# Patent

## Title
Method for quantifying phosphokinase activity on proteins

## Abstract

The invention involves a method for measuring phosphorylation of proteins at specific sites and, as such, is an indicator of the protein kinase activity of enzymes capable of phosphorylating those sites. The method involves the in vitro or in vivo phosphorylation of a target protein at a specific serine, threonine or tyrosine residue, subjecting that protein (non-phosphorylated) to reaction mixture containing all reagents, including phosphokinase which allow the creation of a phosphorylated form of protein. The phosphorylated protein is measured by contacting it with an antibody specific for the phosphorylation site(s). The invention includes antibodies useful in practicing the methods of the invention. The invention particularly relates to all proteins modified by phosphorylation and dephosphorylation as illustrated by Tau, Rb and EGFR proteins and antibodies specific for the site of phosphorylation of the Tau, Rb or EGFR proteins.

## Background
This application is a divisional of U.S. application Ser. No. 12/179,526, filed on Jul. 24, 2008, which is a continuation of U.S. application Ser. No. 09/948,972, filed on Sep. 7, 2001, abandoned, which claims the priority of provisional application No. 60/235,620, filed on Sep. 27, 2000, the disclosures of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to assays and reagents for measuring protein kinase activity in vitro.
2. Background of the Art
Drug development efforts involve a continuum of activities initiated by target selection of a molecule. Since all drugs work at the level of the cell, those targets are usually proteins that somehow are involved in cellular communication pathways. Signal transduction pathways are key to normal cell function. Aberrations in the expression of intracellular molecules and coordinated interactions of signal transduction pathways are associated with a variety of diseases and, thus, are the focus of drug discovery efforts. Phosphorylation of proteins in signal transduction pathways is one of the key covalent modifications that occur in multicellular organisms. The enzymes that carry out this modification arc the protein kinases, which catalyze the transfer of the phosphate from ATP to tyrosine, serine or threonine residues on protein substrates. 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. Because 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. Also, 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.
Currently, neurobiologists are focusing efforts on the proteins in the brain that can be associated with disease. One such protein is called 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). 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-3β, PKA, PKC, CDK5, MARK, JNK, p38MAPK and casein kinase II, can phosphorylate Tau.
Detection of in vitro kinase activity is critical for screening compounds that may be able to inhibit this activity and therefore could be useful in ameliorating various neurodegenerative diseases where Tau phosphorylation is abnormally high. Current efforts exist to identify drugs that might suppress kinase activity towards the Tau protein; however, these methods suffer from poor sensitivity and low specificity. Phosphorylation at individual Serine or Threonine residues within the Tau protein has been shown to correlate with disease. This invention overcomes both of these deficiencies in the described ‘art’.
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 and U.S. Pat. No. 6,066,462 relates to quantitation of individual protein kinase activity. These patents are incorporated herein by reference.
In addition to the detection of Tau phosphorylation in AD, other models exist to show the general applicability of the currently described format for monitoring protein kinase activity. For the purposes of illustration, we have also designed assays around the intranuclear Retinoblastoma (Rb) protein important in cell cycle regulation and a cell surface receptor molecule (EGFR), which are both described in detail below.
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-Abl. 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 contains at least 16 consensus serine/threonine phosphorylation sites for cdks, although the significance of all these sites is still unclear. It has been demonstrated that phosphorylation of threonine 821 on Rb is catalyzed by cdk2/complex such as Cyclin E/cdk2 and Cyclin A/cdk2. The phosphorylation of threonine 821 disrupts the interaction of Rb with the proteins containing the sequence LXCXE, where L=leucine, C=cysteine, E=glutamic acid, and X=any amino acid residue. The dephosphorylation of Rb protein returns Rb to its active, growth suppressive state. 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. The quantitation of Rb phosphorylated at specific amino acid residues gives important information regarding the activity of kinases as well as the functional state of the Rb protein itself. For the purposes of illustration, we designed an assay to quantitate the amount of Rb protein that is specifically phosphorylated at threonine 821 using an ELISA format. This assay does not recognize Rb phosphorylated at sites other than [pT821] or when it is in the non-phosphorylated form. Samples can be controlled for Rb content by parallel measurement of total Rb protein.
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.
As a third example of the utility of this approach, a cell surface receptor was studied and a kinase-dependent ELISA designed. The Epidermal growth factor receptor (EGFR) belongs to the family of receptor tyrosine kinases (RTKs), which regulate cell growth, survival, proliferation and differentiation. 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. For example, 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 α (TGFα), 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. Several sites of in vivo phosphorylation have been identified in the EGFR including Tyr845, Tyr992, Tyr1068, Tyr1086, and Tyr1173. These sites bind and activate a variety of downstream signaling proteins that contain SH2 domains, including growth factor receptor-binding protein 2 (Grb2), Src homology and collagen domain protein (She) and phospholipase C-γ (PLCγ). The binding of these or other signaling proteins to the receptor and/or their phosphorylation results in transmission of subsequent signaling events that culminate in DNA synthesis and cell division.
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.
SUMMARY OF THE INVENTION
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. However, the invention can be applied to all proteins and antibodies that recognize specific phosphorylation sites on these proteins (see Table I).
In each example system, 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). 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. In contrast to existing methods that quantitate phosphorylated proteins as a diagnostic or prognostic indication of disease, 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates specificity of the Anti-phospho Tau [pS199] phosphorylation site-specific antibody (PSSA).
FIG. 2 illustrates Anti-phospho Tau [pS214] PSSA specificity.
FIGS. 3a and 3b illustrate detection of total Tau vs. Tau phosphorylated at the PKA/serine 214 site by ELISA.
FIG. 4a-b illustrate detection of Tau phosphorylated at GSK-3β/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-3β enzymes.
FIG. 7 illustrates that multiple GSK-3β 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 [pS214] and Tau [pS199] PSSAs as detected by ELISA.
FIG. 9 defines the specificity of the anti-Rb [pT821].
FIG. 10 shows studies to determine the specificity of the Rb [pT821] ELISA.
FIG. 11 shows the specificity of the Rb [pT821] ELISA for threonine 821 as determined by peptide competition.
FIG. 12 shows the application of the Rb [pT821] 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 [pY1173].
FIG. 14 shows the specificity of the EGFR [pY1173] 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 [pY1173] ELISA.
FIG. 16 shows the application of the EGFR [pY845] ELISA in evaluating kinase activity in A431 cells were grown in the presence of the tyrosine kinase inhibitor, PD158780.



DETAILED DESCRIPTION OF THE INVENTION
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 Recombinant Protein: Full length Tau-441 protein is purified recombinant protein. derived through cloning of human Tau cDNA and expressed in E. coli. The protein is purified via standard methods. This protein is commercially available from multiple vendors.
Tau pS199 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 ED #1. The Tau pS199 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. This antibody recognizes specifically the Tau protein when phosphorylated on serine 199, as demonstrated by peptide competition analysis in a western blotting assay. Serine 199 is phosphorylatcd in vitro and in vivo by glycogen synthase kinase-3β (GSK-3β), which is commercially available.
The specificity of the anti-Tau [pS199] PSSA Tau specificity is shown in FIG. 1. Cell extracts from African green monkey kidney (CV-1) cells, stably expressing human four repeat tau and a protein phosphatase inhibitor, were resolved by SDS-PAGE on a 10% Tris-glycine gel. The proteins were transferred to nitrocellulose. Membranes were incubated with 0.50 μg/mL anti-phosphoTau [pS199], 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 detection method. The data in FIG. 1 show that only the phosphopeptide corresponding to this site blocks the antibody signal, illustrating the specificity of the Anti-Tau [pS199] antibody for this phosphorylation site.
Tau [pS214] PSSA. The procedures for generating this antibody were similar to those described above for the Tau pS199 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.
Tau pS214 PSSA specificity is show in FIG. 2. SF-9 cell extracts, expressing human four repeat tau, were resolved by SDS PAGE on a 10% Tris-glycine gel. The proteins were transferred to nitrocellulose. Membranes were incubated with 0.50 μg/mL anti-phospho tau [pS214], 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 data in FIG. 2 show that only the phosphopeptide corresponding to this site blocks the antibody signal, illustrating the. specificity of the Anti-Tau [pS214] antibody for this phosphorylation site. PSSAs to other Tau sites [pS202, pS396, pT181, pS199/pS202, pS404] have been characterized using similar methods.
Pan-Tau Polyclonal Antibody
Rabbits were immunized with the recombinant Tau protein and the resulting antibody was purified from the rabbit serum using a protein-A affinity column. This antibody recognizes multiple antigenic sites on Tau protein. This antibody will bind to both non-phosphorylated and phosphorylated forms of Tau protein.
Tau-5 Monoclonal Antibody (mAb)
The mouse mAb to Tau was raised using purified bovine microtubule-associated proteins (MAPs) as the immunogen. 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.
Total Tau ELISA and Phospho-Tau ELISA
A concentration of 2.5 μg/mL of Tau-5 monoclonal antibody in carbonate buffer, pH 9.4, was incubated at 100 μL/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. GSK-3β phosphorylated Tau, PKA phosphorylated Tau, and nonphosphorylated Tau were added to the wells at various concentrations and incubated for 1 hour at room temperature. After washing 3 times with Washing Buffer, the wells were incubated respectively with Tau pS214 PSSA, Tau pS199 PSSA or Pan-Tau antibodies at the optimized concentrations (ranging from 0.1 to 1 μg/mL) for 1 hour at room temperature. The plates then were washed three times with Washing Buffer, followed by the addition of an HRP conjugated anti-rabbit IgG secondary antibody at 1:5000 dilution for 1 hour at room temperature. After washing 3 times, 100 μL of Stabilized Chromogen was added to each well and then incubated for 20 minutes at room temperature in the dark. The OD values at 450 nm were measured following the addition of stop solution to each well.
Kinase Reactions
Phosphorylation of Tau using PKA was performed as follows. PKA was purchased from New England Biolabs. Recombinant Tau protein (1 μg) was incubated with various concentrations of PKA enzyme in buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and 100 μM ATP for 1 hour at 30° C.
Phosphorylation of Tau Using GSK-3β
GSK-3β was purchased from Upstate Biotechnology Inc. Recombinant Tau protein (1 μg) was incubated with various concentrations of the enzyme in buffer containing 40 mM HEPES (pH 7.2), 5 mM MgCl2, 5 mM EDTA, 100 μM ATP, and 50 μg/mL heparin for 1 hour at 30° C. FIGS. 3a and 3b show the assessment of total Tau and selective Tau phosphorylation at the PKA/Ser214 site by ELISA. In FIG. 3a, phosphorylated Tau was detected by an ELISA using a PSSA specific for Tau pS214 or by a pan-Tau antibody. Both antibodies detected the phosphorylated Tau protein with equal signals. In FIG. 3b, non-phosphorylated Tau was placed into the same assay. As expected, the anti-Tau [pS214] 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. 4a and 4b show the assessment of total Tau vs. selective Tau phosphorylation at the GSK-3β/Ser199/202 sites by ELISA. FIG. 4a uses either non-phosphorylated Tau or GSK-3β-phosphorylated Tau in the ELISA with the anti-Tau pS199/202 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. 4b).
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. Various amounts of PKA enzyme were used to phosphorylate the Tau protein. Starting with the highest concentration of PKA, 5 units, (PKA tau 1), 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 [pS214] (a PKA site). These data indicate that lower amounts of protein kinase in the reaction result in a proportionally lower amount of phosphoprotein produced, as detected in the ELISA. Thus, the ELISA signal provides an indirect, yet quantitative, measure of phosphokinase activity.
FIGS. 6a and 6b shows the specificity in detecting Tau protein phosphorylation catalyzed by PKA vs. GSK3β enzymes using the Tau PSSAs and ELISA. The results demonstrate that the Tau pS214 PSSA ELISA only detects Tau when phosphorylated by PKA and the Tau pS199 PSSA ELISA only detects Tau when phosphorylated by GSK3β.
FIG. 7 shows that the GSK3β enzyme can phosphorylate multiple sites on the Tau protein and PSSAs can independently detect the phosphorylated sites at Tau pT181, Tau pS202, Tau pS199/pS202, Tau pS396, and Tau pS404. 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 (pS214) and the other for a GSK site (pS199) on Tau protein. A PKA-specific inhibitor, PKI (heat-stable inhibitor of c-AMPdependent protein kinase; New England Biolabs), was mixed at various ratios of inhibitor to enzyme (either PKA or GSK) and the resultant mixture analyzed by ELISA using the Tau PSSAs. The PKA-specific inhibitor altered the kinase activity of the pS214 site alone. These data again attest to the specificity of the ELISA and the ability to independently monitor kinase activities on the same protein at different sites using the highly specific PSSAs as the assay detectors. These data also illustrate the capability of selectively screening for drug interference of protein kinase activity using this format.
Antibodies to other tau sites shown in Table II are also representative of the invention. Some of the phosphorylated sites are known to be associated with disease as further indicated in Table II.









TABLE II






Disease




Linked


Phospho Site
(Y/N/?)
Notes







T39
?
Phosphorylated by Casein kinase II


T181, hu
?
Involved in pretangle process?


S184
Y
Phosphorylated by GSK-3b; disrupts microtubule network


S195
Y
Phosphorylated by GSK-3b; disrupts microtubule network


S198
Y
Phosphorylated by GSK-3b; disrupts microtubule network


S199
Y
Phosphorylated by GSK-3b; linked to hereditary FTD


S202
Y
Microtubule-dependent phosphorylation by CDK 5 and GSK-3b;




linked to hereditary NGD


T205
?
Microtubule-dependent phosphorylation by CDK 5 and GSK-3b


T212
Y
Specific for NGD processes; phosphorylated by GSK-3b and PKA


S214
Y
Specific for NGD processes; may block aggregation; phos'd by PKA


2T17
?


T231
Y
Involved in pretangle process?; phos'd by GSK-3b and cdc2/CDK1


S235
?
Microtubule-independent phosphorylation by GSK-3b


S262
Y
May block aggregation; phosphorylated by CAM K II and GSK-3b;




major site in AD brain


S320
?


S324
?


S356
Y
Involved in pretangle process?; AD pathway; major site in AD brain;




phosphorylated by GSK-3b


S361
?


S396
N
Phos'd by GSK-3b


S400
?
Phos'd by GSK-3b


T403
?


T404
?
Involved in pretangle process?; microtubule-independent




phosphorylation; phosphorylated by GSK-3b


S409
Y
AD pathway; phosphorylated by PKA


S412
?
AD pathway


S413
Y
AD pathway; phosphorylated by GSK-3b


S416
?
Phosphorylated by CAM K II


S422
Y
Linked with several NGD's; phosphorylated by MAPK





(NGD = Neurodegenerative disease; FTD =)






Rb System: This model system describes a large intra-nuclear protein with multiple phosphorylation sites that arc acted upon by multiple protein kinases. Both phosphoserine and phosphotyrosine residues are examined, for which both mono-phospho and dual-phosphoresidues arc 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 [pT821] 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. 9 defines the specificity of the anti-Rb [pT821]. SDS-PAGE on a 7.5% Tris-glycine gel resolved cell extracts, prepared from MCF-7 cells. The proteins were then transferred to PVDF. Membranes were incubated with 0.5 μg/mL anti-RB [pT821], following prior incubation in the absence (a) or presence of the peptide immunogen (b), the non-phosphopeptide corresponding to the RB phosphopeptide (c), the phosphopeptides corresponding to threonine 356 (d), serine 807/811 (e), serine 249/threonine 252 (f), and serine 751 (g) on phospho-RB. 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 data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the anti-Rb [pT821] antibody for this phosphorylated residue.
Total Rb [pan] Detection Antibody: the 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 nonphosphorylated 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 [pT821] ELISA: A concentration of 1.25 μg/mL of Rb monoclonal antibody in carbonate buffer, pH 9.4, was incubated at 100 μL/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. After three washings with Washing Buffer, the wells were incubated, respectively, with Rb [pT821] PSSA and biotinylated Pan-Rb antibodies at the optimized concentrations (ranging from 0.1 to 1 μg/mL) for 1 hour at room temperature. The plates then were washed three times with Washing Buffer, followed by the addition of an HRP conjugated anti-rabbit IgG secondary antibody at 1:5000 dilution or 0.25 μg/mL of streptavidin-HRP for 1 hour at room temperature. After washing, 100 μL of Stabilized Chromogen was added to each well and then incubated for 20 minutes at room temperature in the dark. The OD values at 450 run were measured following the addition of stop solution to each well.
FIG. 10 shows studies to determine the specificity of the Rb [pT821] ELISA. In the first study, solutions containing Rb protein at a concentration of 20 ng/mL from Jurkat, U2OS, and Colo205 were analyzed with the Rb [pT821] 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. These data provide evidence that appropriate phosphorylation of the Rb protein is requisite for reactivity in this assay.
In the second study, specificity for threonine 821 was determined by peptide competition. The data presented in FIG. 11 show that only the peptide corresponding to the region surrounding threonine 821, containing the phospho-threonine, could block the ELISA signal.
Kinase reactions for Rb: Natural sources for Rb were obtained for these studies from exponentially growing cells. Endogenous cellular kinases provided the phosphorylation of the natural Rb protein. 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 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 [pY1173] 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). The final product is generated by affinity chromatography using an EGFR-derived peptide that is phosphorylated at tyrosine 1173. FIG. 13 illustrates the specificity of the EGFR PSSA [PY1173]. 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 μg/mL anti-EGFR [pY1173) 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 [pY1173] antibody for this phosphorylated residue.
EGFR [pY845] PSSA: Prepared essentially as EGFR [pY1173] 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 [Pan] 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 Fall Length ELISA: A concentration of 2.5 μg/mL of pan-EGFR monoclonal antibody in carbonate buffer, pH 9.4, was incubated at 100 μL/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. After three washings with Washing Buffer, the wells were incubated, respectively, with EGFR [pY845] PSSA, EGFR [pY1173] PSSA, and Pan-EGFR antibodies at the optimized concentrations (ranging from 0.1 to 1 μg/mL) for 1 hour at room temperature. The plates then were washed three times with Washing Buffer, followed by the addition of an HRP conjugated anti-rabbit IgG secondary antibody at 1:2000 dilution for 1 hour at room temperature. After washing, 100 μL of Stabilized Chromogen was added to each well and then incubated for 20 minutes at room temperature in the dark. The OD values at 450 nm were measured following the addition of stop solution to each well.
The specificity of the EGFR [pY1173] ELISA for tyrosine residue 1173 was determined by peptide competition. The data presented in FIG. 14 show that only the peptide corresponding to the region surrounding tyrosine residue 1173 and in the phosphorylated state could block the ELISA signal generated with this PSSA.
Kinase Reactions: (Autophosphorylation)
EGFR was incubated to induce auto-phosphorylation in a buffer of 15 mM HEPES (pH7.4), 6 mM MnCl2 and 15 mM MgCl2 containing 1 μM 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 [pY1173] 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. In this assay, 2 ng/vial of purified human EGFR was incubated (autophosphorylated) in the buffer of 15 mM HEPES (pH7.4), 6 mM MnCl2 and 15 mM MgCl2 containing 1 μM ATP for 30 minutes at 30° C. To inhibit phosphorylation of EGFR [pY845], tyrosine kinase inhibitor PD158780 (Calbiochem, cat #. 513035) was added to the reaction at the indicated concentration (see FIG. 16). EGFR [pY845] phosphorylation was measured using 4 ng/mL of EGFR and the EGFR [pY845] PSSA ELISA.
Table 1: Partial List of signal transduction proteins for which site-specific phosphorylation can be determined by methods of the present invention.
TABLE 1
Examples of Signal Transduction Proteins
Protein
A


    alpha-actinin
    alpha-synuclein
    ABL/c-Abl (Abelson nonreceptor protein tyrosine kinase)
    Acetylcholine Receptor
    Ack nonreceptor protein tyrosine kinase;
    Akt/PKB serine/threonine protein kinase
    AP-1 (Activator protein-1 jun/fos dimeric transcription factors)
    AP-2 (Activator protein-2 transcription factor)
    Apaf-1 (Apoptosis protease-activating factor-1)
    Apaf-2 (Apoptosis protease-activating factor-2/cytochrome C)
    Apaf-3 (Apoptosis protease-activating factor-3/caspase-9
    Arp2/3 (Actin related protein)
    Atf-1 (Activating transcription factor-1)
    Atf-2 (Activating transcription factor-2)
    Atf-3 (Activating transcription factor-3)
    Atf-4 (Activating transcription factor-4)
    ATM (Ataxia Telangiectasia Mutated Protein)

B

    B-ATF nuclear basic leucine zipper protein/transcription factors
    Bad
    Bak
    Bax
    Bcl-2 (B-cell chronic lymphocytic leukemia 2)
    Bcl-xL
    Bcl-xS
    BCR/A13L protein tyrosine kinase
    beta-Catenin
    BID (BH-3 Interacting Death Domain)
    Blk (B Lymphotyte Src non-receptor protein tyrosine kinase family member)
    BMK-1 (Big Map Kinase/ERK5)
    Btk (Bruton's Tyrosine Kinase)

C

    Cadherin
    CADTK (calcium activated protein tyrosine kinase/Cakbeta/Pyk2/FAK2/RAFTK)
    CAK (Cdk-Activating Kinase)
    Cak-beta (Cell adhesion kinase beta/CADTK/Pyk2/FAK2/RAFTK)
    caldesmon
    calmodulin
    calpain cysteine proteases
    CaM kinase II (Calmodulin-dependent protein kinase II)
    CB1 (Cannabinoid Receptor 1)
    CB2 (Cannabinoid Receptor 2)
    caspase-2 (Cysteine Aspartyl Protease-2/ICH-1/NEDD-2)
    caspase-3 (Cysteine Aspartyl Protease-3/LICE/CPP32/YAMA/apopain/SCA-1)
    caspase-8 (Cysteine Aspartyl Protease-8/MACH/FLICE/Mch5)
    caspase-9 (Cysteine Aspartyl Protease-9/ICE-LAP6/Mch6/APAF-3)
    Caveolin 1, 2, and 3
    CD45 transmembrane tyrosine phosphatase
    CD45AP (CD45-associated protein)
    c-fos transcription factor
    CDKI/cdc2 (Cyclin-dependent kinase-1)
    CDK2 (Cyclin dependent kinase-2)
    CDK4 (Cyclin dependent kinase-4)
    CDK5 (Cyclin dependent kinase-5)
    c-Jun transcription factor
    c-myc transcription factor
    Cortactin
    COX-2 (Cyclooxygenase-2/prostaglandin-endoperoxide synthase-2)
    c-kit receptor protein
    c-raf protein serine/threonine kinase
    CREB transcription factor
    Crk SH2 and SH3 domain-containing adaptor protein CSK (Carboxyl-terminal Src Kinase)
    cytochrome-c

D

    DAPK (Death Associated Protein Kinase)
    desmin
    DNA-PK (DNA dependent protein kinase)

E

    E2F-1 DNA binding protein
    EGF-R (Epidermal Growth Factor Receptor)
    erF-2alpha (Eukaryotic translation Initiation Factor 2alpha)
    ERK1/MAPK (Extracellular signal-Regulated/Mitogen-Activated Protein Kinase 1)
    ERK2/MAPK (Extracellular signal-Regulated/Mitogen-Activated Protein Kinase 2)
    ERK3 (Extracellular signal-Regulated/p62 Mitogen-Activated Protein Kinase 3)
    ERK4 (Extracellular signal-Regulated Protein Kinase 4)
    ERK5 (Extracellular signal-Regulated Protein Kinase 5/Big MAP Kinase 1)
    ERK6 (Extracellular signal-Regulated Protein Kinase 6/p38gamma)
    ERK7 (Extracellular signal-Regulated Protein Kinase 7)
    ERK8 (Extracellular signal-Regulated Protein Kinase 8)

F

    F-actin
    FADD (Fas-associated Death Domain)
    FAK (Focal Adhesion Kinase/pp125FAK)
    FAS (FAS-Ligand Receptor)
    Fgr non-receptor Src family tyrosine kinase
    Fos B
    Fra-1 (Fos-related antigen-1)
    Fra-2 (Fos-related Antigen-2)
    FRK (Fos-Regulating Kinase)
    FYB (Fyn binding protein)
    Fyn non-receptor Src family tyrosine kinase

G

    Gab 1 (Grb2-associated binder 1)
    Gab 2 (Grb2-associated binder 2)
    GCK (Germinal Center Kinase)
    GEF (Guanine nucleotide Exchange Factor)
    Giα inhibitory guanine nucleotide regulatory protein
    Giβ inhibitory guanine nucleotide regulatory protein
    Giγ inhibitory guanine nucleotide regulatory protein
    Gq/11 guanine nucleotide-binding protein
    Gq/11β guanine nucleotide-binding protein
    Gq/11γ guanine nucleotide-binding protein
    Grb2 (Growth factor Receptor Binding protein-2)
    Grk2 (G protein-coupled Receptor Kinase)
    GSK-3α (Glycogen Synthase Kinase 3alpha)
    GSK-3β (Glycogen Synthase Kinase 3beta)

H

    Hck (Hematopoietic cell kinase)
    HGF-R (Hepatocyte growth factor receptor)
    Hrk (3-Hydroxy-3-methyl glutaryl-coenzyme A Reductase Kinase)

I

    IkappaB alpha NFκB inhibitory protein
    IkappaB beta NFκB inhibitory protein
    IKKalpha (IκB kinase alpha)
    IKKbeta (IκB kinase beta)
    IKKgamma (IκB kinase gamma/NEMO)
    IGF-1 receptor (Insulin-like growth factor-I receptor)
    Insulin receptor
    Integrins
    Integrin-Associated Protein (IAP/CD47)
    IRAK (Interleukin-1 Receptor-Associated Kinase)
    IRK (Insulin Receptor Kinase)
    IRS-1 (Insulin Receptor Substrate 1)
    IRS-2 (Insulin Receptor Substrate 2)

J

    JAB1 (Jun-Activation domain Binding protein 1)
    JAK1 (Janus Activating Kinase 1)
    JAK2 (Janus Activating Kinase 2)
    JAK3 (Janus Activating Kinase 3)
    JNK1/ISAPKγ (c-Jun amino-terminal kinase 1/Stress-Activated Protein Kinase γ)
    JNK2/SAPKβ (c-Jun amino-terminal kinase 2/Stress-Activated Protein Kinase β)
    JNK3/SAPKα (c-Jun amino-terminal kinase 3/Stress-Activated Protein Kinase α)

L

    LAT (Linker for Activation of T cells)
    Lck non-receptor Src family protein tyrosine kinase
    Lyn non-receptor Src family protein tyrosine kinase

M

    MEF2c transcription factor
    MEK1 (Mitogen-activated ERK-activating Kinase 1)
    MEK2 (Mitogen-activated ERK-activating Kinase 2)
    MEK3 (Mitogen-activated ERK-activating Kinase 3)
    MEK4 (Mitogen-activated ERK-activating Kinase 4)
    MEK5 (Mitogen-activated ERK-activating Kinase 5)
    MEKK1 (MEK kinase 1)
    Met (e-met/HGF-receptor)
    MKP 1 (MAP Kinase Phosphatase 1)
    MKP 2 (MAP Kinase Phosphatase 2)
    MKP 3 (MAP Kinase Phosphatase 3)
    MKP 4 (MAP Kinase Phosphatase 4)
    MKP 5 (MAP Kinase Phosphatase 5)
    MKP 6 (MAP Kinase Phosphatase 6)
    MLCK (Myosin light chain kinase)
    MuSK (Muscle specific serine/threonine kinase)
    Myosin
    MLCK PPase (Myosin Light Chain Kinase Phosphatase)

N

    Beta-NAP (Beta-Neuron Adaptor Protein/AP-3)
    NAT1/DAP-5 (Novel APOBEC-1 Target no. 1/Death-Associated Protein-5)
    NCK SH2 and SH3 domains-containing transforming protein
    Nek2 (Nima-related Kinase2)
    NFAT-1 (Nuclear Factor of Activated T-cells)
    NfkappaB (Nuclear Factor Kappa B transcription factor)
    NIK (NFkappaB Inducing Kinase)
    NTK (Nervous Tissue and T cell Kinase)

P

    p130cas
    p190RhoGAP GTPase
    P2Y2 purinoceptor
    p36 CAK assembly/activation factor
    p38 (ERK6 MAPK/SAPK)
    p38d (SAPK4)
    p53 Tumor suppressor gene.
    p58 IPK (Inhibitor of the interferon-induced double-stranded RNA-activated Protein Kinase, PKR)
    p62dok GAP-associated protein
    p62 lck ligand/ZIP
    p68 kinase
    p96
    PAK1 (p21-Activated protein Kinase 1)
    PAK2 (p21-Activated protein Kinase 2)
    PAK3 (p21-Activated protein Kinase 3)
    PARP (Poly(ADP-Ribose) Polymerase)
    Paxillin
    PCNA (Proliferating Cell Nuclear Antigen)
    PDGF Receptor (Platelet Derived Growth Factor Receptor)
    PDK1 (Phosphoinositide-Dependent Kinase-1)
    PDK-2 (Phosphoinositide-Dependent Kinase-2/integrin-linked kinase)
    PECAM-1 (Platelet-Endothelial Cell Adhesion Molecule-1)
    P13K (Phosphatidyl Inositol-3-Kinase)
    PIAS (Protein Inhibitors of Activated STATs)
    PITP alpha (Phosphatidylinositol Transfer Protein alpha)
    PKA alpha/cAMP-dependent protein kinase
    PKB (Protein kinase B)
    PKC alpha (Protein Kinase C alpha)
    PKC beta (Protein Kinase C beta)
    PKC delta (Protein Kinase C delta)
    PKC gamma (Protein Kinase C gamma)
    PKD (Protein Kinase D)
    PKR (Protein Kinase R or double-stranded RNA-activated protein kinase)
    PLC-gammal (Phospholipase C-gammal)
    PRK (Proliferation Related Kinase)
    PTEN (MMAC1 tumor suppressor gene/protein phosphatase)
    Pyk2 (CAKbeta/FAK2/RAFTK) Protein tyrosine Kinase

R

    Rac/cdc42 GTPase
    Raf 1 (C-raf) serine/threonine protein kinase
    A-Raf serine/Jthreonine protein kinase
    B-raf serine/threonine kinase
    V-Raf viral serine/threonine protein kinase
    RAFTK (Related Adhesion Focal Tyrosine Kinase)
    RAIDD (RIP-Associated ICH-1/CM-3 homologous protein with a Death Domain)
    Rap2 GTPase
    Rap1-GAP (C3G) inactivator of Rap-1
    Rapsyn
    Ras GTPase
    Rb (Retinoblastoma tumor suppressor protein)
    Rho Small molecular weight GTPase
    RIP (Receptor Interacting Protein)
    ROCK (Rho-activated kinase)

S

    S6k (S6 Kinase)
    Shc
    SHIP (SH2 domain containing inositol phosphatase)
    SH-PTP1 Protein Tyrosine Phosphatase
    SH-PTP2 Protein Tyrosine Phosphatase
    SIRPalpha1 (Signal Related Protein Alpha)
    SIP1 (Smad Interacting Protein 1)
    Smad2 (Sma and Mad-related 2)
    Smad3 (Sma and Mad-related 3)
    Smad5 (Sma and Mad-related 5)
    Smad7 (Sma and Mad-related 7)
    SOCS-1 (Suppressor of Cytokine Signaling-1)
    SOCS-2 (Suppressor of Cytokine Signaling-2)
    SOCS-3 (Suppressor of Cytokine Signaling-3)
    SOS (Son of Sevenless)
    Src non-receptor tyrosine kinase
    SRF (Serum Response Factor)
    SRPK1 (SR protein-specific Kinase1)
    SRPK2 (SR protein-specific Kinase2)
    STAT1alpha (Signal Transducer and Activator of Transcription 1)
    STAT2 (Signal Transducer and Activator of Transcription 2)
    STAT3 (Signal Transducer and Activator of Transcription 3)
    STAT4 (Signal Transducer and Activator of Transcription 4)
    STAT5alpha (Signal Transducer and Activator of Transcription 5alpha)
    STAT5beta (Signal Transducer and Activator of Transcription 5 beta)
    STAT6 (Signal Transducer and Activator of Transcription 6)
    Syk (Spleen tyrosine kinase)
    Syndecans transmembrane proteoglycan

T

    Tak1 (TGF-b1 activated kinase)
    Talin
    TANK/1-TRAF (INT Receptor Activating Factor)
    Tau microtubule-associated protein
    TBK-1/T2K (TANK Binding Kinase 1)
    Tensin
    TNF-RI (Tumor Necrosis Factor Receptor I)
    TRADD (TNF-Receptor Associated Death Domain protein)
    TRAF1 (TNF-Receptor Associated Factor 1)
    TRAF2 (TNF-Receptor Associated Factor 2)
    TRAF3 (TNF-Receptor Associated Factor 3)
    TRAF4 (TNF-Receptor Associated Factor 4)
    TRAF5 (TNF-Receptor Associated Factor 5)
    TRAF6 (TNF-Receptor Associated Factor 6)
    TrkA protein tyrosine receptor kinase A
    TrkB protein tyrosine receptor kinase B
    TrkC protein tyrosine receptor kinase C

V

    VEGF-receptor (vascular endothelial growth factor receptor, types 1, 2, 3)
    Vinculin

W

    WASP (Wiskott-Aldrich Syndrome Protein)

Z

    ZIP (Zeta Interacting Protein)
    ZIP kinase (zipper serinelthreonine kinase)
    ZRP-1 (Zyxin Related Protein)
    Zyxin


The examples provided illustrate the present invention and are not intended to limit the invention in spirit or scope. Similarly, the description of these reagents and methods can be used in an inverse function to analyze the activity of protein specific phosphatases, enzymes that remove phosphate groups from specific amino acid residues. In addition, antibodies of the present invention are also useful for inactivating phosphorylated polypeptides for therapeutic purposes.

## Claims

1. A kit for the measurement of different kinase activities on a protein by quantitating phosphorylation site profiles comprising:
(a) a first pan antibody that binds to both phosphorylated and non-phosphorylated forms of the protein;
(b) a second pan antibody that binds to an independent site on the protein from the first pan antibody and wherein the second pan antibody is labeled or detected by a labeled reagent;
(c) protein standards for the non-phosphorylated and phosphorylated forms of the protein;
(d) two or more PSSAs which bind to the protein only when the target sites on the protein which are phosphorylated wherein antibody is labeled or detected by a labeled reagent;
(e) buffers.

2. A kit according to claim 1 where the protein is Tau.

3. A kit according to claim 1 where the proteins are as defined in Table I.