaxis, reduce the excessive generation of mucus, reduce serum IgE level or reducing the respiratory tract Eosinophilia.

[0132] reagent can be administered to mammal by various approach widely, comprises in intestines, parenteral and through surface applied.But for example in the reagent oral administration, nose, in suction, intramuscular, subcutaneous, peritonaeum, in the blood vessel, intravenous, through skin, subcutaneous or they make up arbitrarily and use.

[0133] reagent can be applied in pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier and their formulation are known, and general description is in for example Remington:The Science andPractice of Pharmacy (20th Edition, ed.A.Gennaro (ed.), Lippincott, Williams ﹠amp; Wilkins, 2000).In some embodiments, pharmaceutically acceptable carrier is an aerosol form.Any suitable pharmaceutically acceptable carrier well known in the prior art can use.Carrier can be solid, liquid or solid and mixtures of liquids.When existing with liquid or solid and mixtures of liquids, effectively the carrier of solubilising reagent is preferred.Carrier can be taked form or other form known of capsule, tablet, pill, pulvis, lozenge, suspending liquid, emulsion fluid or syrup.Carrier can comprise the material as aromatic, lubricant, solubilizer, suspending agent, bonding agent, stabilizing agent, tablet disintegrant and encapsulating material.Solid or liquid-carrier can take aerosol form with reagent is transported to their the expectation the position, such as when be used in the suction reagent sprayer in the time.

[0134] whole body or Orally administered tablet can comprise excipient, as be known in the art, such as lime carbonate, sodium carbonate, carbohydrate (as lactose, sucrose, mannitol, D-sorbite), cellulose (as methylcellulose, sodium carboxymethyl cellulose), natural gum (as gum arabic, bassora gum), and disintegrant such as corn, starch or alginic acid, bonding agent such as gelatin, collagen or Arabic gum, and lubricant such as dolomol, stearic acid or talcum.In pulvis, carrier is the solid of fine segmentation, the inhibitor mixed of the fine segmentation of itself and effective dose.In solution, suspending liquid or syrup, with the dissolving of the inhibitor of effective dose and be suspended in carrier such as sterilized water, salt solution or organic solvent such as in the liquid propylene glycol.Other composition can be by being dispersed in inhibitor in the known suitable oil of starch or sodium carboxymethyl cellulose solution or prior art.

[0135] reagent is administered to mammal with the treatment effective dose.Such amount is effective treating asthma or alleviating in the SOA.Whether this amount can be according to the activity of utilization reagent, have any other anti-asthma reagent to use altogether and character, asthma character and the patient health situation of this anti-asthma reagent change.Although this tittle can be determined by those of skill in the art, comprise about 10mg/kg/ days to about 100mg/kg/ days but typically treat effective dose.Certainly, may the lower or higher dosage of needs according to concrete case.When reagent and carrier combinations, they can exist with the amount of about 1% percentage by weight to about 99% percentage by weight, and all the other are made up of pharmaceutically acceptable carrier.

[0136] in certain embodiments, the inhibitor of reagent or PKC-θ generation or catalytic activity can be used in for example comprising one or more anti-asthma combination of agents things altogether.These reagent are that prior art is known, comprise for example β-class adrenergic reagent, comprise isoprel, adrenaline, metaproterenol and terbutaline; Methyl xanthine comprises theophylline, aminophylline and choline theophyllinate; Corticosteroid comprises beclomethasone, betamethasone, hydrocortisone and prednisone; Anticholinergic drug comprises atropine and Ipratropium Bromide; Antihistaminic comprises teldane and astemizole; Calcium channel blocker comprises Verapamil, nifedipine; And mast cell stabilizers, comprise nasmil and nedocromilsodium.

[0137] in some embodiments, reagent is nucleic acid molecules.In certain embodiments, nucleic acid molecules is a ribonucleic acid molecule.In some embodiments, ribonucleic acid molecule comprises the nucleotide sequence with the part complementation of nucleotide sequence shown in the SEQ ID NO:3.In certain embodiments, reagent reduces the amount of the RNA of encoded pkc-θ albumen.In some embodiments, reagent suppresses the translation of the RNA of encoded pkc-θ albumen.In specific embodiments, reagent is the antibody (as polyclone, monoclonal, humanization or chimeric antibody) of specificity in conjunction with PKC-θ albumen or its part.

[0138] further, disclosure of the Invention do not express the cell of endogenous PKC-θ.In certain embodiments, cell is a mast cell.Separate the mouse that this cell can knock out from the PKC-θ that for example describes below (also referring to Sun et al., Nature 404:402-407 (2000)).The method of separating mast cell is known (referring to the method that for example describes below).This cell of not expressing endogenous PKC-θ albumen also can be people's cell, and wherein the gene of encoded pkc-θ is lacked or suddenlys change, so that cell is no longer expressed endogenous PKC-θ.

Whether [0139] mast cell of not expressing endogenous PKC-θ albumen is useful, be to treatment asthma useful reagent for detecting test agent for example.As describing among the following embodiment, the PKC-θ of hemagglutinin (HA)-mark expresses in 293 cells.The PKC-θ of HA-mark can express in mast cell, and the active and/or amount of the PKC-θ albumen of HA-mark is measured in the presence of test agent.Yet because mast cell-expressed endogenous PKC-θ, some test agent may influence endogenous PKC-θ albumen, have therefore shielded its influence to the HA-labelled protein.This shielding can not occur in and lack endogenous PKC-θ and express and express in the mast cell of PKC-θ albumen of HA-mark.And it is useful that these cells influence the different test agent of wild type PKC-θ for those PKC-θ that influence the HA-mark of screening with them.

[0140] in some embodiments, cellular expression external source PKC-θ or its fragment.To mention that now specific embodiment comes illustration above-mentioned composition and method.Should be appreciated that embodiment provides the illustration preferred embodiment, do not plan therefore and the restriction scope of invention.

Embodiment 1

PKC-θ film transposition when TCR stimulates the human T-cell altogether and activation cycli phosphateization

[0141] not having the mouse of PKC-θ (being that PKC-θ knocks out) is to survive, but mature T cells is defective (Sun et al., Nature404:402-407 (2000)) in propagation, IL-2 generation and NF-κ B activation.In people's mast cell (HCMC) of cultivating, confirmed the crosslinked back of IgE acceptor PKC kinase activity rapidly (＜5min) be positioned (Kimata etal., BBRC 3:895-900 (1999)) on the film.Because PKC-θ plays central role and have effect (the Liu et al. of confirmation in rat basophilic chronic myeloid leukemia clone RBL-2H3 cell in the signal transmission of TCR-mediation, J.Leukocyte Biol.69:831-840 (2001)), so detected activation and the function of PKC-θ in BMMC, peritonaeum mast cell and T cell.

[0142] after TCR stimulates, PKC-θ translocates to the center that supermolecule activates compound rapidly, keeps there the most nearly four hours (Huang et al., Proc.Natl.Acad.Sci.USA 99:9369-9373 (2002)).For determine this transposition whether with the phosphorylation of PKC-θ albumen change corresponding, with human T-cell's purifying and analyze PKC-θ transposition and autophosphorylation.

[0143] be purifying T cell, (Colmar PA) obtains monocyte preparation from Biological Specialties.With cell Ficoll-Histopaque (can be commercial available from for example SigmaChemical Co., St.Louis, MO) higher slice is collected buffy coat in centrifugal back.With cell rinsing several in PBS, with 10 ⁶The density of/ml is cultivated in RPMI/10%FCS.Through feminine gender select purifying T cell (Dynal Biotech, Oslo, Norway).Resist-CD3 ε (5 μ g/ml with soluble, with 10 μ g/ml anti--mIgG is crosslinked) and soluble anti--CD28 (5 μ g/ml) stimulate purifying T cell 0,2,10,45 and 60 minutes (anti--CD3 ε and anti--CD28 all commerce available from BD Biosciences, San Jose, CA).

[0144] just analyze, through the cell of centrifugal collection irriate, rinsing once in ice-cold PBS.By cell precipitation being resuspended in the hypotonic lysis buffer [20mMTris-HCl of 100 μ l, pH 7.5,2mM EDTA, 5mM ethylene glycol bis-(B-amino-ether)-N, N, N ', N '-tetraacethyl (EGTA), among every mL leupeptin and press down each 10 μ g of enzyme peptide, proteinase mixture and inhibitors of phosphatases] in the full cell pyrolysis liquid of preparation.Cell suspending liquid is sheared for 30 times by No. 25 syringe needles, then centrifugal 7 minutes of 280xg with precipitate nucleus.Preserving after an aliquot is used for analyzing, with full cell extract through high speed centrifugation (16,000xg) clarification.Collect the cytosol extract, film is deposited in rinsing once is resuspended in the same damping fluid then in the hypotonic lysis buffer, add the 1%NP-40 detergent cracking on ice 30 minutes.Obtain the soluble film fraction of detergent through another time high speed centrifugation step, remaining particle fraction is the insoluble film fraction that contains film microstructure territory (DI fraction) of detergent.This DI fraction boiled in the SDS-PAGE sample buffer analyze.Subcellular fraction protein level lease making 4-20%SDS-PAGE analyzes, transfer on the cellulose nitrate, (commercial with anti--phosphoric acid T538PKC-θ specific antibody available from Cell Signaling Technology, Inc. (Beverly, MA)) Western blotting (seeing Figure 1A) in 5%blotto/TBS-Tween.05%.

[0145] next, the cellulose nitrate trace is peeled off, use to resist-(Santa Cruz CA) detects (seeing Figure 1B) to PKC-θ E7 again for SantaCruz Biotechnology, Inc..At last, shown in Fig. 1 C, for showing the equivalent application of sample at all swimming lanes, once more trace is peeled off, (commerce Inc.) is detected available from Santa Cruz Biotechnology with anti--actin then.

[0146] shown in Figure 1A, stimulate (stimulating) through CD3 and CD28 at TCR after, PKC-θ autophosphorylation on 538 threonine residues in the kinase activator ring.The autophosphorylation incident translocates to supermolecule with PKC-θ and activates compound center consistent (seeing Figure 1B).Shown in Fig. 1 C, in all time processing, found the actin of approximate equivalent.

[0147] therefore, these results show and translocate to supermolecule by PKC-θ to activate the compound center be to follow the inducibility phosphorylation corresponding to the kinase activator ring on amino acid residue threonine 538.

Embodiment 2

It is that kinase activity is required that PKC-θ activates the ring autophosphorylation

[0148] as described in the embodiment 1, when TXi Baoshouti stimulated the human T-cell altogether, the transposition of PKC-θ film was corresponding with the induced phosphorylation followed of kinase activator ring on amino acid residue threonine 538.This activation cycli phosphateization be reported as be kinase function required (Liu et al., Biochemical Journal, 2002,361-255-265).For confirming this report, with terminal hemagglutinin (HA) epi-position of C mark with PKC-θ full-length cDNA subclone in plasmid pcDNA3 (commercial) available from Invitrogen, produce total length (WT) PKC-θ (the nucleotide sequence SEQ ID NO:11 of C end HA epi-position mark; Amino acid sequence SEQ IDNO:12).The PKC-θ of the kinases inactivation of HA-mark is also by being that tryptophane produces with 409 lysine mutations of amino acid.The K409W sudden change of this kinases inactivation produces by subclone PCR product, and confirms (nucleotide sequence SEQ ID NO:13 through order-checking; Amino acid sequence SEQID NO:14), subclone is in the pcDNA3 expression vector.(commerce is available from American type culture collection, Manassas, VA) double with these expression construct transient transfection human embryo kidney (HEK) 293 cells to use fat (use MirusTransIT-LT1 reagent, commercial available from Mirus Corporation, Madison WI).Transfection 24 or after 72 hours collecting cell be used for Western engram analysis and activity.

[0149] with cell cracking under hypotonic cracking condition of results, the centrifugal nucleus (referring to more detailed method among the embodiment 1) of removing.Full cell extract electrophoresis on SDS-PAGE with a replicate determination, transfer on the cellulose nitrate, at first detect, peel off then, detect again with anti--HA antibody (Santa Cruz) with anti--phosphoric acid T538PKC-θ specific antibody (Cell Signaling Technology).Shown in Fig. 2 A, exist the kinases inactivation total length PKC-θ albumen (as by it anti--the HA antibody staining is determined), but on 538 threonine residues not by phosphorylation (determined) as lack anti--pT538PKC-θ antibody staining by it.Therefore, as shown in Fig. 2 A transfection experiment, though wild type kinase activates ring by the available phosphorus acidifying, kinases inactivation type (by 409 catalytic lysine in the mutein is that tryptophane produces, K409W therefore by name) is not by phosphorylation.Although the evidence of other PKC isoform shows that the phosphorylation possibility quilt that activates ring (i.e. 538 threonines) owing to PDK-1 kinases (it is based on the evidence of other PKC isotype), does not have phosphorylation PKC-θ to activate ring yet the result who occurs shows the endogenous PDK-1 that exists in people's embryo 293 nephrocytes here.Also the analytical proof of the phosphoric acid engram analysis of the active kinase domain by bacterial expression and purifying kinase domain activate the autophosphorylation (data not shown goes out) of ring.

[0150] next, use peptide substrates to analyze the vitro kinase activity of the cell liquid extract (being those that use among Fig. 2 A) of same replicate determination.Analyzed the vitro kinase activity of cytosol extract in 96 orifice plates, every hole 5 μ g albumen, final concentration are biotinylation peptide substrates (the amino acid sequence FARKGSLRQ of 83 μ M; SEQ ID NO:15), 166 μ MATP, 0.5 μ l P ³³ATP (specific activity 3000Ci/mmol, 10mCi/ml), 84ng/ μ l phosphatidylserine, 8.4ng/ μ l diacylglycerol, in ADBII damping fluid (20mM MOPS pH7.2,25mM β-glyceraldehyde, 1mM sodium orthovanadate, 1mM DTT, 1mM CaCl ₂) middle final volume 30 μ l, room temperature 30 minutes.Stop the kinases analysis with the damping fluid that contains EDTA, transfer to rinsing on the flicker plate that streptavidin coats, detection of radioactive in the plate readout instrument.From whole counting, deduct having only peptide and having only kinase whose reaction as a setting.

[0151] shown in Fig. 2 B, to compare with wild type PKC-θ albumen, the total length PKC-θ albumen of kinases inactivation has significantly lower kinase activity 24 and 72 hours the time in transfection behind human embryo kidney (HEK) 293 cells.At last, the PKC-θ that has determined wild type and kinases inactivation causes the ability of endogenous substrate IKK (I κ B alpha kinase) phosphorylation.For this reason, with cell cracking in the 1%NP-40 lysis buffer of replicate determination group, the film fraction that detergent is insoluble is transferred on the cellulose nitrate.At first detect the cellulose nitrate trace, peel off then, detect again with anti--IKK α with anti--pIKK α/β, peel off at last and with resist-IKK β detects (all antibody are from Cell SignalingTechnology) again.Shown in Fig. 2 C, wild type PKC-θ, rather than the PKC-θ of kinases inactivation have caused the phosphorylation of IKK-β.

[0152] Fig. 2 B and 2C result displayed confirm to activate ring autophosphorylation (promptly 538 threonine) to be that the active and signal of PKC-θ transmits required, as by as shown in the phosphorylation (Fig. 2 C) of using the cell in vitro lysate kinase activity (Fig. 2 B) that synthesizes substrate and endogenous IKK.These results show that wild type kinase induced the IKK phosphorylation, and kinases inactivation type can't be done like this.These results activate the ring autophosphorylation with PKC-θ and are defined as the uniqueness of therapeutic regulation and novel mechanism.

Embodiment 3

The catalyst mechanism of PKC-θ kinase domain

[0153] next studies to illustrate the catalyst mechanism of new phosphorylation PKC-θ kinase domain (PKC-θ KD).For this reason, expressing also, the PKC-θ KD of purifying catalytic activity is used for the phosphorylation site analysis.For these research, at first express and purifying PKC-θ kinase domain (PKC-θ KD; Amino acid residue 362 to 706)., PKC-θ KD (amino acid residue 362 to 706) is cloned in the pET16b expression vector for this reason, introduces six-histidine mark to the C end.The amino acid sequence of the PKC-θ KD of His-mark is provided at SEQ ID NO:63 (noticing that terminal methionine of N and glycine residue among the SEQ ID NO:63 do not appear among the total length PKC-θ).Plasmid is used for transformed into escherichia coli BL21-DE3 strain with overexpression.With 0.1mM IPTG 10 liters the cell culture 37 ℃ of optical density 0.4 was induced 3 hours at 25 ℃, gather in the crops them then and be resuspended in damping fluid (25mM Tris pH 8.0,25mMNaCl, the 5mM 2 mercapto ethanol, the 5mM imidazoles, 50 μ M ATP and protease inhibitors) in, use the microfluidization device cracking.

[0154] lysate is added on nickel-NTA resin of 20mL, 4 ℃ 1 hour.Subsequently resin is toppled over as chromatographic column, with the thorough rinsing of same damping fluid that comprises the 25mM imidazoles.With the protein of 200mM imidazole buffer elution of bound to the resin.Immediately with the protein application of sample to anion exchanger HQ, with 25mM Tris pH 8.0,25mM NaCl, 5mMDTT, 50 μ M ATP rinsing pillars, differentiate to 500mM NaCl linear gradient by using 25mM then.SDS-PAGE is passed through in the selection that contains the fraction of PKC-θ KD, compiles, and dilutes twice with 25mM Tris pH 8.0,5mM DTT, and application of sample is to the heparin chromatographic column.Flow through hydroxyapatite column immediately, with 25mM Tris pH 8.0, the thorough rinsing of 50mM NaCl, 5mMDTT.0 to 100mM sodium phosphate linear gradient elution target protein.Then protein is carried out size fractionation as monomer on Superdex 200 size exclusion chromatography posts, 4 ℃ of dialysed overnight, concentrate with 25mM Tris pH 8.0,50mM NaCl, 5mM DTT.

[0155] next, carry out mass spectrophotometry.For carrying out this analysis, with PKC-θ KD (at 50mM Hepes pH 7.5,5mM MgCl ₂, among 5mM DTT, 10% glycerine and the 0.0025%Brij-35,0.25 μ g/ μ l) electrophoresis on the 10%Tricine gel (Invitrogen), coomassie brilliant blue staining.Downcut band, (MI) middle (Promega, Madison WI) carry out digestion in the glue with trypsase for GenomicsSolutions, Ann Arbor at ProGest Investigator automat.Reduce sample volume through SpeedVac, reset into the final volume of about 30 μ l with 0.1M acetate.Then peptide being carried out nanoLC/MS/MS analyzes.Briefly, with sample be injected into 75 μ m * 10cm IntegraFrit post (New Objectives, Woburn, MA) on, post be with 10 μ m C18 pearls (YMC, Wilmington, NC) filling.HPLC gradient process 45min is increased to 60% solvent B (solvent orange 2 A, 0.1M acetate/1%CAN by 4% linearly with the flow velocity of 250nL/min; Solvent B, 0.1M acetate/90%ACN).(ThermoFinnigan, San Jose CA) collect mass spectrum with LCQ DECA XP ion trap mass spectrometer.(CA) retrieval MS/MS data are to obtain the otherness phosphorylation modification of PKC-θ on serine, threonine and tyrosine for ThermoFinnigan, San Jose to use the Sequest algorithm.

[0156], use direct mutagenesis (use is commercial available from Stratagene, La Jolla, the kit of CA) in PKC-θ KD expression construct, to carry out various sudden changes for helping to analyze the catalyst mechanism of PKC-θ KD.Sequence through these sudden changes of sequence verification.Expression construct is expressed wild type PKC-θ KD as mentioned above, and (commercial available from BioRad, Hercules CA) behind the estimation protein, analyzes the Escherichia coli lysate of equivalent by Western blotting and kinases analysis analyzing by Bradford.Briefly, by 4-20%SDS-PAGE analytical pyrolysis liquid, transfer on the cellulose nitrate, with commerce available from Cell Signaling Technology (Beverly, MA) (Carlsbad, anti--His antibody CA) carries out Western blotting in 5%blotto/TBS-Tween 0.05% available from Invitrogen for anti--pT538PKC-θ antibody or commerce.

[0157] mass spectrum studies show that PKC-θ KD is by phosphorylation.Carry out in Escherichia coli because PKC-θ KD expresses, wherein do not have serine-threonine kinase, so mass spectrum finds to be exactly the result of expressed kinases autophosphorylation.Predicted molecular weight based on amino acid sequence is 41,615 dalton, yet the molecular weight of determining through ESI-MS is 42,092 dalton and 42,173 dalton (every kind 50%), and this shows 5 or 6 amino acid autophosphorylation in Escherichia coli.Fig. 3 A-3D is the synoptic diagram that shows PKC-θ KD autophosphorylation feature.As shown in Figure 3A, new C2 domain is positioned at the amino terminal of protein, then is two C1 domains in conjunction with co-factor, is the carboxyl terminal kinase domain then.Conservative phosphorylation site (i.e. 538 threonines on synoptic diagram 3A, have been shown, 676 serines, 685 serines, and 695 serines), and below synoptic diagram, shown the terminal and C terminal amino acid residue (respectively at 362 and 706) of PKC-θ KD N.

[0158] in mass spectrophotometry, m/z is than the mass ratio that is peptide, and z (electric charge) is 1.Therefore, m/z is than the quality that has provided fragments of peptides.Mass spectrum product ion analysis of spectrum shows Ser ₆₉₅It is phosphorylation site.

[0159] like this, Fig. 3 B has shown the product ion spectrum of peptide NFpSFMNPGMER (spanning position 693-703) at m/z 705.52, and this confirms Ser ₆₉₅It is phosphorylation site.Fig. 3 C has shown that the product ion spectrum of peptide ALINpSMDQNMFR (spanning position 681-692) at m/z 760.48, shows Ser ₆₈₅It is phosphorylation site.Fig. 3 D has shown that the product ion spectrum of peptide TNTFCGTPDYIAPEILLGQK (spanning position 536-555) is at m/z 1159.71.A phosphoric acid on bright this peptide of the product ion stave of Fig. 3 D shows that also phosphorylation site is Thr ₅₃₆Or Thr ₅₃₈The cysteine residues (representing with # in Fig. 3 D) of noting 540 is alkylating by iodoacetamide institute.

[0160] therefore, hydrophobic motif Ser ₆₉₅With corner motif Ser ₆₈₅Just differentiated and be autophosphorylation site (seeing Fig. 3 B and 3C respectively).Mass spectrum does not detect Ser ₆₆₂And Ser ₆₅₇Any phosphorylation of corner motif residue.According to the homology of other PKC corner motif, Ser ₆₇₆May be autophosphorylation, but this not clear and definite in these researchs, because in the peptide of trypsin treatment, do not detect Ser ₆₇₆

[0161] these researchs have further disclosed the Thr that activates in the ring ₅₃₆Or Thr ₅₃₈Also be (the seeing Fig. 3 D) of autophosphorylation.The X-ray structure of the PKC-θ KD that expresses in the bacterium is measured and is confirmed Thr ₅₃₈Residue is phosphorylation (Xu et al., J.Biol.Chem.279 (48): 50401-50409 (2004)).This result is surprising, because it and former suggestion promptly activate ring by PDK-1 phosphorylation (Balendran et al., FEBS Lett.484:217-223 (2000); LeGood et al., Science 281:2042-2045 (1998)) be opposite.In fact, the previous research of the total length PKC-θ mutant K409W of kinases inactivation has shown that this molecule is at Thr ₅₃₈Not by phosphorylation (Liu et al., Biochem.J.361:255-265 (2002)).The HEK293 cell heterologous expression system of describing among the embodiment 2 above using has also been observed the K409W PKC-θ mutant in the cell and has been lacked Thr ₅₃₈Phosphorylation (data not shown).This discovery means Thr ₅₃₈Phosphorylation is not because K409W kinase mutant body can not autophosphorylation.And, the Thr of K409W PKC-θ molecule ₅₃₈Not phosphorylation with lack cell in vitro lysate kinase activity and endogenous IKK α/β phosphorylation be associated (data not shown).

[0162] because proposed in the past, PKC-θ activates ring by PDK-1 phosphorylation (Balendran et al., FEBS Lett.484:217-223 (2000); LeGood et al., Science 281:2042-2045 (1998)), the Thr that has shown the PKC-θ KD that expresses in the bacterium ₅₃₆Or Thr ₅₃₈By the mass spectrometry results of autophosphorylation is surprising (seeing Fig. 3 B-3D).This part ground explains that by the x-ray structure it has disclosed the Thr of phosphorylation ₅₃₈Thr with the front ₅₃₆Side chain is interaction of hydrogen bond (Xu et al., J.Biol.Chem.279 (48): 50401-50409 (2004)).This interaction may further stablize activate in the ring and with the interaction of α C-spiral, the two all relevant (Johnson et al., Cell 85:149-158 (1996)) with catalytic action.

[0163] Yi Qian research has proposed the conformation that PKC-θ has catalytic capability, wherein activates ring by composing type phosphorylation (Newton, A.C., Biochemical Journal.370:361-371 (2003)).PDK-1 activates cycli phosphate PKCs and other AGC family kinase at kinase domain, as required modification (Newton before the autophosphorylation that occurs in the conservative site on hydrophobic and the corner motif, A.C., Biochemical Journal.370:361-371 (2003); Balendran et al., FEBS Lett.484:217-223 (2000)).Here the result who occurs discloses, and opposite with popular hypothesis, PKC-θ is autophosphorylation uniquely.Here the discovery about PKC-θ KD feature of Ti Chuing has proposed evidence and has supported: the hydrophobic and corner motif in kinase domain, activate ring also by autophosphorylation (seeing Fig. 3 B-3D).PDK-1 has also been got rid of in these researchs can activate the possibility of encircling by phosphorylation PKC-θ in cell.Yet, opposite (Smith et al. with the PKC-θ that expresses in the bacterium, J.Biol.Chem.277:45866-45873 (2002)), the discovery that presents in the present embodiment shows that PKC-θ KD can activate ring place autophosphorylation at PKC-θ, so PDK-1 phosphorylation demand just not necessarily.

[0164] mass spectrometric data show the PKC-θ KD that expresses in the bacterium at 5 or 6 amino acid residue places by autophosphorylation.The phosphorylation site of differentiating in these experiments comprises hydrophobic motif Ser ₆₉₅, corner motif Ser ₆₈₅, activate ring Thr ₅₃₈Or Thr ₅₃₆In the peptide of trypsin treatment, do not detect corner motif Ser ₆₇₆Although, according to sequence homology it also may be by phosphorylation.Ser ₆₈₅It is the new corner motif autophosphorylation site of differentiating.At last, except the above-mentioned phosphorylation site of differentiating, other at least 2 amino acid residues are not still arrived by these technology for detection by autophosphorylation.

[0165] the amino acid residue Thr in the activation ring ₅₃₈Be kinase activity required (Liu etal., Biochem.J.361:255-265 (2002)).Therefore, checked that the point mutation of several phosphorylation sites encircles Thr to activating in the kinase domain ₅₃₈The influence of autophosphorylation.For this reason, use anti--pT ₅₃₈PKC-θ antibody is analyzed the Escherichia coli lysate of PKC-θ KD albumen and various mutant by the Western engram analysis.Shown in Fig. 4 A, the sudden change fragment of having only wild type PKC-θ KD albumen and three tests on 538 tyrosine by phosphorylation.Also use the equivalent application of sample of detecting to determine swimming lane again (seeing Fig. 4 B) of anti--His antibody staining by peeling off trace.Also carry out the lysate kinases analysis of these Escherichia coli lysate fractions.These kinases analyses are with final concentration 83 μ M biotinylation peptide substrates (FARKGSLFQ), 166 μ M ATP, the 0.5 μ l of P of 30 μ l ³³ATP (specific activity 3000Ci/mmol, 10mCi/ml), 84ng/ μ l phosphatidylserine, 8.4ng/ μ l diacylglycerol in 20mM MOPS pH 7.2,25mM β-phosphoglycerol, 1mM DTT, 1mM CaCl ₂Carried out 30 minutes in room temperature.The reactant liquor point sample of five to ten μ l is on cellulose phosphate paper, and rinsing three times in 0.75% phosphoric acid then, rinsing is once in acetone.The potpourri that will glimmer is added on the cellulose phosphate, detects the radioactivity of combination with scintillation counter.Shown in Fig. 4 C, in the various PKC-θ KD mutant of test, have only wild type PKC-θ KD albumen and three kinds sudden change fragment on threonine 538 by phosphorylation, shown the activity in external lysate kinase activity assay.In fact, detected phosphorylation threonine 538 (pThr in the mutant lysate of lysate kinase activity and every kind of expression ₅₃₈) degree be associated (comparison diagram 4A and 4C).

[0166] 695 serine (Ser of the terminal hydrophobic motif of the C of PKC-θ KD ₆₉₅) to activate the ring autophosphorylation for the best also be essential, as by anti--pT ₅₃₈(the seeing the S695A mutant (promptly 695 mutant serines are alanine) among Fig. 4 A) that signal proved that obviously reduces among the Western trace figure.Therefore, 695 serines are essential to PKC-θ KD kinase activity, (the seeing the S695A mutant among Fig. 4 C) that is confirmed as S695A mutant kinase activity disappearance, respectively with inactivation with kinases inactivation mutant T538A and K409W very similar (seeing Fig. 4 A and 4C).On the contrary, corner motif residue Ser ₆₆₂To activity and Thr ₅₃₈Autophosphorylation all dispensable (seeing the S662A mutant among Fig. 4 A), and corner motif residue Ser ₆₇₆And Ser ₆₈₅Some effects (seeing S676A and S685A mutant among Fig. 4 A) are arranged.

[0167] therefore, mutation analysis has confirmed the Ser in the conservative corner motif ₆₇₆And Ser ₆₈₅The kinase function of some effects PKC-θ KD (seeing Fig. 4 A and 4C).Reported in the past that the S676A sudden change in the total length kinases did not influence kinase activity, and the sudden change of the S695A in the full-length molecule makes kinase activity reduce by 80% (Liu et al., Biochem.J.361:255-265 (2002)).The S695A residue activity of reporting among the total length PKC-θ and the phosphoric acid-Thr538 aligned phase signal of moderate, this is here to the S695A in the kinase domain viewed (seeing Fig. 4 C, the S695A mutant).This has hinted Ser ₆₉₅Sudden change has caused best Thr ₅₃₈The forfeiture of autophosphorylation, thus weakening of kinase activity caused.This characteristic also is that PKC-θ is exclusive in other PKC isoform.As for PKC-θ, Ser ₆₉₅And Thr ₅₃₈Autophosphorylation have to a certain degree to interdepend be possible.Be known as by the PKC molecule of phosphorylation and be present in co-factor activating the ring place in conjunction with " conformation that catalytic capability is arranged " (Newton, A.C., the Biochemical Journal.370:361-371 (2003)) before, autophosphorylation and the substrate catalytic step.Be to optimize PKC-θ KD kinase function, activate ring and hydrophobic motif all autophosphorylation PKC-θ " conformation that catalytic capability is arranged " is provided is possible.

[0168] determines the phosphorylation site relation of the active PKC-θ KD that expresses, next carried out detailed enzyme mechanism research checking kinase catalytic reaction.Be used for determining that the peptide substrates of studying of PKC-θ kinetic mechanism is shown in Table I.

Table I: be used for the peptide that PKC-θ KD analyzes

Peptide	Sequence	The source	pI
					1 2 3 4 5	FARKGSLRQ RFARKGSLRQKNV LKRSLSEM RTPKLARQASIELPSM FARKGALRQ	Substrate is intended substrate PKC α substrate and is intended substrate PKC α substrate serum response factor substrate lymphocyte specific protein 1 inhibitor plan substrate PKC α	12.01 12.31 8.75 10.84 12.01

[0169] peptide 1 and peptide 2 are to be derived from the substrate that PKC-α intends substrate zone.Peptide 3 and peptide 4 are derived from serum response factor (Heidenreich et al. respectively, J.Biol.Chem.274:14434-14443 (1999)) phosphorylation site in and the phosphorylation site in the lymphocyte specific protein-1 (Huang et al., J.Biol.Chem.272:17-19 (1997)).

[0170] with regard to enzyme kinetic analysis, ATP, ATP γ S, Ficoll-400, sucrose, ATP, ADP, phosphoenolpyruvate (PEP), NADH, pyruvate kinase (PK), lactic dehydrogenase (LDH), AMP-PNP, acetonitrile and damping fluid HEPES available from SigmaChemical Co. (St.Louis, MO).The peptide substrate of peptide substrates, inhibitor and phosphorylation available from AnaSpec (San Jose, CA), SynPep (Dublin, CA) or OpenBiosystems (Huntsville, AL).Use the PK/LDH of coupling to analyze definite enzymatic activity at 25 ℃, then on Molecular Devices plate readout instrument, carry out spectrophotometric analysis in 340nm.Unless otherwise noted, standard reaction is at 25mM HEPES pH 7.5,10mMMgCl ₂, carry out final volume 0.080mL among 2mM DTT, 0.008%TritonX100,100mM NaCl, 20 PK of unit, 30 LDH of unit, 0.25mM NADH and the 2mM PEP.PKC-θ KD concentration changes between 0.312 μ g/ml at 0.156 μ g/ml.

[0171] next, carry out the solvent viscosity analysis.The stability kinetics parameter is determined at the above-mentioned damping fluid that is used for enzyme kinetic analysis, is wherein contained variable sucrose (0-35%) or Ficoll400 (0-8%).Relative solvent viscosity (η ^Rel) use the OstWald viscosity meter with respect to 25mMHEPES pH 7.5,10mM MgCl ₂, 2mM DTT and 100mM NaCl be 25 ℃ of replicate determinations three times.There is not the damping fluid of mucosin (viscogen) to represent with subscript 0.The influence that the conjugate enzyme system is not existed by these mucosins.The sulphur effect study that carries out with ATP γ S and suppress research with the product that ADP carries out and analyze on Hewlett Packard series 1100HPLC uses Phenomenex Auga 5m C18124 A ⁰50mm * 4.60mM post (00B-4299-E0).20mM phosphate pH 8.8/ acetonitrile (50/50) gradient of use 0% to 100% is separated the peptide of unphosphorylated peptide with phosphorylation.By exciting at 485nm and detecting fluorescently-labeled peptide in 530nm monitoring fluorescent emission.

[0172] next determines substrate kinetics.For this reason, data substitution equation 1 to obtain standard Michaelis-Menten dynamics, is perhaps suppressed to obtain substrate substitution equation 2:

$v=\frac{V_{\max }\left[S\right]}{K_m+\left[S\right]}---\left(1\right)$

$v=\frac{V_{\max }\left[S\right]}{\mathrm{Km}+\left[S\right]+\frac{{\left[S\right]}^2}{K_i}}---\left(2\right)$

Wherein S is a substrate, V _MaxBe the maximum enzyme reaction velocity, K _mBe the Michaelis constant, K _iIt is the inhibition constant (Adams, J.A., Biochemistry 42:601-607 (2003)) that substrate suppresses.Establishing an equation under acquisition initial velocity and the substitution under various fixed concentration peptides and the ATP:

$v=\frac{V_{\max }\left[A\right]\left[B\right]}{K_{\mathrm{ia}}{K}_b+K_b\left[A\right]+K_a\left[B\right]+\left[A\right]\left[B\right]}---\left(3\right)$

$v=\frac{V_{\max }\left[A\right]\left[B\right]}{K_b\left[A\right]+K_a\left[B\right]+\left[A\right]\left[B\right]}---\left(4\right)$

[0173] in the superincumbent equation, [A] and [B] is respectively the concentration of ATP and peptide; K _aAnd K _bBe respectively the Km of ATP and peptide; K _IaIt is the constant that A dissociates from the EA compound.

[0174] initial reaction speed suppresses the function of (ADP or phosphoeptide) as product or suppresses the function of (AMP-PNP) and obtain as inactivation is terminal.In these researchs, it is constant that a kind of substrate keeps, and another kind changes with the inhibitor concentration that increases.Under product inhibition situation, constant substrate remains on saturated or the unsaturation level, and in the terminal inhibition of inactivation, constant substrate remains on saturated level.Competitive model (equation 5), noncompetitive inhibition model (equation 6) or the uncompetitive of suppressing of data substitution suppressed model (equation 7):

$v=\frac{V_{\max }\left[S\right]}{K_m(1+\frac{\left[I\right]}{K_{\mathrm{is}}})+\left[S\right]}---\left(5\right)$

$v=\frac{V_{\max }\left[S\right]}{K_m(1+\frac{\left[I\right]}{K_{\mathrm{is}}})+\left[S\right](1+\frac{\left[I\right]}{K_{\mathrm{ii}}})}---\left(6\right)$

$v=\frac{V_{\max }\left[S\right]}{K_m+\left[S\right](1+\frac{\left[I\right]}{K_{\mathrm{ii}}})}---\left(7\right)$

P is K wherein _IiAnd K _IsBe that intercept and slope suppress constant.(Richmond, Sigma Plot 2000 Enzyme Kinetics Module CA) analyze data to use SPSS Science.

[0175] Table II provides peptide 1-4, ATP and has not had the stability kinetics parameter of the ATP of peptide to gather.

Table II: the stability kinetics parameter gathers

Different substrates a	appK m (μM)	k cat (sec -1)	K The i peptide (μM)	k cat/K m (M -1s -1)
					Peptide 1 peptide 2 peptides 3 peptides 4 ATP b ATP c	6.5±0.8 4.3±0.8 420±21 240±16 49±5 59±8	18±1 16±1 21±1 14±1 18±1 0.16+0.01	＞2000 306±57	2700000 3600000 51000 58000 360000 2600

^a Peptide 1 and peptide 2 substitution equations (2); Peptide 3, peptide 4 and ATP substitution equation (1)

^b Peptide 1 exists in this is analyzed

^cIn analysis, there is not peptide to exist

[0176] as shown in Table II, when not having peptide substrates, PKC-θ KD hydrolysising ATP (0.16sec ^-1) (18sec when having peptide ^-1) slow 110 times.The K of ATP _mBe 59 μ M (not having peptide) and 49 μ M (saturated peptide 1), this shows that ATP is combined in and does not have significant difference when peptide substrates exists.PKC-θ stability kinetics Argument List during saturated ATP is in Table II.Peptide 3 and peptide 4 have shown the highest PKC-θ K _m, be respectively 420 μ M and 240 μ M.The K of peptide 1 and peptide 2 by contrast _mValue is respectively 6.5 μ M and 4.3 μ M, causes that when high concentration enzyme suppresses (Table II).The K that more alkaline peptide 1 and peptide 2 are lower _mValue means that the basic amino acid peptide substrate is that PKC-θ is preferred.

[0177] what is interesting is the shorter peptide 1 more remarkable (Table II) of observed substrate rejection ratio in longer more alkaline peptide 2.Therefore, under the NaCl concentration that increases, checked the kinetic parameter of PKC-θ (peptide 1 and ATP).These researchs the results are shown in Table III.

Table III: NaCl is to the influence of PCK-θ KD stability kinetics parameter

[NaCl] mM	appK m ATP a，b (μM)	k cat b (sec -1)	appK M peptide 1 c (μM)	K I peptide 1 c (μM)
					0 50 100 250	25±5 58±8 76±7 121±16	7.5±0.8 18±1 22±1 24±1	6.4±4.2 6.7±2.8 3.2±1.0 9.0±1.2	129±64 201±85 ＞2000 -

^a0.2mM peptide 1

^bSubstitution equation (1)

^cSubstitution equation (2)

[0178] as shown in Table III, as the increase of NaCl concentration, the K of ATP _mConversion increases with enzyme, and the K of peptide 1 _mStill constant relatively.Also studied the influence of ionic strength to PKC-θ, this be by in compound non-product or the inactivation end when substrate combines with enzyme, check that the NaCl taken place realizes the influence that substrate suppresses.In preferred basic peptide 1 and 2, observed the substrate inhibition, but in the peptide 3 and 4 of non-the best, do not observed (seeing Table II).And, find that also substrate suppresses to depend on the ionic strength of damping fluid.When NaCl concentration was increased to 250mM, the substrate of peptide 1 suppressed just to have reduced (seeing Table III).

[0179] therefore, the increase of Table III display buffer liquid NaCl concentration has just increased the PKC-θ KD K of ATP _mAnd enzyme upgrades.Also observed the influence that ionic strength suppresses peptide 1 substrate.When NaCl concentration increases, observed the minimizing (seeing Table III) that peptide 1 substrate suppresses.These observationss can be understood to the influence that ion pair forms in the character of salt (NaCl) and it.According to kation and anionic Hofmeister sequence, NaCl drops on low from liquid sequence and high in the middle of the liquid sequence (Cacace et al., Quarterly Reviews of Biophysics 30:241-277,1997).Therefore, the NaCl enzyme of should be unable to saltouing can not make enzyme denaturation yet.Yet what the increase of damping fluid ionic strength will be to ion pair is formed with influence (Park C.R.R., J.Am.Chem.Soc.123:11472-11479 (2001)).With regard to tyrosine kinase Csk, the adding of 50mM NaCl has the negative charge of increasing substrate poly-(Gly, K Tyr) _mEffect; Yet, for the K of ATP _mPerhaps not influence (Cole et al., J.Biol.Chem.269:30880-30887,1994) of enzyme conversion.Under the situation of PKC-θ KD, the K of ATP _mIncrease may be the result of two kinds of possibilities: 1) when 250mM NaCl, the product of 1 pair of enzyme of peptide-ATP binary complex is in conjunction with more, viewed K _mBe the true K of ATP _mReflection; Perhaps 2) increase of ionic strength is to influence charged substrate A TP with the same mode of peptide 1.Viewed K _mIncrease may be the combination of above-mentioned two kinds of possibilities.With regard to peptide 1 substrate, it may be important in the combination of this substrate to enzyme that ion pair forms (Columbic interaction).When pH 7.5, will have clean positive charge such as peptide 1 such basic peptide.Therefore, the increase of NaCl concentration will produce ion pair will be formed more hostile environment (Park C, R.R., J.Am.Chem.Soc.123:11472-11479 (2001)).If it is the reason that peptide 1 suppresses that ion pair forms, so with NaCl increase the substrate inhibition that reduces be with Columbic interactional weaken corresponding to.

[0180] in determining the kinetic mechanism of PKC-θ, the fixing peptide substrates concentration that changes has been determined the initial velocity of reaction of different ATP when the 100mM NaCl.Analyze and at first carry out, yet, be difficult to explain resulting Lineweaver-Burk curve (data not shown) because the substrate of peptide 1 suppresses with peptide 1.Then carried out the intercept of Lineweaver-Burk curve (not shown) and slope recast figure to peptide 1.

[0181] shown in Fig. 5 A and 5B, intercept and slope recast figure are non-linear to the peptide 1 of 100mM NaCl respectively.Also carry out the initial velocity analysis, used peptide 3 under same condition, to carry out.Different ATP concentration have produced cross-mode (data not shown) to the curve of fixing peptide 3 concentration that change on the Lineweaver-Burk curve, this has shown sequential dynamics mechanism.The K of ATP _IaValue is 61 ± 22 μ M, the K of ATP _aBe 118 ± 17 μ M.Under 625nMNaCl, determined the initial velocity pattern of peptide 1 then, suppressed (seeing Table III) because the salinity that increases has reduced the substrate of peptide 1.Resulting Lineweaver-Burk curve has also produced cross-mode (data not shown), and the sequential dynamics mechanism when being substrate with peptide 1 is consistent.Under high NaCl concentration, the intercept of Lineweaver-Burk curve (not shown) and slope recast figure are linear (seeing Fig. 5 C and 5D) to peptide 1.The K of the ATP that under high NaCl, obtains _IaValue is 66 ± 32 μ M, finds the K with the ATP of peptide 3 under 100mM NaCl _IaValue 61 ± 22 μ M are similar.This shows that the ionic strength of increase can not influence the constant of ATP from enzyme-ATP complex dissociation.The K of the ATP that under 625mM NaCl, obtains _aBe 321 ± 19 μ M, with the K of ATP under 100mMNaCl _a118 ± 17 μ M differences.The K of ATP when this and ionic strength increase _mIncrease is consistent (seeing Table III).

[0182] the terminal inhibition research of inactivation is differentiated ATP and is first substrate in conjunction with PKC-θ KD.Substrate in like this, then definite order catalyst mechanism is in conjunction with order.Be used for suppressing research with the non-hydrolysable analog AMP-PNP of ATP and with the peptide 5 (seeing Table I) that the serine of peptide 1 becomes alanine.Suppress result of study and be shown in Table IV.

Table IV: suppression mode and constant ^a

Inhibitor	Type	Substrate	Pattern b	K is μM	K ii μM	Equation c
							ADP ADP ADP phosphoeptide 1 phosphoeptide 1 phosphoeptide 1 AMP-PNP AMP-PNP peptide 5 peptides 5 peptides 5	The terminal inactivation end of the terminal inactivation of the terminal inactivation of the terminal inactivation of product product product product product product inactivation	ATP peptide	1 peptide 1 d ATP ePeptide 3 f Peptide 3 d，f ATP peptide 1 peptide 1 ATP peptide 3	c - nc uc nc uc c - c uc c	291±24 494±72 1700±1100 228±29 10±3 4.4±0.3	200±29 1600±100 1200±800 2000±400 1100±100	5 6 7 6 7 5 5 7 5

^aNaCl concentration remains on 100mM

^bC, competitiveness; Nc, noncompetitive; Uc, uncompetitive;-, do not observe inhibition

^cThe equation of data substitution number

^dATP remains on 0.1mM

^e Peptide 3 remains on 0.5mM, because the lower K of peptide 1 _mAnd use peptide 3

^fSuppress to use peptide 3 owing to observed substrate with peptide 1

[0183] as shown in Table IV, find that AMP-PNP is the competitive inhibitor of ATP, K _iValue is 228 μ M.When saturated ATP, peptide is not observed inhibition with AMP-PNP.Inhibitor peptides peptide 5 is shown as the competitive inhibitor of peptide 1 and peptide 3, K _IsValue is respectively 10 μ M and 4.4 μ M (Table IV).Peptide 5 further is shown as the uncompetitive inhibitor of ATP, K _IiValue is 1100 μ M (seeing Table IV).It is conforming that the orderly order of these results and PKC-θ substrate adds, and wherein ATP at first associates with enzyme, then is peptide.

[0184] because the kinases analysis of PK/LDH coupling has consumed catalysate ADP, so use HPLC to analyze to determine the suppression mode (seeing Table IV) of ADP.Find that ADP is the competitive inhibitor of ATP when saturated peptide 1, K _IsBe 291 μ M.When at saturated ATP, analyze ADP, do not observe inhibition peptide 1.When under unsaturation ATP (0.1mM), analyzing, observed the noncompetitive pattern, K _IsBe 494 μ M, K _IiBe 200 μ M (seeing Table IV).These results of ADP have got rid of the chance mechanism of meaning property description as shown in Fig. 6 C, but with order in order (meaning property description as shown in Figure 6A) or Theorell-Chance (schematically describing) kinetic mechanism as Fig. 6 B be conforming, wherein ADP is the end-product of release.For further illustrating kinetic mechanism, carried out the product inhibition analysis with phosphoeptide 1.Owing to observed the substrate inhibition with peptide 1, so carry out the product inhibition analysis with peptide 3.Phosphoeptide 1 is the noncompetitive inhibitor of peptide 3, K under saturated ATP concentration _IsBe 1700 μ M, K _IiBe 1200 μ M (Table IV).Under unsaturation ATP, observed the uncompetitive pattern, K _IiBe 2000 μ M.When unsaturated peptide 3 (0.5mM), phosphoeptide 1 is the uncompetitive inhibitor of ATP, K _IiBe 1600 μ M.Bi-Bi mechanism is more consistent in order with order for The above results, the end-product (see Fig. 6 A) of ADP for discharging.

[0185] use the sulfur analogs ATP γ S of ATP to study the speed of the phosphoric acid transfer step in the catalysis, these researchs the results are shown in Table V.

Table V: sulphur is to the influence of PKC-θ KD

Kinetic parameter	[NaCl]mM	ATP a	ATPγS a	ATP/ATP γ S ratio
					appK m(μM) appK m(μ M) speed bSpeed b	100 250 100 250	251±24 234±19 179±5 190±4	120±9 130±5 1.6±0.1 1.3±0.1	2.1 1.8 112 146

^aThe peptide 1 that uses in the analysis

^bSpeed: peak region/(reaction time, minute) ([enzyme] nM)

[0186] as shown in Table V, ATP γ S is to the alternative k that produces reaction of ATP _CatBig variation.With the reacting phase ratio of ATP, ATP γ S is reflected among 100mM NaCl and the 250mM NaCl and distinguishes slow 112 times and 146 times.Yet, use the ATP of HPLC acquisition and the K of ATP γ S _mOnly differ twice (Table V).

[0187] for determining whether chemical step is unique contributor of reaction velocity, has determined the influence of solvent viscosity to PKC-θ stability kinetics parameter.In this research, adopted two types mucosin, little mucosin sucrose and big mucosin Ficoll-400.Little mucosin directly influences micromolecular diffusion, produces the observed viscosity effect of viscosity meter (Blacklow et al., Biochemistry 27:1158-1167 (1988)) simultaneously.Big mucosin produces the visible viscosity effect of viscosity meter, but and the micromolecular rate of propagation of not obvious influence, thereby with the contrast (Cole et al., J.Biol.Chem.269:30880-30887 (1994)) of observed microviscosity effect in performing an analysis.Under the solvent viscosity that increases and two kinds of different ionic strengths, determined the stability kinetics parameter of peptide 1, peptide 3 and ATP.Solvent viscosity is to kinetic parameter k _CatAnd k _Cat/ K _m, relative effect to the mapping of the relative viscosity of damping fluid, carry out linear regression fit.

[0188] Fig. 7 A-7D has shown the k of solvent viscosity to PKC-θ KD _CatAnd k _Cat/ K _mInfluence.Fig. 7 A has shown the peptide 1 that uses variation, the k when ATP remains on 2.0mM _CatEffect.Fig. 7 B shows the k of ATP at 0.125mM peptide 1 _Cat/ K _mFig. 7 C has shown with the peptide 3 that changes, the k when ATP remains on 2.0mM _CatEffect.Fig. 7 D has shown the k of peptide 3 when 2.0mMATP _Cat/ K _mWith regard to Fig. 7 A-7D, open circles symbol (zero) expression 100mM NaCl is in the sucrose that increases; Hollow inverted triangle symbol () expression 250mM NaCl is in the sucrose that increases; Filled circles symbol (●) expression 100mM NaCl is in the Ficoll 400 that increases; Solid inverted triangle symbol () expression 250mM NaCl is in the Ficoll 400 that increases.Dotted line among Fig. 7 A-7D is represented slope 1.The little mucosin of slope 1 expression is to the ceiling effect of kinetic parameter.The existence of big mucosin is very little to the influence of enzyme speed.Along with the increase of solvent microviscosity, as seen to (k _Cat) the appropriateness influence of η value.The linearity of three kinds of all visible viewed enzyme speed of the research of substrate in 100mM NaCl and 250mMNaCl reduces.The slope [(k of Huo Deing under all conditions _Cat) η] between 0.38 to 0.54, change, mean that it is (Fig. 7 A and the 7C) of part speed limit that product discharges.0.8 the value to 1 shows that it is catalysis rate-limiting step (Adams, J.A., Biochemistry 42:601-607 (2003)) that product discharges.

[0189] substrate viscosity can be determined by the viscosity analysis.Briefly, for the viscosity substrate, it is fast that product forms speed ratio substrate dissociation rate, and the non-sticky substrate will form speed quickly from the enzyme (Cleland that dissociates than product, W.W. (1986) Investigations of Rates andMechanisms of Reactions, Vol.6, Wiley-Interscience Publications, JohnWiley ﹠amp; Sons, New York, NY).The solvent microviscosity that increases is to k _Cat/ K _mRelative influence to relative solvent viscosity map (Fig. 7 B and 7D).When the microviscosity influence was mapped to relative solvent viscosity, concerning peptide 1, the slope in 250mM NaCl had 0.86 (k _Cat/ K _m) η value (data not shown).Peptide 1 does not obtain data when 100mM NaCl, because suppress at the viewed substrate of low ionic strength.On the other hand, peptide 3 does not all demonstrate solvent viscosity effect (Fig. 7 D) under arbitrary ionic strength.These researchs mean that peptide 1 is the viscosity substrate, and peptide 3 is non-stickies.

[0190] reported several kinase whose kinetic mechanism (referring to for example Wu et al., Biochemistry 41:1129-1139 (2003); Trauger et al., Biochemistry 41:8948-8953 (2003); Chen et al., Biochemistry 39:2079-2087 (2000)).Two kinds of peptide substrates are under high and low ionic strength, and PKC-θ KD initial velocity curve has all produced in the drawings at the coordinate left side and the following line that intersects.This pattern is clear to show it is order mechanism, and it keeps not being subjected to the damping fluid ionic strength affect.In addition, ATP K _IaValue does not have any difference, and peptide 3 is 61 μ M when 100mM NaCl, and peptide 1 is 66 μ M when 625mM NaCl, shows further that ATP dissociates from enzyme-ATP compound not to be subjected to ionic strength affect.Under two kinds of conditions, all find K _IaValue is less than the K that is 118 μ M and 321 μ M respectively _aValue has been got rid of quick balancing.

[0191] terminal inhibition of inactivation and product inhibition research (seeing Table IV) are conforming with order ordered mechanism, and wherein ATP is first substrate of combination.Inhibitor peptides (peptide 5 in the Table I) is emulative to two kinds of peptide substrates, is uncompetitive to ATP.Although having found ATP analog AMP-PNP is emulative to ATP, the AMP-PNP that is up to 2.0mM under saturated ATP does not observe the inhibition to peptide 1.When saturated ATP, although ATP has observed competitive pattern when ADP is changed, 1 couple of ADP of peptide does not observe inhibition when changing.When unsaturated ATP, when 1 couple of ADP of peptide changes, observed the noncompetitive pattern.These suppress the chance mechanism that PKC-θ KD has been got rid of in research, prove that ADP is the end product that discharges, as shown in Figure 6A.

[0192] the initial velocity experiment of peptide 1 under 100mM NaCl provided some understandings to observed substrate inhibition type.Briefly, at orderly bireactant system (Segel, I.H. shown in Figure 8 Enzyme Kinetics:Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, Whiely-Interscience, 1975) in, the substrate of observing three types suppresses.Two kinds is that such substrate suppresses, and wherein substrate B forms the terminal EB compound of inactivation, and perhaps substrate A forms the terminal compound of EAA inactivation.The third is that such substrate suppresses, and wherein B forms the terminal compound of EBQ inactivation.The formation of the terminal compound of EAA inactivation has been excluded, because first substrate that associates is ATP, the substrate of not observing ATP suppresses.Fig. 5 A-5D has shown the recast figure of the initial velocity data of peptide 1 under 100mM NaCl and 625mM NaCl.Shown in Fig. 5 A and 5B, depression effect has all been found (curve map that is recast is non-linear) on slope under the 100mM NaCl and intercept recast figure.Yet under 625mM NaCl (shown in Fig. 5 C and 5D), recast figure becomes linear, shows that the inhibition that is up to 0.5mM peptide 1 under high ionic strength all has been eliminated.The competitive substrate of substrate depression effect right and wrong on slope and intercept recast figure suppresses corresponding to (Cleland, W.W., Methods Enzymol.63:500-513 (1979)).Noncompetitive substrate in orderly order mechanism suppress to show formed below the non-product multienzyme complex of type.Two kinds are shown among Fig. 8, EB and EBQ compound.The third possibility will be the EAB compound of non-product.This obviously is possible, because when 0mM NaCl, it is effective (0.129mM) that substrate suppresses, and ATP concentration is about 80x K _m(0.025mM is under 0mM NaCl).In order ordered mechanism, ATP is combination at first, just seldom exists free enzyme to form the terminal compound of EB inactivation.

[0193] use thiophosphate to study dissimilar enzymatic phosphoric acid transfer reactions.Under the situation of Csk kinases (Cole et al., J.Biol.Chem.269:30880-30887 (1994)), ATP γ S reaction rate is reacted slow 15 to 20 times than ATP.Similarly, in the enzymatic Mechanism Study of phosphatidylinositol-specific phospholipase C (PLC), when not becoming key oxygen to be replaced by sulphur, reaction slows down 10 ⁵Doubly (Kravchuk et al., Biochemistry 40:5433-5439 (2001)).No matter use which kind of ATP analog, ATP or ATP γ S, product A DP is identical.Therefore, catalytic rate will keep not being subjected to the influence of product release.For the viewed sulphur effect of PKC-θ KD, mean that phosphoric acid shifts the contribution of chemistry to the total speed of enzymatic reaction.In addition, refuted the orderly sequential dynamics mechanism of the Theorell-Chance that describes among Fig. 6 B (McKay et al., Biochemistry 35:8680-8685 (1996)) with the observed very big sulphur effect of PKC-θ.With regard to the Theorell-Chance kinetic mechanism, ternary complex is of short duration, therefore means that chemical step is very fast.

[0194] the solvent viscosity effect is valuable instrument in definite enzymatic reaction rate-limiting step.Non-chemically be diffused into zymophore such as product by enzyme diffusion and substrate on the step, found solvent viscosity effect (the Blacklow et al. that increases, Biochemistry 27,1158-1167 (1988)), on the unimolecule step, do not find (Adams such as the phosphoric acid transfer step, J.A., Biochemistry 42:601-607 (2003)).Here Bao Dao PKC-theta solvent viscosity effect studies show that, the product inhibition is the part rate-limiting step in the catalytic action.

[0195] the research illustration that carries out here PKC enzymatic feature and kinetic mechanism, thereby provide the PKC isoform that highly is similar to PKC and/or the understanding of AGC family kinase.Given result is corresponding to order ordered mechanism, wherein at first combination of ATP, and ADP discharges at last.Phosphoeptide discharges and the phosphoric acid transfer contributes to rate-limiting step.Importantly, in the kinase domain research that provides, disclosed the potential specific characteristic of PKC-θ here.Consider the architectural feature .see Xu et al. of PKC-θ in the lump, J.Biol.Chem.279 (48): 50401-50409 (2004)), these find to have important implications at the selectivity target that advances this kinases to be used for disease treatment.

Embodiment 4

The discriminating of PKC-θ substrate

[0196] then carries out the pepscan array to differentiate the peptide substrates of PKC-θ.For this reason, as described in example 3 above, with PKC-θ from residue 362 to residue 706 catalysis kinase domain be cloned into the pET-16b expression vector.Introduce C end six-histidine mark in this carrier frame to expression cloning.Plasmid is transformed into overexpression in the BL21-DE3 e. coli strains.Is 0.4 at 37 ℃ up to O.D. with 10 liters of cell culture initial growth, reduces the temperature to 25 ℃ then, uses 0.1mM IPTG abduction delivering then.Before results, cytothesis is grown 3 hours.

[0197], uses microfluidization device cracking in Tris 25mM pH 8.0, NaCl 25mM, 2 mercapto ethanol 5mM, imidazoles 5mM, ATP 50 μ M and protease inhibitors with the cell resuspension.Lysate is added to the nickel of 20ml (bed)-NTA resin last 1 hour at 4 ℃ by batch processing method.Subsequently resin is toppled over as chromatographic column, with a large amount of rinsings of same damping fluid that have the imidazoles that is increased to 25mM.Elution step realizes with the 200mM imidazole buffer.Application of sample is to anion exchanger HQ immediately with protein then, and usefulness Tris 25mM pH 8.0, NaCl 25mM, DTT 5mM, ATP 50 μ M rinsings are used the NaCl linear gradient that is up to 500mM then and differentiated.Merge the fraction that SDS-PAGE selects, carry out the twice wash-out with Tris 25mM pH 8.0, DTT 5mM, application of sample is to the heparin chromatographic column.The protein fraction that flows out is added on the hydroxyapatite column immediately, with Tris 25mM pH 8.0, NaCl 50mM, a large amount of rinsings of DTT 5mM.Sodium phosphate linear gradient elution target protein with 0 to 100mM.Then protein is carried out size fractionation as monomer on Superdex 200 size exclusion chromatography posts,, concentrate Tris 25mM pH 8.0, NaCl 50mM, DTT 5mM dialysed overnight.

[0198] synthetic with regard to the point of peptide, the amino acid modified cellulose membrane of using polyglycol and Fmoc-protection is available from Intavis.The alanine of Fmoc-protection available from Chem-Impex (WoodDale, IL).By the coupling Beta-alanine is basic at interval array is limited on the film, use standard DIC/HOBt (diisopropyl carbodiimides/hydroxybenzotriazole) coupling chemistry as described above (referring to for example Molina et al., Peptide Research 9:151-155 (1996); AndFrank, R., Tetrahedron 48:9217-9232 (1992)) synthetic peptide.Use the amino acid of AbimedASP 222 automat point samples activation.Manually carry out rinsing and deprotection steps, after last synthetic circulation that peptide N is terminated acetylated.

[0199] after the synthetic and side chain deprotection of peptide, carries out the kinases analysis.Analyze with regard to these, with film rinsing 10 minutes in methyl alcohol, at analysis buffer (20mM HEPESpH=7.5,10mM MgCl ₂, 2mM DTT, 100mM NaCl and 20 μ M ATP) in rinsing 10 minutes.Then with film with the terminal His-mark of 50nM PKC-θ (kinase domain amino acid residue 362-706) C-, at expression in escherichia coli and purifying) containing in the analysis buffer of 0.33Ci/mMol γ-32P-ATP incubation 1 hour.Then with film with the 200mM sodium phosphate rinsing 5 times that contains 0.1%Triton-X and the cold ATP of 100 μ M, with ethanol rinsing 3 times.Next with the film drying, with Biorad Fx imaging.

[0200] uses these methods, tested 384 peptide sequences.The phosphorylation of these peptides is shown in Fig. 9 A.In these 384 peptide sequences, following being shown is the substrate of PKC-θ.

FARKGSLRQKN(SEQ ID NO：6)

KKRFSFKKSFK(SEQ ID NO：16)

QKRPSQRSKYL(SEQ ID NO：17)

KIQASFRGHMA(SEQ ID NO：18)

LSRTLSVAAKK(SEQ ID NO：19)

AKIQASFRGHM(SEQ ID NO：20)

VAKRESRGLKS(SEQ ID NO：21)

KAFRDTFRLLL(SEQ ID NO：22)

PKRPGSVHRTP(SEQ ID NO：23)

ATFKKTFKHLL(SEQ ID NO：24)

SPLRHSFQKQQ(SEQ ID NO：25)

KFRTPSFLKKS(SEQ ID NO：26)

IYRASYYRKGG(SEQ ID NO：27)

KTRRLSAFQQG(SEQ ID NO：28)

RGRSRSAPPNL(SEQ ID NO：29)

MYRRSYVFQT(SEQ ID NO：30)

QAWSKTTPRRI(SEQ ID NO：31)

RGFLRSASLGR(SEQ ID NO：32)

ETKKQSFKQTG(SEQ ID NO：33)

DIKRLTPRFTL(SEQ ID NO：34)

APKRGSILSKP(SEQ ID NO：35)

MYHNSSQKRH(SEQ ID NO：36)

MRRSKSPADSA(SEQ ID NO：37)

TRSKGTLRYMS(SEQ ID NO：38)

LMRRNSVTPLA(SEQ ID NO：39)

ITRKRSGEAAV(SEQ ID NO：40)

EEPVLTLVDEA(SEQ ID NO：41)

SQKRPSQRHGS(SEQ ID NO：42)

KPFKLSGLSFK(SEQ ID NO：43)

AFRRTSLAGGG(SEQ ID NO：44)

ALGKRTAKYRW(SEQ ID NO：45)

VVRTDSLKGRR(SEQ ID NO：46)

KRRQISIRGIV(SEQ ID NO：47)

WPWQVSLRTRF(SEQ ID NO：48)

GTFRSSIRRLS(SEQ ID NO：49)

RVVGGSLRGAQ(SEQ ID NO：50)

LRQLRSPRRTQ(SEQ ID NO：51)

KTRKISQSAQT(SEQ ID NO：52)

NKRRATLPHPG(SEQ ID NO：53)

SYTRFSLARQV(SEQ ID NO：54)

NSRRPSRATWL(SEQ ID NO：55)

RLRRLTAREAA(SEQ ID NO：56)

NKRRGSVPILR(SEQ ID NO：57)

GKRRPSRLVAL(SEQ ID NO：58)

QKKRVSMILQS(SEQ ID NO：59)

RLRRLTAREAA(SEQ ID NO：60)

[0201] some in these peptides are shown in Fig. 9 B, are represented with runic by the serine of PKC-θ phosphorylation.

[0202] these peptide sequences by PKC-θ phosphorylation can be comprised among the physiology substrate of PKC-θ, similarly may be by in cell or the inhibition of the body build-in test substrate phosphorylation method of testing the inhibitor physiologically active.In addition, because the mechanism in the PKC-θ signal pipeline, contain any physiology substrate of these amino acid residues and may be the treatment target of potential inhibition that is used for treating asthma or adjusting.

Embodiment 5

When the IgE acceptor was linked on the BMMC, inducibility phosphorylation and the transposition of PKC-θ film that PKC-θ activates ring took place

[0203] activates the effect of autophosphorylation in the ring (promptly 538 threonine on) in asthma and allergic reaction for investigating PKC-θ, determined that after the IgE acceptor is crosslinked in BMMC PKC-θ activates the autophosphorylation in encircling.For these research, separated BMMC.For this reason, from the C57Bl/6J mouse (commercial available from The Jackson Laboratory, Bar Harbor, bone ME) (femur and shin bone) extracts marrow, then with 5 * 10 ⁵Cell/ml places the 10%HI FCS+PS/gln of DMEM and 50 μ M β ME+20ng/ml reorganization mouse IL-3 and 50ng/ml reorganization mouse SCF (commercial available from R﹠amp; D Systems, Minneapolis, MN) in.Every 3-7 days with passage.After 4 weeks, culture is exactly＞95% mast cell (as expressing determined) by IgE expression of receptor and c-kit.At this moment, only be in the above-mentioned nutrient culture media of 50ng/ml with cellular incubation at mouse IL-3.

[0204] BMMC that separates is handled overnight incubation (about 16 hours) with anti--DNP (dinitrophenyl) IgE.Second day, adding DNP-BSA, to trigger the IgE acceptor in 0,2,5,30 and 90 minute in the culture crosslinked.Then, with BMMC cracking in the 1%NP-40 lysis buffer of handling,, transfer on the nitrocellulose filter cytosol extract (prepared) electrophoresis on SDS-PAGE by describing among the embodiment 1.At first detect the cellulose nitrate trace, peel off then, detect with anti--PKC-θ (commercial) again available from Santa Cruz with anti--phosphoric acid T538 PKC-θ specific antibody (Cell Signaling Technology).

[0205] shown in Figure 10 A, in the mast cell effector function of irritated and asthma, discovery when the IgE acceptor is crosslinked, 538 threonines of PKC-θ in the MMC of bone marrow derived rapidly by phosphorylation (Fig. 9 A).Notice that all BMMC express the PKC-θ of approximate equivalent, with processing mode irrelevant (seeing Figure 10 B).Do not resemble observed lasting phosphorylation in the T cell and (see that Figure 1A-1C), discovery phosphorylation in this site in mast cell is rapid and of short duration (Figure 10 A).Shown in Figure 10 A, find to activate cycli phosphateization and occur in early to the IgE acceptor crosslinked back 2 minutes, returned to baseline values in crosslinked back 30 minutes.

[0206] for determining whether the IgE acceptor transposition of PKC-θ film takes place when crosslinked in BMMC, with anti--DNP IgE treatments B MMC (separating as mentioned above) overnight incubation, adding DNP-BSA stimulated 0 minute, 2 minutes, 5 minutes or 30 minutes.Then as top embodiment 1 described in lysis and classification.Differentiate film fraction, insoluble fraction (DI) and the full cell extract (WCE) of detergent by SDS-PAGE, transfer on the cellulose nitrate then.At first detect the cellulose nitrate trace then with anti--PKC-θ (Santa Cruz), peel off then, detect film fraction and DI fraction with anti--Fc ε RI γ subunit again, detect WCE with anti--actin (SantaCruz), express with the equivalent of confirmation IgE acceptor (being Fc ε R1 γ subunit) on film and DI fraction, and confirm the equivalent expression of cell protein actin in WCE.Shown in Figure 11 A, can in the film fraction, find PKC-θ at crosslinked IgE acceptor after 2 minutes, be exactly clear tangible after crosslinked 30 minutes.Similarly, shown in Figure 11 B, although (be among Figure 11 B 0 minute) can be observed low-level PKC-θ in the DI fraction in the BMMC that is not stimulated, but the amount of the protein that exists in the crosslinked back of IgE acceptor has just increased.Notice the IgE acceptor that all has equivalent in all swimming lanes that in Figure 11 A and 11B, show.

[0207] therefore, be similar to the signal transmission in the T cell, find when the IgE acceptor is crosslinked in mast cell, PKC-θ translocates to rapidly on the insoluble fraction of the detergent of film.Figure 11 C has confirmed that this result is not the difference of measuring because of PKC-θ in different swimming lanes fully, because all swimming lanes of BMMC all have the PKC-θ of equivalent in their full cell extract.

Embodiment 6

When the IgE acceptor was crosslinked on BMMCs, PKC-δ and PKC-β distributed and significantly do not change.

[0208] after the IgE acceptor is crosslinked, mediation (Nechushtan et al., Blood 95:1752-1757 (2000) that two kinds of other PKC family member PKC-δ and PKC-β relate to the mast cell function; Kalesnikoff et al., J.Immunol.168:4737-4746 (2002).For determining whether translocate on the BMMC film other PKC family member of the crosslinked back of IgE acceptor, the fraction (being film, DI and WCE fraction) that shows the result in the experiment of describing among the embodiment 5 of Figure 11 A-11C is carried out the Western engram analysis, uses anti--PKC-δ (Figure 12 A) and anti--PKC-β I/ β II (Figure 12 B) (both are all from Santa Cruz Biotechnology Inc.) to detect the PKC-δ and the PKC-β (rather than PKC-θ) of trace.Shown in Figure 12 A and 12B, do not detect PKC-β (Figure 12 A) and the derivable film transposition of PKC-δ (Figure 12 B) because stimulate (being that the IgE acceptor is crosslinked) before and afterwards both's equivalent be present in the insoluble fraction of cell liquid, film and detergent.These results have proved PKC-θ and PKC-β and the PKC-δ important difference in the IgE receptor signal in regulating mast cell.

Embodiment 7

The mast cell of PKC-θ knock-out mice is different with the mast cell of wild-type mice

[0209] research of PKC-θ knock-out mice has shown that PKC-θ is essential (Sun et al., Nature 404:402-407 (2000)) for the t cell activation of TCR-mediation.Whether be different from the BMMC of wild-type mice about the BMMC of PKC-θ knock-out mice, determine.For this reason, according to Sun et al., the method for describing among the Nature 404:402-407 (2000) obtains PKC-θ knock-out mice, has confirmed T cell proliferation defective (data not shown).Checked the effect that PKC-θ lacks in MMC (MMC) and connective tissue mast cell (CTMC) among both.These distinct mast cell phenotypes, granule composition and medium content and their anatomy distribution all different (referring to Beil et al., HistolHistopathol.15:937-946 (2000)).MMC finds in lung and intestinal mucosa, contains high-caliber protease trypsase.Their granule is rich in the proteoglycans chondroitin sulfate, makes that cell can enough alcian blue dyeing.On the contrary, the CTMC that finds in intestines, skin and cavum peritoneale expresses high-caliber trypsinlike enzyme and chymotrypsin, discharges higher levels of relatively histamine than MMC.Their granule contains the proteoglycans Heparan sulfate, makes that they can enough Toluidine blue staining, but can not use alcian blue.Distinct mast cell subgroup is may be in their body different (referring to for example Miller and Pemberton aspect function and the adjusting on these two kinds of phenotypes, Immunology 105:375-90 (2002)), but the definite essence of these differences still under study for action.For fully studying the influence of PKC-θ, in PKC-θ knock-out mice, checked every kind of mast cell subgroup to mast cell.The external derived from bone marrow CFU-GM of MMC.On the contrary, CTMC can reclaim with mature form from the mouse peritoneum chamber.

[0210] at first, the CTMC and the BMMC that on phenotype, have compared wild type and PKC-θ knock-out mice.For this reason,, use cytospin to rotate on the slide, use Toluidine blue staining, redye with sarranine then through peritoneal lavage separation of C TMC.Alternately, cell dyes with Wright ' s-Geimsa.Any dyeing scheme all will identify the mast cell granule body.Aspect the quantity or number percent of peritonaeum mast cell, perhaps aspect the granule density or distribution of each cell, there is not significant difference (data not shown) between wild type and the PKC-θ knock-out mice.As the BMMC of separation wild type as described in the embodiment 5 and PKC-θ knock-out mice, use Cytospin to rotate on the slide, dye 5 minutes with the dyed particles body with 1% alcian blue in 3% acetate then.Cell is redyed with sarranine.As shown in FIG. 13A, the BMMC of wild-type mice demonstrates the BMMC more particlesization than PKC-θ knock-out mice.

[0211] next, be the granulating difference among the quantitative IgE acceptor crosslinked back BMMC, taked cell surface membrane connection protein staining.Cell surface membrane connection protein staining increases with threshing, and it is consistent being exposed to plasma membrane surfaces with fusion of granule film and phosphatidylserine.Be analysis of cells surface film connection protein expression, by with 0.2 μ g/ml IgE anti--DNP handles and spends the night, the BMMC that is derived from wild type or PKC-θ knock-out mice is resisted-the DNP application of sample with IgE.Second day, harvesting also was rinsed in the PACM damping fluid.Add the FITC-annexin, 37 ℃ with cell incubation 3 minutes.Time-based data acquisition begins on the FACScan that is equipped with 37 ℃ of sample chambers, interrupts inducing threshing with the DNP-BSA that adds prescribed concentration, and each sample continued 10 minutes more then.Like this, cell is induced threshing in the presence of the annexin of FITC-mark.The sign that membrana granulosa and cell membrane merged when annexin was threshing in the expression of cell surface.Mean fluorecence density is as the function construction (Figure 13 B) of time.

[0212] shown in Figure 13 B, compare with wild type, still less cell surface membrane connection protein staining when the BMMC of PKC-θ knock-out mice demonstrates threshing, this dyes by alcian blue with them, and to check out lower granule content be consistent (seeing Figure 13 A).

Embodiment 8

PKC-θ knock-out mice has the IgE level of reduction

[0213] then compared the level that CTMC goes up the IgE acceptor of wild type and PKC-θ knock-out mice.For this reason, with the PIPES-EDTA damping fluid lavation of the cavum peritoneale of wild type and PKC-θ knock-out mice.The not classification peritoneal lavage cell of individual mouse is rinsed among the PBS (PBS-BSA) that contains 1%BSA, on ice with 5 μ g/ml IgE anti--DNP or do not have the antibody incubation 30 minutes.With cell rinsing in PBS-BSA, dye with the anti--mouse IgE of FITC-mark and the anti--ckit (BD-Pharmingen) of PE-mark then.Mean fluorecence density is quantitative through flow cytometry.Shown in Figure 14 A, compare with wild type, the IgE that PKC-θ knock-out mice has significantly reduce level is attached on the CTMC surface.On the contrary, there is not what difference (Figure 14 B) on the ckit expression.The IgE level that is attached on the surperficial IgE acceptor of CTMC is relevant with circulation IgE level in the animal.The reduction of the IgE of the last mast cell combination of CTMC shows that PKC-θ knock-out mice may have low-level SERUM IgE.

[0214] in addition, between the serum antibody level of wild-type mice and PKC-θ knock-out mice, observed significant difference.With regard to these research, analyzed IgE, IgG1 and the IgA content of the blood serum sample of wild type and PKC-θ knock-out mice.For this reason, with anti--mouse IgE (commercial available from Pharmingen, San Diego, CA), anti--mouse κ light chain (commercial available from Sigma, St.Louis, IgA MO) or anti--(Fab-is specific for mouse IgG; Sigma) IgG1 bag by the Maxi-Sorp elisa plate (commercial available from Nunc, Rochester, NY).With the PBS rinsing plate that contains 0.05%Tween-20 (PBS-Tween), then with the PBS room temperature sealing that contains 0.5% gelatin 2 hours.Serum dilution is added among the PBS-Tween room temperature incubation 2 to 6 hours.In conjunction with detection use at mouse IgE or IgA (commercial available from Southern Biotechnology Associates, Inc., Birmingham, AL) or the specific biological elementization antibody of IgG1 (Pharmingen), then use HRP-streptavidin (Southern Biotechnology Associates) and Sure-Blue peroxidase substrate (commercial) available from Kirkegaard and Perry Labs.Use the standard (Pharmingen) of the suitable isotype of purifying to come quantitative Ig level.

[0215] like this, use specific ELISA s to analyze the level of suitable antibody isotype of the blood serum sample of individual mouse, and by with the standard control of purifying quantitatively.Shown in Figure 15 A, the IgE level significantly reduces in PKC-θ knock-out mice.Frequent and the coordinately regulated IgG1 of IgE also has been lowered.On the contrary, compare the IgA higher level in the PKC-θ knock-out mice (Figure 15 C) with wild type.

[0216] in fact, the external threshing (data not shown) of the CTMCs that has found PKC-θ knock-out mice has still less band anti--IgE is with the IgE consistent (seeing Figure 14 A and 15A) of the mast cell combination of circulation IgE that reduces level and minimizing.

[0217] different with the t cell activation defective reported in the PKC-θ knock-out mice, do not observe remarkable vitro mast cell functional defect (data not shown) among the BMMC of PKC-θ knock-out mice.Because these cells are external derived from bone marrow CFU-GM, they are the influence of the interior IgE level of acceptor not, can-DNP application of sample anti-with the external IgE of external source.Then study determining when IgE acceptor and DNP-BSA are crosslinked, whether threshing of the BMMC of PKC-θ knock-out mice whether produce leukotriene, and whether the cell factor that BMMC produced of the cell factor of generation and wild-type mice is in similar level.With regard to these research, in the following method, with mast cell place the 10%HI FCS+PS/gln of DMEM and 50 μ M β ME+50ng/ml reorganization mouse IL-3+0.1 μ g/ml anti--DNP-IgE (Sigma) spends the night.With cell rinsing in PACM (25mM PIPES, pH 7.2, contain 110mM NaCl, 5mM KCl, 5mMCaCl2,2mM MgCl2 and 0.05%BSA), with final concentration 2.5 * 10 ⁵Cell/ml places the DNP-BSA titer plate, and (commercial available from Calbiochem, San Diego CA) goes up with crosslinked IgE acceptor or ionomycin.

[0218] research that produces with regard to histamine, B-hexosaminidase and leukotriene is cultivated 30mins in 37 ℃ with cell in the presence of DNP-BSA or ionomycin, gather in the crops supernatant then, tests immediately or freezing.Threshing and leukotriene generation result of experiment show that the BMMC of PKC-θ knock-out mice has the threshing and the leukotriene generation (data not shown) of normal level.Maximum threshing is by determining with a cell of 0.1%Triton X-100 cracking.With regard to Hex, the right-nitrobenzophenone N-acetyl B-D glucosaminide (Sigma) among supernatant and the 0.08M sodium citrate pH 4.5 is incubated overnight at 37 ℃.After 12-18 hour, add 1N NaOH cessation reaction, come quantitative Hex by the absorption of in spectrophotometer, reading 405nm.When maximum threshing, do not observe significant difference (data not shown).

[0219] produce to analyze with regard to cell factor, will be in anti--DNP-IgE cell and the DNP-BSA incubation of overnight incubation with crosslinked 6 hours of triggering IgE acceptor, gather in the crops supernatant then.Use is specific to LT (C4/D4/E4), and (commercial (Windham, the leukotriene in elisa assay supernatant NH) perhaps use specific ELISA analysis (R﹠amp available from ALPCO; DSystems, Minneapolis MN) analyzes IL-6, IL-13 or GM-CSF.Shown in Figure 16 A-16C, the BMMC of PKC-θ knock-out mice produces lower level cytokine TNF-α (Figure 16 A), IL-13 (Figure 16 B) and IL-6 (Figure 16 C) than the BMMC of wild-type mice all the time.

[0220] next, (ME) spleen is made single cell suspension for The Jackson Laboratory, BarHarbor with the C57BL/6 mouse, by anti--CD4 magnetic bead, then by Detach-A-bead (Dynal Biotech) separation of C D4+ cell, carry out according to manufacturer's directions for use.Cell as tranquillization T cell analysis, is perhaps activated and produces the effector cell.Effector cell's generation is by having replenished 10%FCS, 2mM L-glutaminate, 5 * 10 ^-5In the DMEM nutrient culture media of M 2 mercapto ethanol, penicillin, streptomysin, Sodium Pyruvate and nonessential amino acid (equal Gibco LifeTechnologies, Invitrogen, Carlsbad, the subsidiary company of CA) 6 * 10 ⁵CD4+ cell/ml bed board to bag by 1 μ g/ml anti--CD3 and 4 μ g/ml are anti--the 24-orifice plate of CD28 antibody in.With the T cell that is rich in Th1 30ng/ml rmIL-12 (Wyeth, Cambridge, MA), 10U/ml rhIL-2 (Invitrogen, Carlsbad, CA) and 5 μ g/ml anti--existence of mouse IL-4 antibody cultivates down.To be rich in the T cell of TH-2 at 40ng/ml rmIL-4 (R﹠amp; D Systems, Minneapolis, MN), 10U/mlrhIL-2 (Invitrogen) and 5 μ g/ml are anti--existence of mouse IFN-gamma antibodies cultivates down.After stimulating three days, cell is being lacked other 3-4 days of the following amplification of IL-2 (5U/ml).With tranquillization CD4+T cell or Th1 or Th2 effector cell with 1 * 10 ⁵The cells/well bed board to bag by 0.5 μ g/ml anti--the 96-hole flat underside of CD3 (2C11) in.All antibody are all from B-DPharMingen, San Jose, CA.Harvesting culture supernatant after 3 days is analyzed (FACS) through the cell factor pearl and is analyzed IL-4 and IL-5.

[0221] shown in Figure 17 A and 17B, the T cell cytokine data presentation of PKC-θ knock-out mice, these mouse have produced two kinds of these cell factors of reduction level.

[0222] these results show, inmature PKC-θ knock-out mice lacks circulation IgE and IgG1 level, and this is consistent with the effect that PKC-θ keeps the interior IL-4 constant level of body.IL-4 is the Th2 cell factor, in causing synthetic Ig (immunoglobulin (Ig)) the gene conversion of IgE and IgG1, have effect (referring to Bacharier and Geha, J.Allergy Clin.Immunol.105 (2Pt2): S547-58 (2000); And Bergstedt-Lindqvist et al., Eur.J.Immunol.18:1073-1077 (1988)).The T cellular expression proof PKC-θ of dominant gene construct has activated the IL-4 genetic transcription, and this is and GDP/GTP exchange factor Vav synergistic (referring to Hehner et al., J.Immunol.164:3829-3836 (2000)).

Embodiment 9

PKC-θ knock-out mice reacts on anti--IgE or do not have the swollen increase of ear in the pca model that has external source IgE

[0223] for determining that whether PKC-θ participates in the mast cells activation of IgE mediation, investigates passive cutaneous anaphylaxis (PCA) and assesses PKC-θ knock-out mice in the breathing problem mouse model.Therefore, whether relevant for determining PKC-θ with the mast cells activation of IgE mediation, with anti--IgE (Pharmingen; 0.5 μ g/kg is in 20 μ l PBS) left ear intracutaneous attack PKC-θ-/-(being that PKC-θ knocks out) mouse and the contrast of C57BL/6 wild type.In contrast, animal is accepted the PBS of 20 μ l at the auris dextra of offside.Before anti--IgE attacks, use the engineering mircrometer gauge to determine baseline ear thickness, Upright Dial Gauge (commercial available from Mitutoyo (Japan)) measures and can be low to moderate .0001 ".After the attack, collected ear thickness measurement value, be expressed as the increase on the baseline reading at 1 hour, 2 hours, 4 hours and 6 hours.

[0224] as shown in figure 18, PKC-θ knock-out mice reacts on anti--IgE does not have the swollen increase of ear.In fact, after anti--IgE attacks, compare with PKC-θ knock-out animal, the wild type animal is in the ear enlargement big approximately 2.5 times (Fig.18) of 1 hour time point.The swollen reaction of the ear that reduces in these mouse is corresponding to cell surface and circulation IgE level.As mentioned above, PKC-θ lacks and has caused mast cell granule still less (Figure 13 A-13B) and lower IgE level (Figure 14 A).These the possibility of result partly are because to regulating the T cell dependence that the weakens influence (Boyce, J.Allergy Clin.Immunol.111:24-32 (2003)) of mast cell function.

[0225] the mast cell signal transmission for providing PKC-θ whether to participate in the IgE mediation is with the Kit of the mast cell selectivity remediation fertilizer maxicell shortage that is derived from PKC-θ knock-out mice or wild-type mice ^W/ Kit ^W-vThe mast cell of mouse (commercial available from the Jackson Laboratory) lacks.In other words, the mast cell of PKC-θ knock-out mice or normal wild type mouse is transferred to Kit ^W/ Kit ^W-v(this adoptive transfer technology is commented the Lantz in Galli and, Allergy.In in the mouse Fundamental Immunology, W.E.Paul (ed.) .pp.1137-1184.Lippincott-Raven Press, Philadelphia PA 1999; And William and Galli, J.Allergy Clin.Immunol.105 (5): 847-859 (2000)).Briefly, with 1 * 10 ⁶The BMMC of PKC-θ knock-out mice or wild-type mice is resuspended among the 20 μ lDMEM, injects the Kit of the mast cell shortage in 7 ages in week ^W/ Kit ^W-vIn the left and right sides ear of mouse (10 animal/groups) (1 * 10 ⁶The BMCMC/ ear).After 12 weeks (make the mast cell of adoptive transfer can be in connective tissue ripe appropriate time cycle), by intracutaneous injection IgE anti--DNP (5 μ g/kg) sensitized mice in the left ear.In contrast, animal is accepted 0.9% salt solution in auris dextra.Behind the twenty four hours, intravenous is attacked animal with DNP-HSA (10mg/kg).Before attacking and attack and collected baseline ear measured value in back 1 hour, 2 hours, 4 hours and 6 hours.

[0226] result shows, with the Kit of the mast cell reconstruct that lacks PKC-θ ^W/ Kit ^W-vMouse ear swollen aspect, with Kit with the same processing of wild type mast cell reconstruct ^W/ Kit ^W-vMouse is compared, and does not demonstrate difference (data not shown).These results show the ear in the PKC-θ mouse swollen lack be likely since the effector molecules that the T cell relies on directly and/or indirectly-acting on other immunocyte type.Some T cell cytokines repressed and that influence immune cell function comprise IL-4, IL-5, TNF-α (Figure 17 A, 17B, and data presented not).

[0227] yet, the mast cell data show that the inhibition of PKC-θ may directly regulate mast cell and reply.As above inquired into, found that PKC-θ is activating on the ring by phosphorylation (seeing Figure 10 A-10B) rapidly when the IgE acceptor is crosslinked.When the IgE acceptor was crosslinked, the subcellular fraction of PKC-θ distributed and has been changed, and PKC-δ or PKC-β I/ β II do not change (seeing Figure 11 A-12B).The most important thing is, react on IgE, the BMMC that is knocked out by PKC-θ has produced the cell factor (Figure 16 A-16C) that reduces.These cells are external derived from bone marrow CFU-GM, are not subjected to the influence of level in the IgE body.

[0228] in another experiment, in attack preceding 24 hours, by with monoclonal IgE anti--DNP (Sigma; 5 μ g/kg are in 20 μ l, 0.9% salt solution) intracutaneous injection comes in the left ear passive sensitization PKC-θ to knock out (be PKC-θ-/-) mouse and contrast of C57BL/6 wild type.In contrast, animal is accepted 20 μ l, 0.9% salt solution in the offside auris dextra.Behind the twenty four hours, collect baseline ear measured value, then animal is carried out DNP-HSA (10mg/kg is in 100 μ l, 0.9% salt solution) intravenous and attack.(promptly at attack back 1 hour, 2 hours, 4 hours and 6 hours readings) collects ear thickness measurement value as mentioned above during subsequently 6 hours.

[0229] as shown in figure 19, PKC-θ knock-out mice is compared with the wild type contrast of same processing, has significantly littler ear and swells.These PCA results of study (Figure 18 and 19) supported PKC-θ micromolecule antagonist in animal disease model allergy and the purposes in the asthma.

Embodiment 10

Compare with PKC-θ wild-type mice, the TH1 of PKC-θ knock-out mice and TH2T cell are reacting on the propagation that demonstrates minimizing when stimulating

[0230] whether normal reply aspect the ability of stimulation for the differentiation T cell of determining PKC-θ knock-out mice at them, with the splenocyte vitro differentiation of wild type and PKC-θ knock-out mice is TH1 or TH2 group, to determine the propagation defective of t helper cell subgroup when PKC-θ does not express.With regard to these experiments, separate inmature T cell, produce TH1 and TH2 effector cell.For this reason, with the C57/B6 mouse (commercial available from Taconic, Germantown, spleen NY) is made single cell suspension.With red blood cell (RBC) RBC lysis buffer (0.3g/L ammonium chloride is in 0.0M Tris-HCl pH of buffer 7.5) cracking, twice of rinsing.Through anti--CD4 magnetic-particle, then through Detach-A-Bead (Dynal), according to the directions for use separation of C D4+ of manufacturer cell.Cell as inmature T cell analysis, is perhaps activated and produces the effector cell.

[0231] 10%FCS, 2mM L-glutaminate, 5 * 10 have been replenished by being in ^-5In the DMEM nutrient culture media of M2-mercaptoethanol, penicillin, streptomysin, Sodium Pyruvate and nonessential amino acid (all from GibcoLife Technologies) 6 * 10 ⁵CD4+ isolated cell/ml bed board wrap by 1 μ g/ml anti--CD3 and 4 μ g/ml are anti--activate inmature T cell in the 24-orifice plate of CD28 to produce the effector cell.The T cell (promptly great majority are T cell masses of TH1 cell) that is rich in TH1 passes through cell at 30ng/ml reorganization mouse IL-12 (Wyeth, Cambridge, MA), the 10U/ml recombinant human il-2 (Invitrogen, Carlsbad, CA) and 5 μ g/ml anti--mouse IL4 exists and cultivated 3 days down and produce.The T cell that is rich in TH2 passes through cell at 40ng/ml reorganization mouse IL-4 (R﹠amp; D Systems, Minneapolis, MN), 10U/ml recombinant human il-2 (Invitrogen) and 5 μ g/ml are anti--mouse IFN-gamma exists and cultivated 3 days down and produce.All antibody are all from B-D PharMingen, San Jose, CA.

[0232] with regard to proliferation assay, with TH1-or TH2-effector cell with 1 * 10 ⁵Cell/0.2ml/ hole bed board to wrap by various concentration anti--the 96-hole flat underside of CD3 (2C11) in, exist as shown or do not exist soluble 5 μ g/ml anti--CD28 (clone 37.51) and/or 10U/ml recombinant human il-2.At the 2nd day, with 0.5 μ Ci [ ³H] (NJ) pulse culture uses 96-orifice plate cropper to gather in the crops on the filtrator to thymidine after 6-8 hour for Amersham Bioscience, Piscataway.(Wallac, Gaithersburg MD) measure the radioactivity of mixing to use liquid scintillation counter.All antibody are all from B-D PharMingen, San Jose, CA.

[0233] shown in Figure 20 and 21, the TH1 of PKC-θ knock-out mice and TH2 cell to best (0.5 μ g/ml) and suboptimal (0.05 μ g/ml) anti--the TCR stimulation (being respectively Figure 20 C and Figure 21 C) and the TCR/CD28 of CD3 signal intensity stimulate the breeder reaction of (being respectively Figure 20 A and Figure 21 A) significantly to reduce altogether.The adding of external source IL-2 does not rely on the approach of PKC-θ and supports T cell proliferation partly to overcome inmature TH0, TH1 and the reduction of TH2 cell proliferative response of PKC-θ knock-out mice, but just the 0.5 μ g/ml of the best anti--(Figure 20 B has anti--CD28 to the CD3 signal, and Figure 20 D is not anti--stimulate altogether with TCR/CD28 CD28) time and work in coordination with.0.05 μ g/ml of suboptimal anti--during CD3, CD28 stimulates the almost completely shortage (Figure 21 A) of failing to overcome the cell proliferation of PKC-θ knock-out mice altogether.Have only the external source IL-2 effect (Figure 21 B and 21D) of moderate under these conditions, the TH2 cell proliferation of PKC-θ knock-out mice has kept about 30% of wild-type mice TH2 cell proliferation.

[0234] these results with knock out the T cell by PKC-θ and suppress IL-2 and produce (referring to for example Sun et al., Nature 404:402-407 (2000)) together, show that TCR-stimulates the propagation of inducing not kept by TH0, TH1 and TH2 cell, prerequisite is that PKC-θ activity is suppressed in these cells.Produce together with the TH2 cell factor that reduces, therefore these t helper cells will can not work as the optimum effect cell and mediate the approach that the T cell in asthma and the anaphylactia physiology relies on.Because these find, invent further aspect and be the PKC-θ in the target TH2 T cell.Therefore, invention further provides the treatment intervention that prevents and/or alleviate SOA by the PKC-θ in the target TH2 T cell.

[0235] although detailed illustration and described invention in accompanying drawing and aforementioned specification; yet be considered to illustrative equally and do not have restricted in nature; should be understood that to show and what describe only is preferred embodiment that the institute within the spiritual scope of invention changes and correction is that expectation is protected.

Sequence table

SEQ ID NO：1

GenBank：NM_006257

People PKC-θ

MSPFLRIGLSNFDCGSCQSCQGEAVNPYCAVLVKEYVESENGQM

YIQKKPTMYPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGK

TEIWLELKPQGRMLMNARYFLEMSDTKDMNEFETEGFFALHQRRGAIKQAKVHHVKCH

EFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIAKCTGSAINSRE

TMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNVHHRCQTK

VANLCGINQKLMAEALAMIESTQQARCLRDTEQIFREGPVEIGLPCSIKNEARPPCLP

TPGKREPQGISWESPLDEVDKMCHLPEPELNKERPSLQIKLKIEDFILHKMLGKGSFG

KVFLAEFKKTNQFFAIKALKKDVVLMDDDVECTMVEKRVLSLAWEHPFLTHMFCTFQT

KENLFFVMEYLNGGDLMYHIQSCHKFDLSRATFYAAEIILGLQFLHSKGIVYRDLKLD

NILLDKDGHIKIADFGMCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFG

VLLYEMLIGQSPFHGQDEEELFHSIRMDNPFYPRWLEKEAKDLLVKLFVREPEKRLGV

RGDIRQHPLFREINWEELERKEIDPPFRPKVKSPFDCSNFDKEFLNEKPRLSFADRAL

INSMDQNMFRNFSFMNPGMERLIS

SEQ ID NO：3

People PKC-θ nucleotide

1 tgctcgctcc agggcgcaac catgtcgcca tttcttcgga ttggcttgtc caactttgac

61 tgcgggtcct gccagtcttg tcagggcgag gctgttaacc cttactgtgc tgtgctcgtc

121 aaagagtatg tcgaatcaga gaacgggcag atgtatatcc agaaaaagcc taccatgtac

181 ccaccctggg acagcacttt tgatgcccat atcaacaagg gaagagtcat gcagatcatt

241 gtgaaaggca aaaacgtgga cctcatctct gaaaccaccg tggagctcta ctcgctggct

301 gagaggtgca ggaagaacaa cgggaagaca gaaatatggt tagagctgaa acctcaaggc

361 cgaatgctaa tgaatgcaag atactttctg gaaatgagtg acacaaagga catgaatgaa

421 tttgagacgg aaggcttctt tgctttgcat cagcgccggg gtgccatcaa gcaggcaaag

481 gtccaccacg tcaagtgcca cgagttcact gccaccttct tcccacagcc cacattttgc

541 tctgtctgcc acgagtttgt ctggggcctg aacaaacagg gctaccagtg ccgacaatgc

601 aatgcagcaa ttcacaagaa gtgtattgat aaagttatag caaagtgcac aggatcagct

661 atcaatagcc gagaaaccat gttccacaag gagagattca aaattgacat gccacacaga

721 tttaaagtct acaattacaa gagcccgacc ttctgtgaac actgtgggac cctgctgtgg

781 ggactggcac ggcaaggact caagtgtgat gcatgtggca tgaatgtgca tcatagatgc

841 cagacaaagg tggccaacct ttgtggcata aaccagaagc taatggctga agcgctggcc

901 atgattgaga gcactcaaca ggctcgctgc ttaagagata ctgaacagat cttcagagaa

961 ggtccggttg aaattggtct cccatgctcc atcaaaaatg aagcaaggcc gccatgttta

1021 ccgacaccgg gaaaaagaga gcctcagggc atttcctggg agtctccgtt ggatgaggtg

1081 gataaaatgt gccatcttcc agaacctgaa ctgaacaaag aaagaccatc tctgcagatt

1141 aaactaaaaa ttgaggattt tatcttgcac aaaatgttgg ggaaaggaag ttttggcaag

1201 gtcttcctgg cagaattcaa gaaaaccaat caatttttcg caataaaggc cttaaagaaa

1261 gatgtggtct tgatggacga tgatgttgag tgcacgatgg tagagaagag agttctttcc

1321 ttggcctggg agcatccgtt tctgacgcac atgttttgta cattccagac caaggaaaac

1381 ctcttttttg tgatggagta cctcaacgga ggggacttaa tgtaccacat ccaaagctgc

1441 cacaagttcg acctttccag agcgacgttt tatgctgctg aaatcattct tggtctgcag

1501 ttccttcatt ccaaaggaat agtctacagg gacctgaagc tagataacat cctgttagac

1561 aaagatggac atatcaagat cgcggatttt ggaatgtgca aggagaacat gttaggagat

1621 gccaagacga ataccttctg tgggacacct gactacatcg ccccagagat cttgctgggt

1681 cagaaataca accactctgt ggactggtgg tccttcgggg ttctccttta tgaaatgctg

1741 attggtcagt cgcctttcca cgggcaggat gaggaggagc tcttccactc catccgcatg

1801 gacaatccct tttacccacg gtggctggag aaggaagcaa aggaccttct ggtgaagctc

1861 ttcgtgcgag aacctgagaa gaggctgggc gtgaggggag acatccgcca gcaccctttg

1921 tttcgggaga tcaactggga ggaacttgaa cggaaggaga ttgacccacc gttccggccg

1981 aaagtgaaat caccatttga ctgcagcaat ttcgacaaag aattcttaaa cgagaagccc

2041 cggctgtcat ttgccgacag agcactgatc aacagcatgg accagaatat gttcaggaac

2101 ttttccttca tgaaccccgg gatggagcgg ctgatatcct gaatcttgcc cctccagaga

2161 caggaaagaa tttgccttct ccctgggaac tggttcaaga gacactgctt gggttccttt

2221 ttcaacttgg aaaaagaaag aaacactcaa caataaagac tgagacccgt tcgcccccat

2281 gtgactttat ctgtagcaga aaccaagtct acttcactaa tgacgatgcc gtgtgtctcg

2341 tctcctgaca tgtctcacag acgctcctga agttaggtca ttactaacca tagttattta

2401 cttgaaagat gggtctccgc acttggaaag gtttcaagac ttgatactgc aataaattat

2461 ggctcttcac ctgggcgcca actgctgatc aacgaaatgc ttgttgaatc aggggcaaac

2521 ggagtacaga cgtctcaaga ctgaaacggc cccattgcct ggtctagtag cggatctcac

2581 tcagccgcag acaagtaatc actaacccgt tttattctat cctatctgtg gatgtataaa

2641 tgctgggggc cagccctgga taggttttta tgggaattct ttacaataaa catagcttgt

2701 acttgll

SEQ ID NO：2

NM_008859

Mouse PKC-θ amino acid sequence

MSPFLRIGLSNFDCGTCQACQGEAVNPYCAVLVKEYVESENGQM

YIQKKPTMYPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGR

TEIWLELKPQGRMLMNARYFLEMSDTKDMSEFENEGFFALHQRRGAIKQAKVHHVKCH

EFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIAKCTGSAINSRE

TMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQGLKCDACGMNVHHRCQTK

VANLCGINQKLMAEALAMIESTQQARSLRDSEHIFREGPVEIGLPCSTKNETRPPCVP

TPGKREPQGISWDSPLDGSNKSAGPPEPEVSMRRTSLQLKLKIDDFILHKMLGKGSFG

KVFLAEFKRTNQFFAIKALKKDVVLMDDDVECTMVEKRVLSLAWEHPFLTHMFCTFQT

KENLFFVMEYLNGGDLMYHIQSCHKFDLSRATFYAAEVILGLQFLHSKGIVYRDLKLD

NILLDRDGHIKIADFGMCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFG

VLVYEMLIGQSPFHGQDEEELFHSIRMDNPFYPRWLEREAKDLLVKLFVREPEKRLGV

RGDIRQHPLFREINWEELERKEIDPPFRPKVKSPYDCSNFDKEFLSEKPRLSFADRAL

INSMDQNMFSNFSFINPGMETLICS″

SEQ ID NO：4

Mouse PKC-θ nucleotide sequence

1 cttgggtcgc caggcccgcg ccagtccccg ccatccgagc aacagcggcg ctgctctggg

61 accgcggccg cgacaccagg gaacaaccat gtcaccgttt cttcgaatcg gtttatccaa

121 ctttgactgt gggacctgcc aagcttgtca gggagaggca gtgaacccct actgcgctgt

181 gcttgtcaaa gagtatgtgg aatcagaaaa tgggcagatg tacatccaga aaaagccaac

241 catgtaccca ccttgggaca gcacctttga cgcccacatt aacaagggaa gggtgatgca

301 gatcatcgtg aagggcaaga atgtagacct catctcagaa acaaccgtgg aactctactc

361 cctggcggag agatgccgca agaacaatgg gcggacagaa atatggttag agctgaaacc

421 tcaaggccga atgctaatga atgcaagata ctttctggaa atgagtgaca caaaggacat

481 gagtgagttt gagaatgaag gattctttgc actgcatcag cgccgaggag ccatcaaaca

541 ggccaaagtc caccatgtca agtgtcacga gttcacggcc acctttttcc ctcaacccac

601 attttgctct gtctgccatg aatttgtctg gggcctgaac aagcagggtt accagtgccg

661 acagtgtaat gcagcgattc acaagaagtg cattgataaa gtgatagcca agtgcacagg

721 atccgcaatc aatagccgag aaaccatgtt ccataaggag agattcaaga tcgacatgcc

781 acacagattc aaagtctaca actacaagag tccaaccttc tgtgagcact gtggtaccct

841 gctctggggg ctggcgaggc aaggactcaa atgtgatgca tgtggcatga acgtccacca

901 ccgatgccag acaaaggttg ccaatctttg tggtataaac cagaagctaa tggctgaagc

961 actagcgatg attgaaagca cccaacaggc tcgctcctta cgagattcag aacacatctt

1021 ccgagaaggc ccagttgaaa ttggtctccc atgctccacc aaaaacgaaa ccaggccacc

1081 atgcgtacca acacctggga aaagagaacc ccagggcatt tcctgggatt cccctttgga

1141 tgggtcaaat aaatcggccg gtcctcctga acccgaagtg agcatgcgca ggacttcact

1201 gcagctgaaa ctgaagatcg atgacttcat cctgcacaag atgttgggaa aaggaagttt

1261 tggcaaggtc ttcctggcag agttcaagag aaccaatcag tttttcgcaa taaaagcctt

1321 aaagaaagat gtggtgttga tggatgatga cgtcgagtgt acaatggtgg aaaagagggt

1381 tctgtccttg gcatgggagc atccatttct aacacacatg ttctgcacat tccagaccaa

1441 ggaaaatctc tttttcgtga tggagtatct caatggaggc gacttaatgt accacatcca

1501 aagttgccac aaatttgatc tttccagagc cacgttttat gctgctgagg tcatccttgg

1561 tctgcagttc cttcattcca aaggaattgt ctacagggac ctgaagcttg ataatatcct

1621 gttagacaga gatggacata tcaaaatagc agactttggg atgtgcaaag agaacatgct

1681 aggagatgcg aagacaaata ctttctgtgg aactcctgac tacattgctc cggagatctt

1741 gctgggtcag aagtacaacc attccgtcga ctggtggtcc ttcggggtgc tcgtttatga

1801 gatgctgatt ggccagtccc ccttccacgg gcaggacgag gaggagctgt tccactccat

1861 ccgcatggac aaccccttct acccgaggtg gctcgaaagg gaggccaagg accttctagt

1921 gaagcttttt gtgagagaac ctgagaagag gctgggagtg agaggagaca tccgccagca

1981 tcctttgttt cgagagatca actgggaaga gcttgaaagg aaagagattg acccaccctt

2041 cagaccaaaa gtgaaatcac catatgactg tagcaatttc gacaaggaat tcctaagtga

2101 gaaaccccgg ctatcattcg ccgacagagc actcatcaac agcatggacc agaacatgtt

2161 cagcaacttt tccttcatta acccagggat ggagactctc atttgctcct gaacctcatc

2221 tgtctccaga ctggaaggga tttgccttct ctctgggaac tggttcaagt aacacttctg

2281 ggggtggggg tctctttttc acgttagaga agaaaagaaa cactgcaaag gcagggagga

2341 ctcctgagct ccttgtgtga cttgttacct acagcacaaa ccacgcctac ttcactaatg

2401 acatcatccc taatgacatc atcccgttat atctcctgga atctctcaca gcagcccttg

2461 aagttagatc attattaact ctagtcattt acttgaaaga tggttcccga tgctgtgaaa

2521 gattcgaaat gcagttctgc tcttgcccta gacaacagct gctggttggt gatgaaccaa

2581 ggcgcaagtg gaacagattt ctcaagactg gagcagtgat cgcctgttat agaagtcaat

2641 tccactcaac cacagagaag gaaccactaa gccacgttga tgtgtgcatg tctgtggaaa

2701 tgtcgatgac agaagggagg gaaaggggaa gctctgagca gattgtaatg ggaagctctc

2761 caataaacat agcatgaaac ttgaaattta caaatctgtt cattctggct agccccaaaa

2821 ttcccaaggc agaggaaagt aaagggcagt gagcttagca gagccctttg tcgccaacag

2881 ggaagggtaa ggatgtcgcc tacgtggaac aacttataca cacagaagga aagtataacc

2941 aacaagggca gggtggttta cagctgccaa tcaaacctgc cctcccccct ctgttctcag

3001 ttgatctctc tgtcagcgta ggtaggcact cattaccatc ctcccatcat acaagaaata

3061 aaatgcatga ctcttctaag ataaagaaaa ccaatccctt atcacgttgt tcccagtgat

3121 ttgatggcaa ataagtccct ccttaggcat cctgcaagac aacccaaccc atgcatgcta

3181 tttgcagtag tcagtcctgt tgagttagag tcctaactat acacaatatc gtgcgatgtt

3241 tatatatgtt gatgagatgt tgtgatgata acgtggatat gtaaaaggga ataaaagaag

3301 aaagaaagat gcc

KKRFSFKKSFK(SEQ ID NO：5)

FARKGSLRQKN(SEQ ID NO：6)

CAGAATATGTTCAGGAACTTTTCCTTCATGAACCCCG(SEQ ID NO：7)

QNMFRNFSFMNP(SEQ ID NO：8)，

GGAGATGCCAAGACGAATACCTTCTGTGGGACACCT(SEQ ID NO：9)

GDAKTNTFCGTP(SEQ ID NO：10)，

SEQ ID NO：11

The nt sequence of the terminal HA mark of PKC θ total length C-

atgtcgccatttcttcggattggcttgtccaactttgactgcgggtcctgccagtcttgtcagggcgaggctgttaacccttact

gtgctgtgctcgtcaaagagtatgtcgaatcagagaacgggcagatgtatatccagaaaaagcctaccatgtacccaccctg

ggacagcacttttgatgcccatatcaacaagggaagagtcatgcagatcattgtgaaaggcaaaaacgtggacctcatctct

gaaaccaccgtggagctctactcgctggctgagaggtgcaggaagaacaacgggaagacagaaatatggttagagctga

aacctcaaggccgaatgctaatgaatgcaagatactttctggaaatgagtgacacaaaggacatgaatgaatttgagacgga

aggcttctttgctttgcatcagcgccggggtgccatcaagcaggcaaaggtccaccacgtcaagtgccacgagttcactgc

caccttcttcccacagcccacattttgctctgtctgccacgagtttgtctggggcctgaacaaacagggctaccagtgccgac

aatgcaatgcagcaattcacaagaagtgtattgataaagttatagcaaagtgcacaggatcagctatcaatagccgagaaac

catgttccacaaggagagattcaaaattgacatgccacacagatttaaagtctacaattacaagagcccgaccttctgtgaac

actgtgggaccctgctgtggggactggcacggcaaggactcaagtgtgatgcatgtggcatgaatgtgcatcatagatgcc

agacaaaggtggccaacctttgtggcataaaccagaagctaatggctgaagcgctggccatgattgagagcactcaacag

gctcgctgcttgagagatactgaacagatcttcagagaaggtccggttgaaattggtctcccatgctccatcaaaaatgaagc

aaggccgccatgtttaccgacaccgggaaaaagagagcctcagggcatttcctgggagtctccgttggatgaggtggata

aaatgtgccatcttccagaacctgaactgaacaaagaaagaccatctctgcagattaaactaaaaattgaggattttatcttgc

acaaaatgttggggaaaggaagttttggcaaggtcttcctggcagaattcaagaaaaccaatcaatttttcgcaataaaggcc

ttaaagaaagatgtggtcttgatggacgatgatgttgagtgcacgatggtagagaagagagttctttccttggcctgggagca

tccgtttctgacgcacatgttttgtacattccagaccaaggaaaacctcttttttgtgatggagtacctcaacggaggggactta

atgtaccacatccaaagctgccacaagttcgacctttccagagcgacgttttatgctgctgaaatcattcttggtctgcagttcc

ttcattccaaaggaatagtctacagggacctgaagctagataacatcctgttagacaaagatggacatatcaagatcgcggat

tttggaatgtgcaaggagaacatgttaggagatgccaagacgaataccttctgtgggacacctgactacatcgccccagag

atcttgctgggtcagaaatacaaccactctgtggactggtggtccttcggggttctcctttatgaaatgctgattggtcagtcgc

ctttccacgggcaggatgaggaggagctcttccactccatccgcatggacaatcccttttacccacggtggctggagaagg

aagcaaaggaccttctggtgaagctcttcgtgcgagaacctgagaagaggctgggcgtgaggggagacatccgccagca

ccctttgtttcgggagatcaactgggaggaacttgaacggaaggagattgacccaccgttccggccgaaagtgaaatcacc

atttgactgcagcaatttcgacaaagaattcttaaacgagaagccccggctgtcatttgccgacagagcactgatcaacagc

atggaccagaatatgttcaggaacttttccttcatgaaccccgggatggagcggctgatatcctacccatacgatgttccagat

tacgcttag

SEQ ID NO：12

The aa sequence of the terminal HA mark of PKC θ total length C-

MSPFLRIGLSNFDCGSCQSCQGEAVNPYCAVLVKEYVESENGQMYIQKKPTM

YPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGKTEI

WLELKPQGRMLMNARYFLEMSDTKDMNEFETEGFFALHQRRGAIKQAKVH

HVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIA

KCTGSAINSRETMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQG

LKCDACGMNVHHRCQTKVANLCGINQKLMAEALAMIESTQQARCLRDTEQI

FREGPVEIGLPCSIKNEARPPCLPTPGKREPQGISWESPLDEVDKMCHLPEPELN

KERPSLQIKLKIEDFILHKMLGKGSFGKVFLAEFKKTNQFFAIKALKKDVVLM

DDDVECTMVEKRVLSLAWEHPFLTHMFCTFQTKENLFFVMEYLNGGDLMYH

IQSCHKFDLSRATFYAAEIILGLQFLHSKGIVYRDLKLDNILLDKDGHIKIADFG

MCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFGVLLYEMLIG

QSPFHGQDEEELFHSIRMDNPFYPRWLEKEAKDLLVKLFVREPEKRLGVRGDI

RQHPLFREINWEELERKEIDPPFRPKVKSPFDCSNFDKEFLNEKPRLSFADRALI

NSMDQNMFRNFSFMNPGMERLISYPYDVPDYA

SEQ ID NO：13；

The nt sequence of the terminal HA mark of PKC θ total length K409W kinases inactivation C-

atgtcgccatttcttcggattggcttgtccaactttgactgcgggtcctgccagtcttgtcagggcgaggctgttaacccttact

gtgctgtgctcgtcaaagagtatgtcgaatcagagaacgggcagatgtatatccagaaaaagcctaccatgtacccaccctg

ggacagcacttttgatgcccatatcaacaagggaagagtcatgcagatcattgtgaaaggcaaaaacgtggacctcatctct

gaaaccaccgtggagctctactcgctggctgagaggtgcaggaagaacaacgggaagacagaaatatggttagagctga

aacctcaaggccgaatgctaatgaatgcaagatactttctggaaatgagtgacacaaaggacatgaatgaatttgagacgga

aggcttctttgctttgcatcagcgccggggtgccatcaagcaggcaaaggtccaccacgtcaagtgccacgagttcactgc

caccttcttcccacagcccacattttgctctgtctgccacgagtttgtctggggcctgaacaaacagggctaccagtgccgac

aatgcaatgcagcaattcacaagaagtgtattgataaagttatagcaaagtgcacaggatcagctatcaatagccgagaaac

catgttccacaaggagagattcaaaattgacatgccacacagatttaaagtctacaattacaagagcccgaccttctgtgaac

actgtgggaccctgctgtggggactggcacggcaaggactcaagtgtgatgcatgtggcatgaatgtgcatcatagatgcc

agacaaaggtggccaacctttgtggcataaaccagaagctaatggctgaagcgctggccatgattgagagcactcaacag

gctcgctgcttgagagatactgaacagatcttcagagaaggtccggttgaaattggtctcccatgctccatcaaaaatgaagc

aaggccgccatgtttaccgacaccgggaaaaagagagcctcagggcatttcctgggagtctccgttggatgaggtggata

aaatgtgccatcttccagaacctgaactgaacaaagaaagaccatctctgcagattaaactaaaaattgaggattttatcttgc

acaaaatgttggggaaaggaagttttggcaaggtcttcctggcagaattcaagaaaaccaatcaatttttcgcaatatgggcc

ttaaagaaagatgtggtcttgatggacgatgatgttgagtgcacgatggtagagaagagagttctttccttggcctgggagca

tccgtttctgacgcacatgttttgtacattccagaccaaggaaaacctcttttttgtgatggagtacctcaacggaggggactta

atgtaccacatccaaagctgccacaag tcgacctttccagagcgacgttttatgctgctgaaatcattcttggtctgcagttcc

ttcattccaaaggaatagtctacagggacctgaagctagataacatcctgttagacaaagatggacatatcaagatcgcggat

tttggaatgtgcaaggagaacatgttaggagatgccaagacgaataccttctgtgggacacctgactacatcgccccagag

atcttgctgggtcagaaatacaaccactctgtggactggtggtccttcggggttctcctttatgaaatgctgattggtcagtcgc

ctttccacgggcaggatgaggaggagctcttccactccatccgcatggacaatcccttttacccacggtggctggagaagg

aagcaaaggaccttctggtgaagctcttcgtgcgagaacctgagaagaggctgggcgtgaggggagacatccgccagca

ccctttgtttcgggagatcaactgggaggaacttgaacggaaggagattgacccaccgttccggccgaaagtgaaatcacc

atttgactgcagcaatttcgacaaagaattcttaaacgagaagccccggctgtcatttgccgacagagcactgatcaacagc

atggaccagaatatgttcaggaacttttccttcatgaaccccgggatggagcggctgatatcctacccatacgatgttccagat

tacgcttag

SEQ I DNO：14

The aa sequence of the terminal HA mark of PKC θ total length K409W kinases inactivation C-

MSPFLRIGLSNFDCGSCQSCQGEAVNPYCAVLVKEYVESENGQMYIQKKPTM

YPPWDSTFDAHINKGRVMQIIVKGKNVDLISETTVELYSLAERCRKNNGKTEI

WLELKPQGRMLMNARYFLEMSDTKDMNEFETEGFFALHQRRGAIKQAKVH

HVKCHEFTATFFPQPTFCSVCHEFVWGLNKQGYQCRQCNAAIHKKCIDKVIA

KCTGSAINSRETMFHKERFKIDMPHRFKVYNYKSPTFCEHCGTLLWGLARQG

LKCDACGMNVHHRCQTKVANLCGINQKLMAEALAMIESTQQARCLRDTEQI

FREGPVEIGLPCSIKNEARPPCLPTPGKREPQGISWESPLDEVDKMCHLPEPELN

KERPSLQIKLKIEDFILHKMLGKGSFGKVFLAEFKKTNQFFAIWALKKDVVLM

DDDVECTMVEKRVLSLAWEHPFLTHMFCTFQTKENLFFVMEYLNGGDLMYH

IQSCHKFDLSRATFYAAEIILGLQFLHSKGIVYRDLKLDNILLDKDGHIKIADFG

MCKENMLGDAKTNTFCGTPDYIAPEILLGQKYNHSVDWWSFGVLLYEMLIG

QSPFHGQDEEELFHSIRMDNPFYPRWLEKEAKDLLVKLFVREPEKRLGVRGDI

RQHPLFREINWEELERKEIDPPFRPKVKSPFDCSNFDKEFLNEKPRLSFADRALI

NSMDQNMFRNFSFMNPGMERLISYPYDVPDYA.

FARKGSLRQ；SEQ ID NO15)

KKRFSFKKSFK(SEQ ID NO：16)

QKRPSQRSKYL(SEQ ID NO：17)

KIQASFRGHMA(SEQ ID NO：18)

LSRTLSVAAKK(SEQ ID NO：19)

AKIQASFRGHM(SEQ ID NO：20)

VAKRESRGLKS(SEQ ID NO：21)

KAFRDTFRLLL(SEQ ID NO：22)

PKRPGSVHRTP(SEQ ID NO：23)

ATFKKTFKHLL(SEQ ID NO：24)

SPLRHSFQKQQ(SEQ ID NO：25)

KFRTPSFLKKS(SEQ ID NO：26)

IYRASYYRKGG(SEQ ID NO：27)

KTRRLSAFQQG(SEQ ID NO：28)

RGRSRSAPPNL(SEQ ID NO：29)

MYRRSYVFQT(SEQ ID NO：30)

QAWSKTTRRI(SEQ ID NO：31)

RGFLRSASLGR(SEQ ID NO：32)

ETKKQSFKQTG(SEQ ID NO：33)

DIKRLTPRFTL(SEQ ID NO：34)

APKRGSILSKP(SEQ ID NO：35)

MYHNSSQKRH(SEQ ID NO：36)

MRRSKSPADSA(SEQ ID NO：37)

TRSKGTLRYMS(SEQ ID NO：38)

LMRRNSVTPLA(SEQ ID NO：39)

ITRKRSGEAAV(SEQ ID NO：40)

EEPVLTLVDEA(SEQ ID NO：41)

SQKRPSQRHGS(SEQ ID NO：42)

KPFKLSGLSFK(SEQ ID NO：43)

AFRRTSLAGGG(SEQ ID NO：44)

ALGKRTAKYRW(SEQ ID NO：45)

VVRTDSLKGRR(SEQ ID NO：46)

KRRQISIRGIV(SEQ ID NO：47)

WPWQVSLRTRF(SEQ ID NO：48)

GTFRSSIRRLS(SEQ ID NO：49)

RVVGGSLRGAQ(SEQ ID NO：50)

LRQLRSPRRTQ(SEQ ID NO：51)

KTRKISQSAQT(SEQ ID NO：52)

NKRRATLPHPG(SEQ ID NO：53)

SYTRFSLARQV(SEQ ID NO：54)

NSRRPSRATWL(SEQ ID NO：55)

RLRRLTAREAA(SEQ ID NO：56)

NKRRGSVPILR(SEQ ID NO：57)

GKRRPSRLVAL(SEQ ID NO：58)

QKKRVSMILQS(SEQ ID NO：59)

RLRRLTAREAA(SEQ ID NO：60)

SEQ ID NO：61

PELNKERP SLQIKLKIED FILHKMLGKG SFGKVFLAEF KKTNQFFAIK

ALKKDVVLMD DDVECTMVEK RVLSLAWEHP FLTHMFCTFQ TKENLFFVME

YLNGGDLMYH IQSCHKFDLS RATFYAAEII LGLQFLHSKG IVYRDLKLDN

ILLDKDGHIK IADFGMCKEN MLGDAKTNTF CGTPDYIAPE ILLGQKYNHS

VDWWSFGVLL YEMLIGQSPF HGQDEEELFH SIRMDNPFYP RWLEKEAKDL

LVKLFVREPE KRLGVRGDIR QHPLFREINW EELERKEIDP PFRPKVKSPF

DCSNFDKEFL NEKPRLSFAD RALINSMDQN MFRNFSFMNP GMERLIS

SEQ ID NO：62

MGPELNKERP SLQIKLKIED FILHKMLGKG SFGKVFLAEF KKTNQFFAIK

ALKKDVVLMD DDVECTMVEK RVLSLAWEHP FLTHMFCTFQ TKENLFFVME

YLNGGDLMYH IQSCHKFDLS RATFYAAEII LGLQFLHSKG IVYRDLKLDN

ILLDKDGHIK IADFGMCKEN MLGDAKTNTF CGTPDYIAPE ILLGQKYNHS

VDWWSFGVLL YEMLIGQSPF HGQDEEELFH SIRMDNPFYP RWLEKEAKDL

LVKLFVREPE KRLGVRGDIR QHPLFREINW EELERKEIDP PFRPKVKSPF

DCSNFDKEFL NEKPRLSFAD RALINSMDQN MFRNFSFMNP GMERLIS

SEQ ID NO：63

MGPELNKERP SLQIKLKIED FILHKMLGKG SFGKVFLAEF KKTNQFFAIK

ALKKDVVLMD DDVECTMVEK RVLSLAWEHP FLTHMFCTFQ TKENLFFVME

YLNGGDLMYH IQSCHKFDLS RATFYAAEII LGLQFLHSKG IVYRDLKLDN

ILLDKDGHIK IADFGMCKEN MLGDAKTNTF CGTPDYIAPE ILLGQKYNHS

VDWWSFGVLL YEMLIGQSPF HGQDEEELFH SIRMDNPFYP RWLEKEAKDL

LVKLFVREPE KRLGVRGDIR QHPLFREINW EELERKEIDP PFRPKVKSPF

DCSNFDKEFL NEKPRLSFAD RALINSMDQN MFRNFSFMNP GMERLISHHH

HHH

NFSFMNPGMER(SEQ ID NO：64)

ALINSMDQNMFR(SEQ ID NO：65)

TNTFCGTPDYIAPEILLGQK(SEQ ID NO：66)

Claims (61)

1. be used to differentiate the method for the correctives of PKC-θ albumen, comprise:

(a) PKC-θ albumen or its functional fragment are contacted with test agent; And

(b) determine whether test agent regulates the kinase activity of PKC-θ albumen or its functional fragment,

Wherein in the presence of test agent, the variation of the kinase activity of PKC-θ albumen or its functional fragment shows the correctives of PKC-θ albumen.

2. the process of claim 1 wherein that the correctives of the PKC-θ albumen that reduces kinase activity is the inhibitor of PKC-θ albumen or its functional fragment.

3. the process of claim 1 wherein that increasing the correctives of the PKC-θ albumen of kinase activity is the activator of PKC-θ albumen or its functional fragment.

4. the process of claim 1 wherein that PKC-θ albumen is total length PKC-θ albumen.

5. the process of claim 1 wherein that PKC-θ albumen is the functional variant of total length PKC-θ albumen.

6. the process of claim 1 wherein that functional fragment is a PKC-θ kinase domain.

7. the process of claim 1 wherein that determining step comprises that compare test reagent is with respect to the kinase activity under the condition that does not have test agent.

8. the process of claim 1 wherein that contact procedure is to realize by the reaction mixture that PKC-θ albumen or its functional fragment and test agent are provided.

9. the method for claim 8, wherein reaction mixture is in the damping fluid of the NaCl that contains the concentration that is selected from 50mM-100mM, 100-150mM, 150-200mM and 200-250mM and 250-300mM.

10. the method for claim 9, wherein NaCl concentration is 250mM.

11. the process of claim 1 wherein that PKC-θ albumen or its fragment obtain from prokaryotic.

12. the method for claim 11, wherein prokaryotic is Escherichia coli.

13. the process of claim 1 wherein that contact procedure realizes in cell.

14. the process of claim 1 wherein that the correctives of PKC-θ albumen is useful for treatment mammal asthma.

15. the method for claim 14, wherein mammal is the people.

16. the method for claim 14, wherein asthma is the asthma of IgE-mediation.

17. the process of claim 1 wherein that kinase activity is the autophosphorylation of PKC-θ albumen or its functional fragment.

18. the method for claim 17, wherein the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues of SEQ ID NO:1.

19. the method for claim 18, wherein autophosphorylation occurs on 538 threonine residues of SEQ ID NO:1.

20. the step of the process of claim 1 wherein (a) further comprises PKC-θ albumen or its functional fragment are contacted with test agent and PKC-θ substrate.

21. the method for claim 20, wherein kinase activity is the phosphorylation of PKC-θ substrate.

22. the method for claim 20, wherein PKC-θ substrate contains R-X-X-S motif or R-X-X-T motif, and wherein R is an arginine, and X is unknown or any known amino acid, and S is a serine, and T is a threonine.

23. the method for claim 22, wherein PKC-θ substrate has and is selected from following amino acid sequence: KKRFSFKKSFK (SEQ ID NO:5), FARKGSLRQKN (SEQ IDNO:6), FARKGSLRQ (SEQ ID NO:15), KKRFSFKKSFK (SEQ IDNO:16), QKRPSQRSKYL (SEQ ID NO:17), KIQASFRGHMA (SEQID NO:18), LSRTLSVAAKK (SEQ ID NO:19), AKIQASFRGHM (SEQ ID NO:20), VAKRESRGLKS (SEQ ID NO:21), KAFRDTFRLLL (SEQ ID NO:22), PKRPGSVHRTP (SEQ IDNO:23), ATFKKTFKHLL (SEQ ID NO:24), SPLRHSFQKQQ (SEQID NO:25), KFRTPSFLKKS (SEQ ID NO:26), IYRASYYRKGG (SEQ ID NO:27), KTRRLSAFQQG (SEQ ID NO:28), RGRSRSAPPNL (SEQ ID NO:29), MYRRSYVFQT (SEQ ID NO:30), QAWSKTTPRRI (SEQ ID NO:31), RGFLRSASLGR (SEQ IDNO:32), ETKKQSFKQTG (SEQ ID NO:33), DIKRLTPRFTL (SEQID NO:34), APKRGSILSKP (SEQ ID NO:35), MYHNSSQKRH (SEQID NO:36), MRRSKSPADSA (SEQ ID NO:37), TRSKGTLRYMS (SEQ ID NO:38), LMRRNSVTPLA (SEQ ID NO:39), ITRKRSGEAAV (SEQ ID NO:40), EEPVLTLVDEA (SEQ IDNO:41), SQKRPSQRHGS (SEQ ID NO:42), KPFKLSGLSFK (SEQID NO:43), AFRRTSLAGGG (SEQ ID NO:44), ALGKRTAKYRW (SEQ ID NO:45), VVRTDSLKGRR (SEQ ID NO:46), KRRQISIRGIV (SEQ ID NO:47), WPWQVSLRTRF (SEQ ID NO:48), GTFRSSIRRLS (SEQ ID NO:49), RVVGGSLRGAQ (SEQ IDNO:50), LRQLRSPRRTQ (SEQ ID NO:51), KTRKISQSAQT (SEQID NO:52), NKRRATLPHPG (SEQ ID NO:53), SYTRFSLARQV (SEQ ID NO:54), NSRRPSRATWL (SEQ ID NO:55), RLRRLTAREAA (SEQ ID NO:56), NKRRGSVPILR (SEQ IDNO:57), GKRRPSRLVAL (SEQ ID NO:58), QKKRVSMILQS (SEQID NO:59), and RLRRLTAREAA (SEQ ID NO:60).

24. the process of claim 1 wherein that the correctives of PKC-θ albumen makes the kinase activity of PKC-θ albumen or its functional fragment reduce twice at least.

25. the process of claim 1 wherein that PKC-θ albumen or its functional fragment are in cell.

26. the method for claim 25, wherein cell is selected from mast cell and CD4+T cell.

27. the method for claim 14 further is included in the effectiveness of the test agent of differentiating in the appraisal procedure (b) in the asthmatic model in the external or body,

Wherein demonstrating the test agent that render a service to increase with respect to contrast agents in external or body in the asthmatic model is just differentiated to being useful for treatment asthma.

28. be used to differentiate the method for the correctives of PKC-θ albumen, comprise:

(a) cell that will express PKC-θ albumen or its functional fragment contacts with test agent; And

(b) determine whether test agent reduces PKC-θ albumen or the autophosphorylation of its functional fragment in cell,

Wherein in the presence of test agent, the variation of the autophosphorylation of PKC-θ albumen or its functional fragment shows the correctives of PKC-θ albumen.

29. the method for claim 28, the correctives that wherein reduces the PKC-θ albumen of kinase activity is the inhibitor of PKC-θ albumen or its functional fragment.

30. the method for claim 28, the correctives that wherein increases the PKC-θ albumen of kinase activity is the activator of PKC-θ albumen or its functional fragment.

31. the method for claim 28, wherein PKC-θ albumen is total length PKC-θ albumen.

32. the method for claim 28, wherein PKC-θ albumen is the functional variant of total length PKC-θ albumen.

33. the method for claim 28, wherein functional fragment is a PKC-θ kinase domain.

34. the method for claim 28, wherein determining step comprises that compare test reagent is with respect to the kinase activity under the condition that does not have test agent.

35. the method for claim 28, wherein the correctives of PKC-θ albumen is useful for treatment asthma.

36. the method for claim 35 further is included in the effectiveness of the test agent of differentiating in the appraisal procedure (b) in the asthmatic model in the external or body,

Wherein demonstrating the test agent that render a service to increase with respect to contrast agents in external or body in the asthmatic model is just differentiated to being useful for treatment asthma.

37. the method for claim 28, wherein cell is a prokaryotic.

38. the method for claim 37, wherein prokaryotic is Escherichia coli.

39. the method for claim 28, wherein the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues of SEQ ID NO:1.

40. the method for claim 39, wherein autophosphorylation occurs on 538 threonine residues of SEQ ID NO:1.

41. the method for treatment asthma comprises to suffering from asthma or suffering from the reduction PKC-θ albumen of administration treatment effective dose of SOA or the kinase activity of its functional fragment or reduce the reagent that functional PKC-θ albumen produces.

42. the method for claim 41, wherein reagent is used in pharmaceutically acceptable carrier.

43. the method for claim 42, wherein carrier is an aerosol form.

44. the method for claim 41, wherein reagent by be selected from intravenous, oral, use through skin and intramuscular approach.

45. the method for claim 41, wherein reagent is used through suction.

46. the method for claim 41, wherein asthma is the asthma of IgE-mediation.

47. the method for claim 41, wherein reagent is to use altogether with the medicine that is selected from beta-adrenergic reagent, theophylline compound, corticosteroid, anticholinergic, antihistaminic, calcium channel blocker and nasmil.

48. the method for claim 41, wherein reagent is the antibody of specificity in conjunction with PKC-θ albumen or its fragment.

49. the method for claim 48, wherein antibody is polyclonal antibody.

50. the method for claim 48, wherein antibody is monoclonal antibody.

51. the method for claim 41, wherein reagent is nucleic acid molecules.

52. the method for claim 51, wherein nucleic acid molecules is a ribonucleic acid molecule.

53. the method for claim 52, wherein ribonucleic acid molecule comprises the nucleotide sequence with the part complementation of the described nucleotide sequence of SEQ ID NO:3.

54. the method for claim 41, wherein kinase activity is the autophosphorylation of PKC-θ albumen or its functional fragment.

55. the method for claim 54, wherein the autophosphorylation of PKC-θ albumen or its functional fragment occurs on the amino acid residue that is selected from 695 serine residues, 685 serine residues, 538 threonine residues and 536 threonine residues of SEQ ID NO:1.

56. the method for claim 55, wherein the autophosphorylation of PKC-θ albumen or its functional fragment occurs on 538 threonine residues of SEQ ID NO:1.

57. the method for claim 41, wherein kinase activity is the phosphorylation of PKC-θ substrate.

58. the method for claim 57, wherein PKC-θ substrate contains R-X-X-S motif or R-X-X-T motif, and wherein R is an arginine, and X is unknown or any known amino acid, and S is a serine, and T is a threonine.

59. the method for claim 58, wherein PKC-θ substrate has and is selected from following amino acid sequence: KKRFSFKKSFK (SEQ ID NO:5), FARKGSLRQKN (SEQ IDNO:6), FARKGSLRQ (SEQ ID NO:15), KKRFSFKKSFK (SEQ IDNO:16), QKRPSQRSKYL (SEQ ID NO:17), KIQASFRGHMA (SEQID NO:18), LSRTLSVAAKK (SEQ ID NO:19), AKIQASFRGHM (SEQ ID NO:20), VAKRESRGLKS (SEQ ID NO:21), KAFRDTFRLLL (SEQ ID NO:22), PKRPGSVHRTP (SEQ IDNO:23), ATFKKTFKHLL (SEQ ID NO:24), SPLRHSFQKQQ (SEQID NO:25), KFRTPSFLKKS (SEQ ID NO:26), IYRASYYRKGG (SEQ ID NO:27), KTRRLSAFQQG (SEQ ID NO:28), RGRSRSAPPNL (SEQ ID NO:29), MYRRSYVFQT (SEQ ID NO:30), QAWSKTTPRRI (SEQ ID NO:31), RGFLRSASLGR (SEQ IDNO:32), ETKKQSFKQTG (SEQ ID NO:33), DIKRLTPRFTL (SEQID NO:34), APKRGSILSKP (SEQ ID NO:35), MYHNSSQKRH (SEQID NO:36), MRRSKSPADSA (SEQ ID NO:37), TRSKGTLRYMS (SEQ ID NO:38), LMRRNSVTPLA (SEQ ID NO:39), ITRKRSGEAAV (SEQ ID NO:40), EEPVLTLVDEA (SEQ IDNO:41), SQKRPSQRHGS (SEQ ID NO:42), KPFKLSGLSFK (SEQID NO:43), AFRRTSLAGGG (SEQ ID NO:44), ALGKRTAKYRW (SEQ ID NO:45), VVRTDSLKGRR (SEQ ID NO:46), KRRQISIRGIV (SEQ ID NO:47), WPWQVSLRTRF (SEQ ID NO:48), GTFRSSIRRLS (SEQ ID NO:49), RVVGGSLRGAQ (SEQ IDNO:50), LRQLRSPRRTQ (SEQ ID NO:51), KTRKISQSAQT (SEQID NO:52), NKRRATLPHPG (SEQ ID NO:53), SYTRFSLARQV (SEQ ID NO:54), NSRRPSRATWL (SEQ ID NO:55), RLRRLTAREAA (SEQ ID NO:56), NKRRGSVPILR (SEQ IDNO:57), GKRRPSRLVAL (SEQ ID NO:58), QKKRVSMILQS (SEQID NO:59), and RLRRLTAREAA (SEQ ID NO:60).

60. do not express the mast cell of the separation of endogenous PKC-θ albumen.

61. the mast cell of claim 40, wherein cellular expression external source PKC-θ albumen or its fragment.

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