US20030162230A1 - Method for quantifying phosphokinase activity on proteins - Google Patents

Method for quantifying phosphokinase activity on proteins Download PDF

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US20030162230A1
US20030162230A1 US09/948,972 US94897201A US2003162230A1 US 20030162230 A1 US20030162230 A1 US 20030162230A1 US 94897201 A US94897201 A US 94897201A US 2003162230 A1 US2003162230 A1 US 2003162230A1
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protein
phosphorylated
antibody
tau
kinase
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Kevin Reagan
Erik Schaefer
Jimin Wang
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Life Technologies Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein

Definitions

  • This invention relates to assays and reagents for measuring protein kinase activity in vitro.
  • Phosphorylation of these amino acid residues can alter the function and/or location of the protein within the cell. This change can involve changes in the enzymatic activity of the affected protein and/or create binding sites for the recruitment of other signaling proteins.
  • protein kinases are critical components of many cellular signaling pathways, their catalytic activity is often tightly regulated. Abnormalities in protein kinase activity result in different patterns of phosphorylation that can dramatically alter cell function. Indeed, many drug discovery efforts involve the identification of therapeutic agents that selectively suppress or augment protein kinase activity in order to treat a disease.
  • This invention is designed to provide assays and reagents to monitor protein kinase activity.
  • the targeted residues for phosphorylation can be contained in a full-length, biologically active molecule of recombinant or natural origin.
  • Most methods currently employed for measuring protein kinase activity use peptide substrates, which include the targeted phosphorylation residue.
  • This art is taught in U.S. Pat. No. 6,066,462 (Quantitation of individual protein kinase activity) incorporated herein by reference.
  • This method differs from the present invention in that the peptide substrate does not contain all possible phosphorylation sites that can be acted on by kinases and thus may not truly reflect activity on a natural protein.
  • the invention described herein can be used with whole molecule or fragments, of natural or recombinant origin.
  • the delineation of activity at different phosphorylation sites requires in the invention, a different PSSA for detection as opposed to a different peptide in U.S. Pat. No. 6,066,462.
  • Another method for detection of kinase activity involves use of a generic antibody that binds to all phospho-tyrosine residues. This method is described in U.S. Pat. No. 5,766,863 (Kinase receptor activation assay) incorporated herein by reference. This method suffers from an inability to discriminate among phosphorylated tyrosine residues on a molecule. This method does not address detection of phospho-serine or phospho-threonine events since the anti-phospho-tyrosine antibody does not detect such phosphorylated residues. In contrast, the method described herein uses antibodies, which bind to the sequence specific residues surrounding the phosphorylated amino acid plus the phospho-residue itself. The reagents used in this invention are capable of detecting phosphorylated threonine, serine or tyrosine molecules.
  • the current invention and related methods are applicable to a wide range of signal transduction proteins (see Table I for a partial list). Three examples are illustrated below using important molecular targets of current interest in basic research and disease-oriented pharmaceutical study.
  • Tau a neuronal microtubule-associated protein found predominantly in axons.
  • the function of Tau is to promote tubulin polymerization and stabilize microtubules, but it also serves to link certain signaling pathways to the cytoskeleton.
  • Tau phosphorylation regulates both normal and pathological functions of this protein.
  • Tau in its hyper-phosphorylated form, is the major component of paired helical filaments (PHF), the building block of neurofibrillary lesions that are often found in the brains of individuals with Alzheimer's disease (AD).
  • PHF paired helical filaments
  • Hyperphosphorylation impairs the microtubule binding function of Tau, resulting in the destabilization of microtubules in AD brains, ultimately leading to neuronal degeneration.
  • Hyperphosphorylated Tau is also found in a range of other central nervous system disorders. Numerous serine/threonine kinases, including GSK-3 ⁇ , PKA, PKC, CDK5, MARK, JNK, p38MAPK and casein kinase II, can phosphorylate Tau.
  • U.S. Pat. No. 5,601,985 relates to methods of detecting abnormally phosphorylated Tau Protein
  • U.S. Pat. No. 5,843,779 relates to monoclonal antibodies directed against the microtubule-associated protein, Tau, and hybridomas secreting these antibodies
  • U.S. Pat. No. 5,733,734 relates to methods of screening for Alzheimer's disease or disease associated with the accumulation of paired helical filaments
  • U.S. Pat. No. 6,066,462 relates to quantitation of individual protein kinase activity.
  • Retinoblastoma protein (Rb), the tumor suppressor product of the retinoblastoma susceptibility gene, is a 110 kDa protein which plays an important role in regulating cell growth and differentiation. Loss of its function leads to uncontrolled cell growth and tumor development. Mutational inactivation of the Rb gene is found in all retinoblastomas and in a variety of other human malignancies including cancers of breast, lung, colon, prostate, osteosarcomas, soft tissue sarcomas, and leukemia. Central to the role of the Rb protein as a tumor suppressor is the ability of Rb to suppress inappropriate proliferation by arresting cells in the G1 phase of the cell cycle.
  • Rb protein exerts its growth suppressive function by binding to transcription factors including E2F-1, PU.1, ATF-2, UBF, Elf-1, and c-Ab1.
  • the binding of Rb protein is governed by its phosphorylation state.
  • Hypo- or under-phosphorylated forms of Rb bind and sequester transcription factors, most notably those of the E2F/DP family, inhibiting the transcription of genes required to traverse the G1 to S phase boundary of the cell cycle.
  • This cell cycle inhibitory function is abrogated when Rb undergoes phosphorylation catalyzed by specific complexes of cyclins and cyclin-dependent protein kinases (cdks).
  • Rb Removal of phosphates on Rb appears to be carried out by a multimeric complex of protein phosphatase type 1 (PP1) and noncatalytic regulatory subunits at the completion of mitosis.
  • PP1 protein phosphatase type 1
  • WO 01/11367 (Assay of the phosphorylation activity of cyclin/CDK complex on retinoblastoma (RB) protein for identifying compounds which modify this activity) describes a method for detecting kinase activity by ELISA using a synthetic peptide and a monoclonal antibody that recognizes the phosphorylated form of the peptide.
  • the basis of this method is the coating of a solid phase with a synthetic peptide containing the consensus sequence of a region upon which a kinase acts. The peptide is allowed to come in contact with a kinase that allows a specific residue on that peptide to become phosphorylated.
  • the activity of the kinase then is estimated by the binding of the generic monoclonal antibody to the target phosphopeptide.
  • Our invention differs from WO 01/11367 in that it uses a natural protein as the substrate for kinase activity. This feature is superior to the use of peptides since all naturally occurring phosphorylation sites would be present and the protein would be presented in its normal conformation.
  • the use of a single monoclonal antibody recognizing phosphoserine (clone 2B9) also does not allow any discrimination of the many phosphorylation sites that naturally occur on Rb protein.
  • Our use of specific PSSAs allows that distinction as well as the detection of phosphothreonine and phosphotyrosine residues allowing a profile of Rb phosphorylation sites to be constructed.
  • EGFR Epidermal growth factor receptor
  • RTKs receptor tyrosine kinases
  • EGFR is expressed at full length as a 170 kDa type I transmembrane glycoprotein which consists of an extracellular ligand-binding domain, a single hydrophobic transmembrane region, and an intracellular domain that exhibits tyrosine enzymatic activity and which is involved in signal transduction.
  • Several deletions in the extra- and intracellular domain of the EGFR have been found in a number of tumors.
  • EGFRvIII is a 145 kDa protein with a deletion of exons 2-7 in EGFR mRNA.
  • a 100 kDa truncated EGFR without the cytoplasmic domain is observed in the culture supernatant from A431 cells, a human epidermal carcinoma cell line.
  • EGFR is activated by binding of a number of ligands such as EGF, transforming growth factor ⁇ (TGF ⁇ ), amphiregulin, betacellulin, heparin binding EGF-like growth factor (HB-EGF) and epiregulin.
  • ligands such as EGF, transforming growth factor ⁇ (TGF ⁇ ), amphiregulin, betacellulin, heparin binding EGF-like growth factor (HB-EGF) and epiregulin.
  • the binding causes EGFR homo- and heterodimerization and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain, which involves rapid activation of its intrinsic tyrosine kinase activity.
  • Phosphorylation of tyrosine residues in the COOH-terminal tail of the EGFR serve as binding sites for cytosolic signaling proteins containing Src homology 2 (SH2) domains.
  • SH2 Src homology 2
  • Elevated expression and/or amplification of the EGFR have been found in breast, bladder, glioma, colon, lung, squamous cell, head and neck, ovarian, and pancreatic cancers.
  • Selective compounds have been developed that target either the extracellular ligand-binding domain of EGFR or the intracellular tyrosine kinase region, resulting in interference with the signaling pathways that modulate mitogenic and other cancer-promoting responses.
  • These potential anticancer agents include a number of small molecule, tyrosine kinase inhibitors.
  • the invention describes assays and reagents for quantitating phosphorylation of proteins.
  • the method involves subjecting a protein to a protein kinase that will phosphorylate the protein and binding this specific phosphoryated form of the protein with an antibody specific for the amino acid sequence containing the phosphorylated site and detecting the primary antibody bound to the phosphorylated site.
  • the invention includes antibodies useful in practicing the methods of the invention.
  • the invention particularly relates to phosphorylation of Tau, Rb, and EGFR proteins and antibodies specific for the sites of phosphorylation within the Tau, Rb, and EGFR proteins.
  • the invention can be applied to all proteins and antibodies that recognize specific phosphorylation sites on these proteins (see Table I).
  • the targeted protein (Tau, Rb or EGFR) is phosphorylated in vitro or in vivo and the specific phosphorylation event is detected using a highly selective phosphorylation site-specific antibody (PSSA).
  • PSSA highly selective phosphorylation site-specific antibody
  • the appearance or disappearance of the targeted phosphorylation event can be quantified as a percentage of total protein that may be phosphorylated at each site.
  • the highly specific nature of the PSSAs allows parallel independent measurement of multiple phosphorylation sites on one protein. Moreover, different kinases can be measured simultaneously by using different PSSAs that selectively target different sites in the protein, thereby providing an avenue for generating phosphorylation site profiles.
  • this invention measures protein kinase enzymatic activity that results in the phosphorylation of proteins at a specific site(s). This method is also amenable to large-scale ‘High Throughput Screening’ formats currently being used by pharmaceutical and biotech companies to discover new drugs that block specific phosphorylation events.
  • FIG. 1 illustrates specificity of the Anti-phospho Tau [pS 199 ] phosphorylation site-specific antibody (PSSA).
  • FIG. 2 illustrates Anti-phospho Tau [pS 214 ] PSSA specificity.
  • FIGS. 3 a and 3 b illustrate detection of total Tau vs. Tau phosphorylated at the PKA/serine 214 site by ELISA.
  • FIGS. 4 a - b illustrate detection of Tau phosphorylated at GSK-3 ⁇ /serine 199/202 (4a) vs. total Tau (4b) sites by ELISA.
  • FIG. 5 illustrates a dose-response curve generated in an ELISA using the Tau serine 214 PSSA.
  • FIG. 6 illustrates the specificity of the Tau PSSAs in an ELISA to detect Tau phosphorylation catalyzed by PKA vs. GSK-3 ⁇ enzymes.
  • FIG. 7 illustrates that multiple GSK-3 ⁇ phosphorylation sites on Tau can be detected by ELISA using Tau PSSAs.
  • FIG. 8 illustrates that a specific inhibitor of PKA activity selectively inhibits the phosphorylation on serine 214 of Tau but does not interfere with GSK enzyme activity as demonstrated using Tau [pS 214 ] and Tau [pS 199 ] PSSAs as detected by ELISA.
  • FIG. 9 defines the specificity of the anti-Rb [pT 821 ].
  • FIG. 10 shows studies to determine the specificity of the Rb [pT 821 ] ELISA.
  • FIG. 11 shows the specificity of the Rb [pT 821 ] ELISA for threonine 821 as determined by peptide competition.
  • FIG. 12 shows the application of the Rb [pT 821 ] ELISA in evaluating kinase activity in Jurkat cells were grown in the presence of the kinase inhibitor, staurosporine.
  • FIG. 13 illustrates the specificity of the EGFR PSSA [pY 1173 ].
  • FIG. 14 shows the specificity of the EGFR [pY 1173 ] ELISA for tyrosine residue 1173 as determined by peptide competition.
  • FIG. 15 demonstrates the response curve of phosphorylation of EGFR in A431 cells after treatment with EGF using the EGFR [pY 1173 ] ELISA.
  • FIG. 16 shows the application of the EGFR [pY 845 ] ELISA in evaluating kinase activity in A431 cells were grown in the presence of the tyrosine kinase inhibitor, PD158780.
  • Tau System The Tau protein system demonstrates the utility of this invention on a protein that is found both intracellularly and extracellularly in normal and pathological conditions.
  • the Tau protein has multiple phosphorylation sites acted upon by multiple protein kinases. Phosphoserine and phosphotyrosine residues exist. Both mono-phospho and dual-phosphoresidues are distinguishable in this model system.
  • Tau pS 199 PSSA Rabbits were immunized with a chemically synthesized and KLH conjugated phosphopeptide corresponding to the region of the longest isoform of the Tau protein that includes serine 199.
  • the chemically synthesized phosphopeptides (RSGYS(pS)PGSPG) is sequence ID #1.
  • the Tau pS 199 PSSA was purified from rabbit serum by sequential epitope-specific chromatography. The antibody was negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Tau. The final product was generated by affinity chromatography using the peptide that is phosphorylated at serine 199.
  • FIG. 1 show that only the phosphopeptide corresponding to this site blocks the antibody signal, illustrating the specificity of the Anti-Tau [pS 199 ] antibody for this phosphorylation site.
  • Tau [pS 214 ] PSSA The procedures for generating this antibody were similar to those described above for the Tau pS 199 PSSA.
  • the chemically synthesized phosphopeptide was derived from the region of the longest isoform of Tau protein that includes serine 214 (GSRSRTP(pS)LPTPP) sequence ID#2.
  • This antibody recognizes specifically the Tau protein when phosphorylated on serine 214, as demonstrated by peptide competition analysis in a western blotting assay.
  • Serine 214 is phosphorylated in vitro and in vivo by cAMP-dependent protein kinase (PKA), which is commercially available from Biosource International.
  • PKA cAMP-dependent protein kinase
  • FIG. 2 Tau pS 214 PSSA specificity is show in FIG. 2.
  • Membranes were incubated with 0.50 ⁇ g/mL anti-phospho tau [pS 214 ], following prior incubation in the absence (a) or presence of the peptide immunogen (b), or the non-phosphopeptide corresponding to the tau phosphopeptide (c). After washing, membranes were incubated with goat F(ab′) 2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStarTM method.
  • the mouse mAb to Tau was raised using purified bovine microtubule-associated proteins (MAPs) as the immunogen.
  • MAPs bovine microtubule-associated proteins
  • the resulting hybridoma was produced by fusing immunized BALB/c mouse splenocytes and mouse myeloma Sp2/0-Ag14 cells. It shows no cross-reaction with other MAPs or tubulin. It reacts with the non-phosphorylated as well as the phosphorylated forms of Tau and the reactive epitope maps to residues 210-230. This reagent is commercially available from Biosource International.
  • Phosphorylation of Tau using PKA was performed as follows.
  • PKA was purchased from New England Biolabs.
  • Recombinant Tau protein (1 ⁇ g) was incubated with various concentrations of PKA enzyme in buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 and 100 ⁇ M ATP for 1 hour at 30° C.
  • GSK-3 ⁇ was purchased from Upstate Biotechnology Inc. Recombinant Tau protein (1 ⁇ g) was incubated with various concentrations of the enzyme in buffer containing 40 mM HEPES (pH 7.2), 5 mM MgCl 2 , 5 mM EDTA, 100 ⁇ M ATP, and 50 ⁇ g/mL heparin for 1 hour at 30° C.
  • FIGS. 3 a and 3 b show the assessment of total Tau and selective Tau phosphorylation at the PKA/ Ser 214 site by ELISA.
  • phosphorylated Tau was detected by an ELISA using a PSSA specific for Tau pS 214 or by a pan-Tau antibody. Both antibodies detected the phosphorylated Tau protein with equal signals.
  • non-phosphorylated Tau was placed into the same assay.
  • the anti-Tau [pS 214 ] antibody failed to detect the Tau protein lacking the phosphate group at serine 214, whereas the pan-Tau antibody did detect the Tau protein.
  • FIGS. 4 a and 4 b show the assessment of total Tau vs. selective Tau phosphorylation at the GSK-3 ⁇ / Ser 199/202 sites by ELISA.
  • FIG. 4 a uses either non-phosphorylated Tau or GSK-3 ⁇ - phosphorylated Tau in the ELISA with the anti-Tau pS 199/202 antibody. Non-phosphorylated Tau does not react in the ELISA, whereas the phosphorylated Tau shows strong signals. If the pan-Tau antibody is used as the detector, both proteins are readily detected (FIG. 4 b ).
  • FIG. 5 shows the direct relationship between the amount of phospho-Tau protein detected by ELISA and the quantity of protein kinase activity in the in vitro reaction.
  • PKA tau 1 5 units, the PKA enzyme was then serially diluted 1:2 as shown, followed by a 1:1000 dilution and then applied to each well of the ELISA. Detection of phosphoTau was performed using the anti-Tau [pS 214 ] (a PKA site).
  • FIGS. 6 a and 6 b shows the specificity in detecting Tau protein phosphorylation catalyzed by PKA vs. GSK3 ⁇ enzymes using the Tau PSSAs and ELISA.
  • the results demonstrate that the Tau pS 214 PSSA ELISA only detects Tau when phosphorylated by PKA and the Tau pS 199 PSSA ELISA only detects Tau when phosphorylated by GSK3 ⁇ .
  • FIG. 7 shows that the GSK3 ⁇ enzyme can phosphorylate multiple sites on the Tau protein and PSSAs can independently detect the phosphorylated sites at Tau pT 181 , Tau pS 202 , Tau pS 199/ pS 202 , Tau pS 396 , and Tau pS 404 .
  • This provides evidence that the ELISA is useful in creating a profile of phosphorylation events on the protein subjected to kinase enzyme activity.
  • FIG. 8 shows the specificity of kinase reaction when tested as a profile with two antibodies, one specific for a PKA phosphorylation site (pS 214 ) and the other for a GSK site (pS 199 ) on Tau protein.
  • a PKA-specific inhibitor PKI (heat-stable inhibitor of c-AMP-dependent protein kinase; New England Biolabs)
  • PKI heat-stable inhibitor of c-AMP-dependent protein kinase
  • the PKA-specific inhibitor altered the kinase activity of the pS 214 site alone.
  • Rb System This model system describes a large intra-nuclear protein with multiple phosphorylation sites that are acted upon by multiple protein kinases. Both phosphoserine and phosphotyrosine residues are examined, for which both mono-phospho and dual-phosphoresidues are distinguishable in this model system.
  • Rb protein Full length Rb protein is purified recombinant protein derived through cloning of human Rb cDNA and expressed in E. coli. The protein is purified via standard methods. This protein is commercially available from multiple vendors.
  • Rb [pT 821 PSSA: The rabbit antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human Rb that contains threonine 821. Antibody was purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Rb. The final product is generated by affinity chromatography using a Rb-derived peptide that is phosphorylated at threonine 821. FIG.
  • Detection Antibody is a monoclonal, clone G3-245, available commercially from BD/Pharmingen (San Diego, Calif.). It recognizes an epitope between amino acids 332-344 of Rb protein. This antibody will bind to both non-phosphorylated and phosphorylated forms of Rb protein.
  • Rb monoclonal antibody the capture antibody [linked to the solid phase] is a monoclonal, clone 3C8, available commercially from QED Biosciences (San Diego, Calif.). It reacts with epitope on near the C-terminal end of the Rb protein (aa886-aa905). This antibody will bind to both non-phosphorylated and phosphorylated forms of Rb protein.
  • Total Rb and Rb [pT 821 ELISA: A concentration of 1.25 ⁇ g/mL of Rb monoclonal antibody in carbonate buffer, pH 9.4, was incubated at 100 ⁇ L/well in microtiter plates at 4° C. overnight. The wells were washed with a PBS/Tween-20 solution three times followed by blocking on other sites on the plastic surface with a buffered solution containing unrelated proteins such as BSA for 2 hours at room temperature. Jurkat cell lysate containing phosphorylated Rb or non-phosphorylated recombinant Rb were added to the wells at various concentrations and incubated for 2 hour at room temperature.
  • FIG. 10 shows studies to determine the specificity of the Rb [pT 821 ] ELISA.
  • solutions containing Rb protein at a concentration of 20 ng/mL from Jurkat, U2OS, and Colo205 were analyzed with the Rb [pT 821 ] ELISA kit, along with a solution containing 20 ng/mL purified full length Rb protein expressed in E. coli (non-phosphorylated).
  • FIG. 11 shows that the Rb protein isolated from the cell lines was strongly recognized.
  • FIG. 12 shows the application of this ELISA to study kinase reactions.
  • Jurkat cells were grown in the presence of the kinase inhibitor, staurosporine, at various concentrations for 36 hours prior to lysis. Lysates were normalized for total Rb content using the Total Rb ELISA (BioSource International catalog #KHO0011). Levels of Rb phosphorylation at threonine 821 were determined. These data show that staurosporine inhibits the phosphorylation of Rb at threonine 821, presumably through the inhibition of cdks.
  • EGFR System This model system presents an analysis of the cell surface receptor Epidermal Growth Factor Receptor (EGFR). This protein is a large transmembrane signaling protein with multiple phosphorylation sites consisting of phospho-threonine, phospho-serine and phospho-tyrosine residues.
  • EGFR Epidermal Growth Factor Receptor
  • EGFR protein Human EGFR protein was purified from human carcinoma A431 cells by affinity purification. The product is purchased from Sigma (St. Louis, Mo.; cat # E-2645).
  • EGFR [pY 1173 ]
  • PSSA Rabbit antiserum was produced against a chemically synthesized phosphopeptide derived from the region of EGFR that contains tyrosine 1173. The sequence is conserved in human, mouse, and rat. Antibody was purified from serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using (i) a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated EGFR enzyme, and (ii) a generic tyrosine phosphorylated peptide to remove antibody that is reactive with phospho-tyrosine (irrespective of the sequence).
  • FIG. 13 illustrates the specificity of the EGFR PSSA [pY 1173 ].
  • Cell extracts prepared from NIH3T3 cells expressing EGFR were starved for 30 hours, then stimulated for 10 minutes with 30 ng/mL EGF (+), or left unstimulated ( ⁇ ), then resolved by SDS-PAGE on a 6% Tris-glycine gel, and transferred to nitrocellulose.
  • Membranes were incubated with 0.50 ⁇ g/mL anti-EGFR [pY 1173 ] antibody, following prior incubation in the absence (lanes 1& 2), or presence of the peptide immunogen (lanes 3 & 4), or the non-phosphopeptide corresponding to the EGFR phosphopeptide (lanes 5 & 6). After washing, membranes were incubated with goat F(ab′) 2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStarTM detection method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the anti-EGFR [pY 1173 ] antibody for this phosphorylated residue.
  • EGFR [pY 845 ] PSSA Prepared essentially as EGFR [pY 1173 ] PSSA but using chemically synthesized phosphopeptides from the region that contains tyrosine 845.
  • EGFR [Pan] monoclonal antibody The capture antibody is a mouse monoclonal antibody, clone 199.12, available commercially from Neomarkers, Inc. (Union City, Calif.). It is specific for human EGFR and does not react with HER2/neu, HER3 and HER4. This antibody will bind to both non-phosphorylated and phosphorylated forms of EGFR protein and therefore is used as an initial capture antibody in the EGFR ELISA
  • EGFR Detection Antibody: This rabbit antibody was prepared by immunization with a synthetic peptide corresponding to C-terminus of human EGFR. The antibody was purified using protein A affinity column. It shows no cross-reactivity with HER2/neu, HER3 and HER4.
  • EGFR PSSA and Full Length ELISA A concentration of 2.5 ⁇ g/mL of pan-EGFR monoclonal antibody in carbonate buffer, pH 9.4, was incubated at 100 ⁇ L/well in microtiter plates at 4° C. overnight. The wells were washed with a PBS/Tween-20 solution three times followed by blocking on other sites on the plastic surface with a buffered solution containing unrelated proteins such as BSA for 2 hours at room temperature. Autophosphorylated EGFR or non-phosphorylated EGFR were added to the wells at various concentrations and incubated for 1 hour at room temperature.
  • EGFR was incubated to induce auto-phosphorylation in a buffer of 15 mM HEPES (pH7.4), 6 mM MnCl 2 and 15 mM MgCl 2 containing 1uM ATP for 30 minutes at 30° C.
  • FIG. 15 demonstrates the dose-response curve of phosphorylation of EGFR in A431 cells after treatment with EGF at 1-500 ng/mL for 10 minutes.
  • the level of tyrosine phosphorylation of EGFR at tyrosine 1173 was detected with the EGFR [pY 1173 ] ELISA.
  • FIG. 16 demonstrates use of the described invention to detect protein kinase activity associated with EGFR at tyrosine residue 845 and inhibition of that activity by a protein kinase inhibitor.
  • 2 ng/ vial of purified human EGFR was incubated (auto-phosphorylated) in the buffer of 15 mM HEPES (pH7.4), 6 mM MnCl 2 and 15 mM MgCl 2 containing 1uM ATP for 30 minutes at 30° C.
  • tyrosine kinase inhibitor PD158780 (Calbiochem, cat #. 513035) was added to the reaction at the indicated concentration (see FIG.
  • PAK1 p21-Activated protein Kinase 1
  • PAK2 p21-Activated protein Kinase 2
  • PAK3 p21-Activated protein Kinase 3
  • PARP Poly(ADP-Ribose) Polymerase)
  • PCNA Proliferating Cell Nuclear Antigen
  • PDGF Receptor Platinum Derived Growth Factor Receptor
  • PDK1 Phosphoinositide-Dependent Kinase-1)
  • PDK-2 Phosphoinositide-Dependent Kinase-2/ Integrin-linked kinase
  • PECAM-1 Platinum-Endothelial Cell Adhesion Molecule-1) PI3

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WO2008147687A1 (fr) * 2007-05-21 2008-12-04 Mayo Foundation For Medical Education And Research Traitement de la pancréatite
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US7888050B2 (en) 2000-09-27 2011-02-15 Life Technologies Corporation Method for quantifying phosphokinase activity on proteins
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US20040014082A1 (en) * 2000-08-11 2004-01-22 Invitrogen Corporation Highly homogeneous molecular markers for electrophoresis
US20110165588A1 (en) * 2000-09-27 2011-07-07 Life Technologies Corporation Method for quantifying phosphokinase activity on proteins
US8114617B2 (en) 2000-09-27 2012-02-14 Life Technologies Corporation Method for quantifying phosphokinase activity on proteins
US7888050B2 (en) 2000-09-27 2011-02-15 Life Technologies Corporation Method for quantifying phosphokinase activity on proteins
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
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DE60136840D1 (de) 2009-01-15
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US20120135426A1 (en) 2012-05-31
JP2004516823A (ja) 2004-06-10
EP2298814A3 (fr) 2012-05-30
EP1328812A2 (fr) 2003-07-23
CA2423979C (fr) 2012-03-06
US8114617B2 (en) 2012-02-14
US20090104628A1 (en) 2009-04-23
WO2002027017A3 (fr) 2003-01-16
EP2040078A1 (fr) 2009-03-25
EP2298814A2 (fr) 2011-03-23
ATE416385T1 (de) 2008-12-15
WO2002027017A2 (fr) 2002-04-04
US7888050B2 (en) 2011-02-15
US20110165588A1 (en) 2011-07-07

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