U.S. patent application number 09/948972 was filed with the patent office on 2003-08-28 for method for quantifying phosphokinase activity on proteins.
Invention is credited to Reagan, Kevin J., Schaefer, Erik, Wang, Jimin.
Application Number | 20030162230 09/948972 |
Document ID | / |
Family ID | 22886277 |
Filed Date | 2003-08-28 |
United States Patent
Application |
20030162230 |
Kind Code |
A1 |
Reagan, Kevin J. ; et
al. |
August 28, 2003 |
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.
Inventors: |
Reagan, Kevin J.; (Agoura
Hills, CA) ; Schaefer, Erik; (Hopkinton, MA) ;
Wang, Jimin; (Thousand Oaks, CA) |
Correspondence
Address: |
John J. McDonnell
McDonnell Boehnen Hulbert & Berghoff
300 S. Wacker Drive
Chicago
IL
60606
US
|
Family ID: |
22886277 |
Appl. No.: |
09/948972 |
Filed: |
September 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60235620 |
Sep 27, 2000 |
|
|
|
Current U.S.
Class: |
435/7.4 |
Current CPC
Class: |
C07K 16/2863 20130101;
C12Q 1/485 20130101; C07K 2317/34 20130101; C07K 16/18 20130101;
G01N 2333/4709 20130101 |
Class at
Publication: |
435/7.4 |
International
Class: |
G01N 033/53 |
Claims
What is claimed:
1. A method for measuring phosphokinase activity on a protein
comprising: (a) subjecting a protein to a phosphokinase to
phosphorylate a phosphorylation site on the protein (b) providing
an antibody specific to a phosphorylated phosphorylation site on
the protein; (c) contacting the protein from (a) with the antibody
of (b); and (d) detecting the antibody bound to the phosphorylation
site.
2. A method according to claim 1 wherein the protein is the Tau
protein.
3. A method according to claim 2 wherein the phosphokinase is
GSK-3.beta., PKA, PKC, CDK-5, MARK, JNK, p38MAPK, or casein kinase
II.
4. The method according to claim 2 wherein the antibody is specific
to a phosphorylated site at a specific location in the Tau protein,
selected from the sites shown in Table II.
5. A method according to claim 1 wherein the protein is selected
from the proteins in Table 1.
6. The method of claim 1 wherein the protein is EGFR.
7. The method of claim 1 wherein the protein is Rb.
8. An antibody prepared from a polypeptide immunogen having a
phosphorylated serine.
9. An antibody raised to a polypeptide immunogen having a
phosphorylated threonine.
10. An antibody raised to a polypeptide immunogen having a
phosphorylated tyrosine.
11. An antibody specific for sequence ID #1.
12. An antibody specific for sequence ID #2.
13. An antibody to EGFR phosphorylated tyrosine 1173 or tyrosine
845 site.
14. An antibody to phosphorylated threonine 821 site.
15. A kit for the measurement of phosphokinase activity on a
protein comprising (a) a first pan antibody specific and 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, wherein the second pan
antibody is labeled or detected by a labeled reagent; (c)
non-phosphorylated and phosphorylated protein standards; (d) a
phosphorylation site-specific antibody (PSSA) which binds to the
protein only when the target site on the protein is phosphorylated
and wherein the PSSA antibody is labeled or detected by a labeled
reagent; and (e) buffers.
16. A kit according to claim 11 where the protein is Tau.
17. A kit according to claim 11 where the protein is selected from
the proteins listed in Table I.
18. 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.
19. A kit according to claim 14 where the protein is Tau.
20. A kit according to claim 14 where the proteins are as defined
in Table I.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims the priority of provisional
application No. 60/235,620 filed on Sep. 27, 2000.
FIELD OF THE INVENTION
[0002] This invention relates to assays and reagents for measuring
protein kinase activity in vitro.
BACKGROUD OF THE ART
[0003] 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 are 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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-3.beta., PKA, PKC, CDK5,
MARK, JNK, p38MAPK and casein kinase II, can phosphorylate Tau.
[0008] 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`.
[0009] 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.
[0010] 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.
[0011] 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).
[0012] 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
[pT.sup.821] or when it is in the non-phosphorylated form. Samples
can be controlled for Rb content by parallel measurement of total
Rb protein.
[0013] 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.
[0014] 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.
[0015] EGFR is activated by binding of a number of ligands such as
EGF, transforming growth factor .alpha.(TGF.alpha.), 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 Tyr.sup.845, Tyr.sup.992,
Tyr.sup.1068, Tyr.sup.1086, and Tyr.sup.1173. 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 (Shc) and
phospholipase C-.gamma.(PLC.gamma.). 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.
[0016] 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
[0017] 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).
[0018] 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.
[0019] 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
[0020] FIG. 1 illustrates specificity of the Anti-phospho Tau
[pS.sup.199] phosphorylation site-specific antibody (PSSA).
[0021] FIG. 2 illustrates Anti-phospho Tau [pS.sup.214] PSSA
specificity.
[0022] FIGS. 3a and 3b illustrate detection of total Tau vs. Tau
phosphorylated at the PKA/serine 214 site by ELISA.
[0023] FIGS. 4a-b illustrate detection of Tau phosphorylated at
GSK-3.beta./serine 199/202 (4a) vs. total Tau (4b) sites by
ELISA.
[0024] FIG. 5 illustrates a dose-response curve generated in an
ELISA using the Tau serine 214 PSSA.
[0025] FIG. 6 illustrates the specificity of the Tau PSSAs in an
ELISA to detect Tau phosphorylation catalyzed by PKA vs.
GSK-3.beta. enzymes.
[0026] FIG. 7 illustrates that multiple GSK-3.beta. phosphorylation
sites on Tau can be detected by ELISA using Tau PSSAs.
[0027] 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.sup.214] and Tau [pS.sup.199] PSSAs as detected by
ELISA.
[0028] FIG. 9 defines the specificity of the anti-Rb
[pT.sup.821].
[0029] FIG. 10 shows studies to determine the specificity of the Rb
[pT.sup.821] ELISA.
[0030] FIG. 11 shows the specificity of the Rb [pT.sup.821] ELISA
for threonine 821 as determined by peptide competition.
[0031] FIG. 12 shows the application of the Rb [pT.sup.821] ELISA
in evaluating kinase activity in Jurkat cells were grown in the
presence of the kinase inhibitor, staurosporine.
[0032] FIG. 13 illustrates the specificity of the EGFR PSSA
[pY.sup.1173].
[0033] FIG. 14 shows the specificity of the EGFR [pY.sup.1173]
ELISA for tyrosine residue 1173 as determined by peptide
competition.
[0034] FIG. 15 demonstrates the response curve of phosphorylation
of EGFR in A431 cells after treatment with EGF using the EGFR
[pY.sup.1173] ELISA.
[0035] FIG. 16 shows the application of the EGFR [pY.sup.845] ELISA
in evaluating kinase activity in A431 cells were grown in the
presence of the tyrosine kinase inhibitor, PD158780.
DETAILED DESCRIPTION OF THE INVENTION
[0036] 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.
[0037] 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.
[0038] Tau pS.sup.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.sup.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. 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
phosphorylated in vitro and in vivo by glycogen synthase
kinase-3.beta. (GSK-3.beta.),which is commercially available.
[0039] 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 .mu.g/mL anti-phosphoTau
[pS.sup.199], 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').sub.2 anti-rabbit IgG
alkaline phosphatase and bands were detected using the Tropix
WesternStar.TM. 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 [pS.sup.199]
antibody for this phosphorylation site.
[0040] Tau [pS.sup.214] PSSA. The procedures for generating this
antibody were similar to those described above for the Tau
pS.sup.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.
[0041] Tau pS.sup.214 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 .mu.g/mL
anti-phospho tau [pS.sup.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').sub.2
anti-rabbit IgG alkaline phosphatase and bands were detected using
the Tropix WesternStar.TM. 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
[pS.sup.214] antibody for this phosphorylation site. PSSAs to other
Tau sites [pS.sup.202, ps.sup.396, pT.sup.181,
pS.sup.199/pS.sup.202, pS.sup.404] have been characterized using
similar methods.
[0042] Pan-Tau Polyclonal Antibody
[0043] 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.
[0044] Tau-5 Monoclonal Antibody (mAb)
[0045] 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.
[0046] Total Tau ELISA and Phospho-Tau ELISA
[0047] A concentration of 2.5 .mu.g/mL of Tau-5 monoclonal antibody
in carbonate buffer, pH 9.4, was incubated at 100 .mu.L/well in
microtiter plates at 4.degree. 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-3.beta. phosphorylated Tau, PKA phosphorylated
Tau, and non-phosphorylated 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 pS.sup.214 PSSA, Tau pS.sup.199 PSSA or
Pan-Tau antibodies at the optimized concentrations (ranging from
0.1 to 1 .mu.g/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 .mu.L 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.
[0048] Kinase Reactions
[0049] Phosphorylation of Tau using PKA was performed as follows.
PKA was purchased from New England Biolabs. Recombinant Tau protein
(1 .mu.g) was incubated with various concentrations of PKA enzyme
in buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2 and
100 .mu.M ATP for 1 hour at 30.degree. C.
[0050] Phosphorylation of Tau Using GSK-3.beta.
[0051] GSK-3.beta. was purchased from Upstate Biotechnology Inc.
Recombinant Tau protein (1 .mu.g) was incubated with various
concentrations of the enzyme in buffer containing 40 mM HEPES (pH
7.2), 5 mM MgCl.sub.2, 5 mM EDTA, 100 .mu.M ATP, and 50 .mu.g/mL
heparin for 1 hour at 30.degree. C.
[0052] FIGS. 3a and 3b show the assessment of total Tau and
selective Tau phosphorylation at the PKA/ Ser.sup.214 site by
ELISA. In FIG. 3a, phosphorylated Tau was detected by an ELISA
using a PSSA specific for Tau pS.sup.214 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 [pS.sup.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.
[0053] FIGS. 4a and 4b show the assessment of total Tau vs.
selective Tau phosphorylation at the GSK-3.beta./ Ser.sup.199/202
sites by ELISA. FIG. 4a uses either non-phosphorylated Tau or
GSK-3.beta.- phosphorylated Tau in the ELISA with the anti-Tau
pS.sup.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. 4b).
[0054] 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 [pS.sup.214] (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.
[0055] FIGS. 6a and 6b shows the specificity in detecting Tau
protein phosphorylation catalyzed by PKA vs. GSK3.beta. enzymes
using the Tau PSSAs and ELISA. The results demonstrate that the Tau
pS.sup.214 PSSA ELISA only detects Tau when phosphorylated by PKA
and the Tau pS.sup.199 PSSA ELISA only detects Tau when
phosphorylated by GSK3.beta..
[0056] FIG. 7 shows that the GSK3.beta. enzyme can phosphorylate
multiple sites on the Tau protein and PSSAs can independently
detect the phosphorylated sites at Tau pT.sup.181, Tau pS.sup.202,
Tau pS.sup.199/pS.sup.202, Tau pS.sup.396, and Tau pS.sup.404. This
provides evidence that the ELISA is useful in creating a profile of
phosphorylation events on the protein subjected to kinase enzyme
activity.
[0057] FIG. 8 shows the specificity of kinase reaction when tested
as a profile with two antibodies, one specific for a PKA
phosphorylation site (pS.sup.214) and the other for a GSK site
(pS.sup.199) on Tau protein. A PKA-specific inhibitor, PKI
(heat-stable inhibitor of c-AMP-dependent 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 pS.sup.214 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.
[0058] Antibodies to other tau sites shown in Table II are also
representative of the invention. Som of the phosphorylated sites
are known to be associated with disease as further indicated in
Table II.
1TABLE II Disease Linked Notes Phospho Site (Y/N/?) (NGD =
Neurodegenerative disease; FTD =) 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 ? S404 ?
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
[0059] 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.
[0060] 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.
[0061] Rb [pT.sup.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. 9
defines the specificity of the anti-Rb [pT.sup.821], 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 .mu.g/mL anti-RB [pT.sup.821], 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').sub.2 anti-rabbit IgG alkaline phosphatase and bands were
detected using the Tropix WesternStar.TM. method. The data show
that only the phosphopeptide corresponding to this site blocks the
antibody signal, demonstrating the specificity of the anti-Rb
[pT.sup.821] antibody for this phosphorylated residue.
[0062] 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
non-phosphorylated and phosphorylated forms of Rb protein.
[0063] 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.
[0064] Total Rb and Rb [pT.sup.821 ELISA: A concentration of 1.25
.mu.g/mL of Rb monoclonal antibody in carbonate buffer, pH 9.4, was
incubated at 100 .mu.L/well in microtiter plates at 4.degree. 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 [pT.sup.821] PSSA and
biotinylated Pan-Rb antibodies at the optimized concentrations
(ranging from 0.1 to 1 .mu.g/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 .mu.g/mL of
streptavidin-HRP for 1 hour at room temperature. After washing, 100
.mu.L 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.
[0065] FIG. 10 shows studies to determine the specificity of the Rb
[pT.sup.821] 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 [pT.sup.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.
These data provide evidence that appropriate phosphorylation of the
Rb protein is requisite for reactivity in this assay.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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).
[0070] EGFR [pY.sup.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). 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 [pY.sup.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 .mu.g/mL anti-EGFR [pY.sup.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').sub.2 anti-rabbit IgG alkaline phosphatase and bands were
detected using the Tropix WesternStar.TM. 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.sup.1173] antibody for this phosphorylated
residue.
[0071] EGFR [pY.sup.845] PSSA: Prepared essentially as EGFR
[pY.sup.1173] PSSA but using chemically synthesized phosphopeptides
from the region that contains tyrosine 845.
[0072] 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
[0073] 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.
[0074] EGFR PSSA and Full Length ELISA: A concentration of 2.5
.mu.g/mL of pan-EGFR monoclonal antibody in carbonate buffer, pH
9.4, was incubated at 100 .mu.L/well in microtiter plates at
4.degree. 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 [pY.sup.845 ] PSSA,
EGFR [pY.sup.1173] PSSA, and Pan-EGFR antibodies at the optimized
concentrations (ranging from 0.1 to 1 .mu.g/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 .mu.L 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.
[0075] The specificity of the EGFR [pY.sup.1173] 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.
[0076] Kinase Reactions: (autophosphorylation)
[0077] EGFR was incubated to induce auto-phosphorylation in a
buffer of 15 mM HEPES (pH7.4), 6 mM MnCl.sub.2 and 15 mM MgCl.sub.2
containing 1uM ATP for 30 minutes at 30.degree. C.
[0078] 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.sup.1173]
ELISA.
[0079] 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 (auto-phosphorylated) in the buffer of 15 mM HEPES
(pH7.4), 6 mM MnCl.sub.2 and 15 mM MgCl.sub.2 containing 1uM ATP
for 30 minutes at 30.degree. C. To inhibit phosphorylation of EGFR
[pY.sup.845], tyrosine kinase inhibitor PD158780 (Calbiochem, cat
#. 513035) was added to the reaction at the indicated concentration
(see FIG. 16). EGFR [pY.sup.845] phosphorylation was measured using
4ng/mL of EGFR and the EGFR [pY.sup.845] PSSA ELISA.
2TABLE 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/ABL protein tyrosine kinase beta-Catenin BID
(BH-3 Interacting Death Domain) Blk (B Lymphocyte 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 CDK1/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) eIF-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)
ERK5 (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)
Gi.alpha. inhibitory guanine nucleotide regulatory protein Gi.beta.
inhibitory guanine nucleotide regulatory protein Gi.gamma.
inhibitory guanine nucleotide regulatory protein Gq/11 guanine
nucleotide-binding protein Gq/11.beta. guanine nucleotide-binding
protein Gq/11.gamma. guanine nucleotide-binding protein Grb2
(Growth factor Receptor Binding protein-2) Grk2 (G protein-coupled
Receptor Kinase) GSK-3.alpha. (Glycogen Synthase Kinase 3alpha)
GSK-3.beta. (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 NFkB inhibitory protein IkappaB beta NFkB inhibitory protein
IKKalpha (IkB kinase alpha) IKKbeta (IkB kinase beta) IKKgamma (IkB
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/SAPK.gamma. (c-Jun amino-terminal kinase 1/Stress-Activated
Protein Kinase .gamma.) JNK2/SAPK.beta. (c-Jun amino-terminal
kinase 2/Stress-Activated Protein Kinase .beta.) JNK3/SAPK.alpha.
(c-Jun amino-terminal kinase 3/Stress-Activated Protein Kinase
.alpha.) 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 (c-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) PI3K (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-gamma1 (Phospholipase C-gamma1) PRK (Proliferation Related
Kinase) PTEN (MMAC1 tumor suppressor gene/protein phosphatase) Pyk2
(CAKbeta/FAK2/RAFTK) Protein tyrosine Kinase R Rac/cdc42 GTPase
Raf1 (C-raf) serine/threonine protein kinase A-Raf serine/threonine
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/CED-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/I-TRAF (TNF 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 serine/threonine kinase) ZRP-1 (Zyxin Related
Protein Zyxin
[0080] 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.
Sequence CWU 1
1
2 1 11 PRT Artificial Sequence MISC_FEATURE (1)..(11) chemically
synthesized phosphopeptide corresponding to the region of the
longest isoform of the Tau protein that includes serine 199 1 Arg
Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly 1 5 10 2 13 PRT Artificial
Sequence MISC_FEATURE (1)..(13) chemically synthesized
phosphopeptide derived from the region of the longest isoform of
Tau protein that includes serine 214 2 Gly Ser Arg Ser Arg Thr Pro
Ser Leu Pro Thr Pro Pro 1 5 10
* * * * *