U.S. patent application number 10/382017 was filed with the patent office on 2003-10-30 for compositions and methods for monitoring the phosphorylation of natural binding partners.
This patent application is currently assigned to Cyclacel, Ltd.. Invention is credited to Colyer, John, Craig, Roger K..
Application Number | 20030203407 10/382017 |
Document ID | / |
Family ID | 29254216 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203407 |
Kind Code |
A1 |
Craig, Roger K. ; et
al. |
October 30, 2003 |
Compositions and methods for monitoring the phosphorylation of
natural binding partners
Abstract
This invention relates to methods and compositions for
monitoring the interaction of binding partners as a function of the
addition or subtraction of a phosphate group to or from one of the
binding partners by a protein kinase or phosphatase.
Inventors: |
Craig, Roger K.; (Smallwood,
GB) ; Colyer, John; (Bardsey, GB) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
Cyclacel, Ltd.
|
Family ID: |
29254216 |
Appl. No.: |
10/382017 |
Filed: |
March 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10382017 |
Mar 5, 2003 |
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09511204 |
Feb 23, 2000 |
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09511204 |
Feb 23, 2000 |
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09258981 |
Feb 26, 1999 |
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Current U.S.
Class: |
435/7.1 ;
530/388.26 |
Current CPC
Class: |
C12Q 1/485 20130101;
C12Q 1/42 20130101; G01N 33/542 20130101; G01N 33/573 20130101 |
Class at
Publication: |
435/7.1 ;
530/388.26 |
International
Class: |
G01N 033/53; C07K
016/40 |
Claims
What is claimed is:
1. An isolated natural binding domain and a binding partner
therefor, wherein said isolated natural binding domain includes a
site for post-translational phosphorylation and binds said binding
partner in a manner dependent upon phosphorylation or
dephosphorylation of said site.
2. The isolated natural binding domain and binding partner therefor
of claim 1, wherein said phosphorylation or dephosphorylation is
performed by an enzyme which is a kinase or a phosphatase,
respectively.
3. The isolated natural binding domain and binding partner therefor
of claim 1, wherein phosphorylation of said site prevents binding
of said isolated natural binding domain to said binding
partner.
4. The isolated natural binding domain and binding partner therefor
of claim 1, wherein phosphorylation of said site promotes binding
of said isolated natural binding domain to said binding
partner.
5. The isolated natural binding domain and binding partner therefor
of claim 1, wherein dephosphorylation of said site prevents binding
of said isolated natural binding domain to said binding
partner.
6. The isolated natural binding domain and binding partner therefor
of claim 1, wherein dephosphorylation of said site promotes binding
of said isolated natural binding domain to said binding
partner.
7. The isolated natural binding domain and binding partner therefor
of claim 1, wherein at least one of said isolated natural binding
domain and said binding partner comprises a detectable label.
8. The isolated natural binding domain and binding partner therefor
of claim 7, wherein said detectable label emits light.
9. The isolated natural binding domain and binding partner therefor
of claim 8, wherein said light is fluorescent.
10. The isolated natural binding domain and binding partner
therefor of claim 9, wherein one of said isolated natural binding
domain and said binding partner comprises a quencher for said
detectable label.
11. A kit comprising an isolated natural binding domain and a
binding partner therefor, wherein said isolated natural binding
domain includes a site for post-translational phosphorylation and
binds said binding partner in a manner dependent upon
phosphorylation of said site, and packaging material therefor.
12. The kit of claim 11, wherein said kit further comprises a
buffer which permits phosphorylation-dependent binding of said
isolated natural binding domain and said binding partner.
13. The kit of claim 12, wherein said buffer permits
phosphorylation or dephosphorylation of said site by a kinase or a
phosphatase, respectively.
14. The kit of claim 11, wherein said kit further comprises one or
both of a kinase and a phosphatase.
15. The kit of claim 14, wherein said kit further comprises a
substrate for said phosphatase or kinase, said substrate being
MgATP.
16. The kit of claim 14, wherein said kit further comprises a
cofactor for one or both of said kinase and phosphatase.
17. The kit of claim 11, wherein at least one of said isolated
natural binding domain and said binding partner comprises a
detectable label.
18. The kit of claim 17, wherein said detectable label emits
light.
19. The kit of claim 18, wherein said light is fluorescent.
20. A method for monitoring activity of an enzyme comprising
performing a detection step to detect binding of an isolated
natural binding domain and a binding partner therefor as a result
of contacting one or both of said isolated natural binding domain
and said binding partner with said enzyme, wherein said isolated
natural binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner
dependent upon phosphorylation of said site and wherein detection
of binding of said isolated natural binding domain and said binding
partner as a result of said contacting is indicative of enzyme
activity.
21. A method for monitoring activity of an enzyme comprising
performing a detection step to detect dissociation of an isolated
natural binding domain from a binding partner therefor as a result
of contacting one or both of said isolated natural binding domain
and said binding partner with said enzyme, wherein said isolated
natural binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner
dependent upon phosphorylation of said site and wherein detection
of dissociation of said isolated natural binding domain from said
binding partner as a result of said contacting is indicative of
enzyme activity.
22. The method of claim 20 or 21, wherein at least one of said
isolated natural binding domain and said binding partner is labeled
with a detectable label.
23. The method of claim 22, wherein said label emits light.
24. The method of claim 23, wherein said light is fluorescent.
25. The method of claim 22 wherein said detection step is to detect
a change in signal emission by said detectable label.
26. The method according to claim 25, wherein said method further
comprises exciting said detectable label and monitoring
fluorescence emission.
27. The method according to claim 25, wherein said method further
comprises the step, prior to or after said detection step, of
contacting said isolated natural binding domain and said binding
partner with an agent which modulates the activity of said
enzyme.
28. A method for monitoring the activity of a modulator of the
activity of an enzyme comprising: a) mixing an isolated natural
binding domain, a binding partner of said isolated natural binding
domain, said enzyme, and a candidate modulator which binds to said
isolated natural binding domain, wherein said isolated natural
binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner
dependent upon phosphorylation of said site, and wherein the
combination of said isolated natural binding domain and said
binding partner comprises detection means for monitoring
association or dissociation of said isolated natural binding domain
and said binding partner, and wherein detection of binding of said
isolated natural binding domain and said binding partner as a
result of said mixing is indicative of enzyme activity, and wherein
said phosphorylation of said site occurs prior to said mixing step;
and b) monitoring association or dissociation of said isolated
natural binding domain and said binding partner, said association
or dissociation being indicative of modulation by said candidate
modulator of said activity, wherein said modulator reduces binding
of said isolated natural binding domain and said binding partner
and wherein a reduction in binding is detected by said detection
means.
29. A method for monitoring the activity of a modulator of the
activity of an enzyme comprising: a) mixing an isolated natural
binding domain, a binding partner of said isolated natural binding
domain, said enzyme, and a candidate modulator which binds to said
isolated natural binding domain, wherein said isolated natural
binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner
dependent upon phosphorylation of said site, and wherein the
combination of said isolated natural binding domain and said
binding partner comprises detection means for monitoring
association or dissociation said isolated natural binding domain
and said binding partner, and wherein detection of binding of said
isolated natural binding domain and said binding partner as a
result of said mixing is indicative of enzyme activity, and wherein
said phosphorylation of said site occurs during said mixing step;
and b) monitoring association or dissociation of said isolated
natural binding domain and said binding partner, said association
or dissociation being indicative of modulation by said candidate
modulator of said activity, wherein said modulator reduces binding
of said isolated natural binding domain and said binding partner
and wherein a reduction in binding is detected by said detection
means.
30. A method for monitoring the activity of a modulator of the
activity of an enzyme comprising: a) mixing an isolated natural
binding domain, a binding partner of said isolated natural binding
domain, said enzyme, and a candidate modulator which binds to said
binding partner, wherein said isolated natural binding domain
includes a site for post-translational phosphorylation and binds
said binding partner in a manner dependent upon phosphorylation of
said site, and wherein the combination of said isolated natural
binding domain and said binding partner comprises detection means
for monitoring association or dissociation of said isolated natural
binding domain and said binding partner, and wherein detection of
binding of said isolated natural binding domain and said binding
partner as a result of said mixing is indicative of enzyme
activity, and wherein said phosphorylation of said site occurs
prior to said mixing step; and b) monitoring association or
dissociation of said isolated natural binding domain and said
binding partner, said association or dissociation being indicative
of modulation by said candidate modulator of said activity, wherein
said modulator reduces binding of said isolated natural binding
domain and said binding partner and wherein a reduction in binding
is detected by said detection means.
31. A method for monitoring the activity of a modulator of the
activity of an enzyme comprising: a) mixing an isolated natural
binding domain, a binding partner of said isolated natural binding
domain, said enzyme, and a candidate modulator which binds to said
binding partner, wherein said isolated natural binding domain
includes a site for post-translational phosphorylation and binds
said binding partner in a manner dependent upon phosphorylation of
said site, and wherein the combination of said isolated natural
binding domain and said binding partner comprises detection means
for monitoring association or dissociation of said isolated natural
binding domain and said binding partner, and wherein detection of
binding of said isolated natural binding domain and said binding
partner as a result of said mixing is indicative of enzyme
activity, and wherein said phosphorylation of said site occurs
during said mixing step; and b) monitoring association or
dissociation of said isolated natural binding domain and said
binding partner, said association or dissociation being indicative
of modulation by said candidate modulator of said activity, wherein
said modulator reduces binding of said isolated natural binding
domain and said binding partner and wherein a reduction in binding
is detected by said detection means.
32. The method of claim 28, 29, 30 or 31, wherein at least one of
said isolated natural binding domain and said binding partner is
labeled with a detectable label.
33. The method of claim 32, wherein said label emits light.
34. The method of claim 33, wherein said light is fluorescent.
35. The method of claim 34, wherein said detection step is to
detect a change in signal emission by said detectable label.
36. The method of claim 35, wherein said method further comprises
exciting said detectable label and monitoring fluorescence
emission.
37. The method of claim 35, wherein said method further comprises
the step, prior to or after said detection step, of contacting said
isolated natural binding domain and said binding partner with an
agent which modulates the activity of said enzyme.
38. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an isolated natural binding domain, a binding partner
therefor and an enzyme with a candidate modulator of said kinase or
phosphatase which binds said isolated natural binding domain,
wherein said natural binding domain includes a site for
post-translational phosphorylation and binds said binding partner
in a manner that is dependent upon phosphorylation or
dephosphorylation of said site by said kinase or phosphatase and
wherein the combination of said isolated natural binding domain and
said binding partner comprises a detection means for monitoring
association or dissociation between said isolated natural binding
domain and said binding partner, and wherein said phosphorylation
or dephosphorylation occurs prior to said mixing, and b) monitoring
the association or dissociation of said isolated natural binding
domain to said binding partner, wherein association or dissociation
of said isolated natural binding domain and said binding partner as
a result of said contacting is indicative of modulation of
enzymatic activity by said candidate modulator of said kinase or
phosphatase.
39. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an isolated natural binding domain, a binding partner
therefor and an enzyme with a candidate modulator of said kinase or
phosphatase which binds to said isolated natural binding domain,
wherein said natural binding domain includes a site for
post-translational phosphorylation and binds said binding partner
in a manner that is dependent upon phosphorylation or
dephosphorylation of said site by said kinase or phosphatase and
wherein the combination of said isolated natural binding domain and
said binding partner comprises a detection means for monitoring
association or dissociation between said isolated natural binding
domain and said binding partner, and wherein said phosphorylation
or dephosphorylation occurs during said mixing, and b) monitoring
the association or dissociation of said isolated natural binding
domain to said binding partner, wherein association or dissociation
of said isolated natural binding domain and said binding partner as
a result of said contacting is indicative of modulation of
enzymatic activity by said candidate modulator of said kinase or
phosphatase.
40. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an isolated natural binding domain, a binding partner
therefor and an enzyme with a candidate modulator of said kinase or
phosphatase which binds said binding partner, wherein said natural
binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner that is
dependent upon phosphorylation or dephosphorylation of said site by
said kinase or phosphatase and wherein the combination of said
isolated natural binding domain and said binding partner comprises
a detection means for monitoring association or dissociation
between said isolated natural binding domain and said binding
partner, and wherein said phosphorylation or dephosphorylation
occurs prior to mixing, and b) monitoring the binding of said
isolated natural binding domain to said binding partner, wherein
binding or dissociation of said isolated natural binding domain and
said binding partner as a result of said contacting is indicative
of modulation of enzymatic activity by said candidate modulator of
said kinase or phosphatase.
41. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an isolated natural binding domain, a binding partner
therefor and an enzyme with a candidate modulator of said kinase or
phosphatase which binds said binding partner, wherein said natural
binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner that is
dependent upon phosphorylation or dephosphorylation of said site by
said kinase or phosphatase and wherein the combination of said
isolated natural binding domain and said binding partner comprises
a detection means for monitoring association or dissociation
between said isolated natural binding domain and said binding
partner, and wherein said phosphorylation or dephosphorylation
occurs during said mixing, and b) monitoring the association or
dissociation of said isolated natural binding domain to said
binding partner, wherein association or dissociation of said
isolated natural binding domain and said binding partner as a
result of said contacting is indicative of modulation of enzymatic
activity by said candidate modulator of said kinase or
phosphatase.
42. The method of claim 38, 39, 40 or 41, wherein said detectable
label emits light.
43. The method of claim 42, wherein said light is fluorescent.
44. The method of claim 43, wherein said monitoring comprises
measuring a change in energy transfer between a detectable label
present on said isolated natural binding domain and a detectable
label present on said binding partner.
45. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an assay system comprising an isolated natural binding
domain and a binding partner for said isolated natural binding
partner with a candidate modulator of enzymatic activity of a said
kinase or phosphatase which binds to said isolated natural binding
domain, and b) monitoring association or dissociation of an
isolated natural binding domain and a binding partner therefor in
said assay system, wherein said isolated natural binding domain
includes a site for post-translational phosphorylation and binds
said binding partner in a manner that is dependent upon
phosphorylation or dephosphorylation of said site by a said kinase
or phosphatase in said assay system, wherein the combination of
said isolated natural binding domain and said binding partner
comprises a detection means for monitoring association or
dissociation between said isolated natural binding domain and said
binding partner and wherein said phosphorylation or
dephosphorylation occurs prior to said mixing, and wherein
association or dissociation of said isolated natural binding domain
and said binding partner as a result of said contacting is
indicative of modulation of enzymatic activity by said candidate
modulator of a said kinase or phosphatase.
46. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an assay system comprising an isolated natural binding
domain and a binding partner for said isolated natural binding
domain with a candidate modulator of enzymatic activity of a said
kinase or phosphatase which binds to said isolated natural binding
domain, and b) monitoring association or dissociation of an
isolated natural binding domain and a binding partner therefor in
said assay system, wherein said isolated natural binding domain
includes a site for post-translational phosphorylation and binds
said binding partner in a manner that is dependent upon
phosphorylation or dephosphorylation of said site by a said kinase
or phosphatase in said assay system, wherein the combination of
said isolated natural binding domain and said binding partner
comprises a detection means for monitoring association or
dissociation between said isolated natural binding domain and said
binding partner and wherein said phosphorylation or
dephosphorylation occurs during said mixing, and wherein
association or dissociation of said isolated natural binding domain
and said binding partner as a result of said contacting is
indicative of modulation of enzymatic activity by said candidate
modulator of a said kinase or phosphatase.
47. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an assay system comprising an isolated natural binding
domain and a binding partner with a candidate modulator of
enzymatic activity of a said kinase or phosphatase which binds to
said binding partner, and b) monitoring association or dissociation
of an isolated natural binding domain and a binding partner
therefor in said assay system, wherein said isolated natural
binding domain includes a site for post-translational
phosphorylation and binds said binding partner in a manner that is
dependent upon phosphorylation or dephosphorylation of said site by
a said kinase or phosphatase in said assay system, wherein the
combination of said isolated natural binding domain and said
binding partner comprises a detection means for monitoring
association or dissociation between said isolated natural binding
domain and said binding partner and wherein said phosphorylation or
dephosphorylation occurs prior to said mixing, and wherein
association or dissociation of said isolated natural binding domain
and said binding partner as a result of said contacting is
indicative of modulation of enzymatic activity by said candidate
modulator of a said kinase or phosphatase.
48. A method of screening for a candidate modulator of enzymatic
activity of a kinase or a phosphatase, the method comprising a)
mixing an assay system comprising an isolated natural binding
domain and a binding partner with a candidate modulator of
enzymatic activity of a said kinase or phosphatase which binds to
said binding partner, and b) monitoring binding of an isolated
natural binding domain and a binding partner therefor in said assay
system, wherein said isolated natural binding domain includes a
site for post-translational phosphorylation and binds said binding
partner in a manner that is dependent upon phosphorylation or
dephosphorylation of said site by a said kinase or phosphatase in
said assay system, wherein the combination of said isolated natural
binding domain and said binding partner comprises a detection means
for monitoring association or dissociation between said isolated
natural binding domain and said binding partner and wherein said
phosphorylation or dephosphorylation occurs during said mixing, and
wherein association or dissociation of said isolated natural
binding domain and said binding partner as a result of said
contacting is indicative of modulation of enzymatic activity by
said candidate modulator of a said kinase or phosphatase.
49. The method of claim 28, 29, 30, 31, 38, 39, 40, 41, 45, 46, 47,
or 48, wherein said method comprises real-time observation of
association or dissociation of a said isolated natural binding
domain and its binding partner.
Description
FIELD OF THE INVENTION
[0001] The invention relates to monitoring of phosphorylation or
dephosphorylation of a protein.
BACKGROUND OF THE INVENTION
[0002] The post-translational modification of proteins has been
known for over 40 years and since then has become a ubiquitous
feature of protein structure. The addition of biochemical groups to
translated polypeptides has wide-ranging effects on protein
stability, protein secondary/tertiary structure, enzyme activity
and in more general terms on the regulated homeostasis of cells.
Such additions include, but are not limited to, protein
phosphorylation and dephosphorylation.
[0003] Phosphorylation is a well-studied example of a
post-translational modification of proteins. There are many cases
in which polypeptides form higher order tertiary structures with
like polypeptides (homo-oligomers) or with unalike polypeptides
(hetero-oligomers). In the simplest scenario, two identical
polypeptides associate to form an active homodimer. An example of
this type of association is the natural association of myosin II
molecules in the assembly of myosin into filaments.
[0004] The dimerization of myosin II monomers is the initial step
in seeding myosin filaments. The initial dimerization is regulated
by phosphorylation, the effect of which is to induce a
conformational change in myosin II secondary structure resulting in
the folded 10S monomer subunit extending to a 6S molecule. This
active molecule is able to dimerize and subsequently to form
filaments. The involvement of phosphorylation of myosin II in this
priming event is somewhat controversial. Although in higher
eukaryotes the conformational change is dependant on
phosphorylation, in Ancanthoamoeba, a lower eukaryote, the
post-translational addition of phosphate is not required to effect
the initial dimerization step. It is of note that the dimerization
domains in myosin II of higher eukaryotes contain the sites for
phosphorylation and it is probable that phosphorylation in this
region is responsible for enabling myosin II to dimerize and
subsequently form filaments. In Dictyostelium this situation is
reversed in that the phosphorylation sites are outside the
dimerization domain and phosphorylation at these sites is required
to effect the disassembly of myosin filaments. In contrast to both
these examples, Acanthoamoeba myosin II is phosphorylated in the
dimerization domain but this modification is not necessary to
enable myosin II monomers to dimerize in this species.
[0005] By far the most frequent example of post-translational
modification is the addition of phosphate to polypeptides by
specific enzymes known as protein kinases. These enzymes have been
identified as important regulators of the state of phosphorylation
of target proteins and have been implicated as major players in
regulating cellular physiology. For example, the
cell-division-cycle of the eukaryotic cell is primarily regulated
by the state of phosphorylation of specific proteins, the
functional state of which is determined by whether or not the
protein is phosphorylated. This is determined by the relative
activity of protein kinases which add phosphate and protein
phosphatases which remove the phosphate moiety from these proteins.
Clearly dysfunction of either the kinases or phosphatases may lead
to a diseased state. This is best exemplified by the uncontrolled
cellular division shown by tumor cells. The regulatory pathway is
composed of a large number of genes that interact in vivo to
regulate the phosphorylation cascade that ultimately determines if
a cell is to divide or arrest cell division.
[0006] Currently there are several approaches to analysing the
state of modification of target proteins in vivo:
[0007] 1. In vivo incorporation of labeled (for example,
radiolabeled) phosphate, which is added to target proteins.
According to one common procedure, intracellular ATP pools are
labeled with .sup.32PO.sub.4, which is subsequently incorporated
into protein. Analysis of modified proteins is typically performed
by electrophoresis and autoradiography, with specificity enhanced
by immunoprecipitation of proteins of interest prior to
electrophoresis.
[0008] 2. Back-labeling. The incorporation of a labeled phosphate
(e.g., .sup.32P) into a protein in vitro to estimate the state of
modification in vivo.
[0009] 3. The use of cell membrane-permeable protein kinase
inhibitors (e.g., Wortmannin, staurosporine) to block
phosphorylation of target proteins.
[0010] 4. Western blotting, of either 1- or 2-dimensional gels
bearing test protein samples, in which phosphorylation is detected
using antibodies specific for phosphorylated forms of target
proteins.
[0011] 5. The exploitation of eukaryotic microbial systems to
identify mutations in protein kinases and/or protein
phosphatases.
[0012] These strategies have certain limitations. Monitoring states
of phosphorylation by pulse or steady-state labeling is merely a
descriptive strategy to show which proteins are phosphorylated when
samples are separated by gel electrophoresis and visualized by
autoradiography. This is unsatisfactory, due to the inability to
identify many of the proteins that are phosphorylated. A degree of
specificity is afforded to this technique if it is combined with
immunoprecipitation; however, this is of course limited by the
availability of antibodies to target proteins. Moreover, only
highly-expressed proteins are readily detectable using this
technique, which may fail to identify many low-abundance proteins,
which are potentially important regulators of cellular
functions.
[0013] The use of kinase inhibitors to block activity is also
problematic. For example, very few kinase inhibitors have adequate
specificity to allow for the unequivocal correlation of a given
kinase with a specific kinase reaction. Indeed, many inhibitors
have a broad inhibitory range. For example, staurosporine is a
potent inhibitor of phospholipid/Ca.sup.+2 dependant kinases.
Wortmannin is some what more specific, being limited to the
phosphatidylinositol-3 kinase family. This is clearly
unsatisfactory because more than one biochemical pathway may be
affected during treatment making the assignment of the effects
almost impossible.
[0014] Monoclonal antibodies directed against phosphorylated
epitopes, except in specific cases, exhibit a limitation of
specificity comparable to that observed when in vivo labeling is
undertaken. Immunological methods can only detect phosphorylated
proteins globally (e.g., an anti-phosphotyrosine antibody will
detect all tyrosine-phosphorylated proteins) and can only describe
a steady state, rather provide a real-time assessment of
protein:protein interactions. Such assays also require considerable
manpower for processing.
[0015] Finally, yeast (Saccharomyces cervisiae and
Schizosaccharomyces pombe) has been exploited as a model organism
for the identification of gene function using recessive mutations.
It is through research on the effects of these mutations that the
functional specificities of many protein kinases have been
elucidated. However, these molecular genetic techniques are not
easily transferable to higher eukaryotes, which are diploid and
therefore not as genetically tractable as these lower
eukaryotes.
[0016] Recent research into the sites of protein phosphorylation
has revealed a number of sequence specific motifs which, when
phosphorylated or dephosphorylated, promote interaction with
selected target proteins to either induce or inhibit activity of
either the phosphorylated polypeptide or the target
polypeptide.
[0017] For example, and not by way of limitation, many proteins
involved in intracellular signal transduction have been shown to
contain a domain comprising a sequence of approximately 100 amino
acids; this sequence is termed the Src homology two (SH2) domain.
SH2 domains bind target polypeptides that contain phosphorylated
tyrosine. This binding is dependent on the primary amino acid
sequence around the phosphotyrosine in the target protein and
several peptide sequences which, when phosphorylated, bind to an
SH2 domain have been identified (see e.g., Songyang et al., 1993,
Cell, 72: 767-778). Non-limiting examples of such sequences include
FLPVPEYINQSV, a sequence found in human ECF receptor, and
AVGNPEYLNTVQ, a sequence found in human EGF receptor, both of which
are autophosphorylated growth factor receptors which stimulate the
biochemical signaling pathways that control gene expression,
cytoskeletal architecture and cell metabolism. Both of these
sequences interact with SH2 domains found in the Sen-5 adapter
protein.
[0018] The tumor suppressor protein p53, becomes activated by a
transcription factor in response to DNA damage. A DNA-dependent
protein kinase (DNA-PK) that is activated in response to breaks in
DNA is thought to be regulator of p53 activity (Woo et al., 1998,
Nature, 394: 700-704). The data described by Woo et al. indicate
that the phosphorylation of p53 by DNA-PK serves a dual purpose
insofar as phosphorylation promotes the binding of p53 to DNA and
also prevents p53 inactivation by MDM2. A p53-derived peptide
sequence EPPLSOEAFADLWKK is identified as the site of
phosphorylation in p53 that (when phosphorylated) prevents the
interaction of p53 with MDM2.
[0019] An example of heterodimer association is described in patent
application number WO92/00388. It describes an adenosine 3:5 cylic
monophosphate (cAMP) dependent protein kinase which is a
four-subunit enzyme being composed of two catalytic polypeptides
(C) and two regulatory polypeptides (R). In nature the polypeptides
associate in a stoichiometry of R.sub.2C.sub.2. In the absence of
cAMP the R and C subunits associate and the enzyme complex is
inactive. In the presence of cAMP the R subunit functions as a
ligand for cAMP resulting in dissociation of the complex and the
release of active protein kinase. The invention described in
WO92/00388 exploits this association by adding fluorochromes to the
R and C subunits.
[0020] The polypeptides are labeled (or `tagged`) with fluorophores
having different excitation/emission wavelengths. The excitation
and emission of one such fluorophore effects a second
excitation/emission event in the second fluorophore. By monitoring
the fluorescence emission of each fluorophore, which reflects the
presence or absence of fluorescence energy transfer between the
two, it is possible to derive the concentration of cAMP as a
function of the level of association between the R and C.
Therefore, the natural affinity of the C subunit for the R subunit
has been exploited to monitor the concentration of a specific
metabolite, namely cAMP.
[0021] The prior art teaches that intact, fluorophore-labeled
proteins can function as reporter molecules for monitoring the
formation of multi-subunit complexes from protein monomers;
however, in each case, the technique relies on the natural ability
of the protein monomers to associate.
[0022] Tsien et al. (WO97/28261) teach that fluorescent proteins
having the proper emission and excitation spectra that are brought
into physically close proximity with one another can exhibit
fluorescence resonance energy transfer ("FRET"). The invention of
WO97/28261 takes advantage of that discovery to provide tandem
fluorescent protein constructs in which two fluorescent protein
labels capable of exhibiting FRET are coupled through a linker to
form a tandem construct. In the assays of Tsien et al., protease
activity is monitored using FRET to determine the distance between
fluorophores controlled by a peptide linker and subsequent
hydrolysis thereof. Other applications rely on a change in the
intrinsic fluorescence of the protein as in the kinase assays of
WO98/06737.
[0023] The present invention instead encompasses monitoring of the
association of polypeptides, as described herein, which are labeled
with fluorescent (protein and chemical) or other labels. FRET, a
non-limiting example of a detection method of use in the invention,
indicates the proximity of two labeled polypeptide binding
partners, which labeled partners associate either in the presence
or absence of post-translational addition/removal of a phosphate
group to/from a natural binding domain present in at least one of
the partners, but not into the fluorophore, reflecting the
phosphorylation state of one or both of the binding partners and,
consequently, the level of activity of a protein kinase or
phosphatase.
[0024] There is a need in the art for efficient means of monitoring
and/or modulating post-translational protein phosphorylation and/or
dephosphorylation. Further, there is a need to develop a technique
whereby the addition/removal of a phosphate group can be monitored
continuously during real time to provide a dynamic assay system
that also has the ability to resolve spatial information.
SUMMARY OF THE INVENTION
[0025] The invention provides natural binding domains, sequences
and polypeptides, as well as kits comprising these molecules and
assays of enzymatic function in which they are employed as reporter
molecules. As used herein in reference to a polypeptide component
of assays of the invention, the term "natural" refers both to the
existence of such an amino acid sequence, whether contiguous or
non-contiguous, in nature as well as to the
phosphorylation-dependent binding of that component to a second
polypeptide or binding partner, and does not relate to attributes
of such a polypeptide other than such binding.
[0026] One aspect of the invention is an isolated natural binding
domain and a binding partner therefor, wherein the isolated natural
binding domain includes a site for post-translational
phosphorylation and binds the binding partner in a manner dependent
upon phosphorylation or dephosphorylation of the site.
[0027] The invention also provides a method for monitoring activity
of an enzyme comprising performing a detection step to detect
binding of an isolated natural binding domain and a binding partner
therefor as a result of contacting one or both of the isolated
natural binding domain and the binding partner with the enzyme,
wherein the isolated natural binding domain includes a site for
post-translational phosphorylation and binds the binding partner in
a manner dependent upon phosphorylation of the site and wherein
detection of binding of the isolated natural binding domain and the
binding partner as a result of the contacting is indicative of
enzyme activity.
[0028] An enzyme to be assayed according to the invention is a
protein kinase or a phosphatase.
[0029] The invention additionally encompasses a method for
monitoring activity of an enzyme comprising performing a detection
step to detect dissociation of an isolated natural binding domain
from a binding partner therefor as a result of contacting one or
both of the isolated natural binding domain and the binding partner
with the enzyme, wherein the isolated natural binding domain
includes a site for post-translational phosphorylation and binds
the binding partner in a manner dependent upon phosphorylation of
the site and wherein detection of dissociation of the isolated
natural binding domain from the binding partner as a result of the
contacting is indicative of enzyme activity.
[0030] As used herein, the term "binding domain" in a
three-dimensional sense refers to the amino acid residues of a
first polypeptide required for phosphorylation-dependent binding
between the first polypeptide and its binding partner. The amino
acids of a "binding domain" may be either contiguous or
non-contiguous and may form a binding pocket for
phosphorylation-dependent binding. A domain must include at least 1
amino acid, but may include 2 or more amino acids, preferably at
least 4 amino acids, which are contiguous or non-contiguous, but
are necessary for phosphorylation-dependent binding to the binding
partner. A binding domain will not include a natural full-length
polypeptide, but will include a subset of the amino acids of a
full-length polypeptide, wherein the subset may include a number of
amino acids as high as one fewer than the length of a given natural
full-length polypeptide.
[0031] A binding domain which is of use in the invention is a
"natural binding domain" (i.e., a binding domain that exhibits
phosphorylation-dependent binding to a binding partner in nature).
A natural binding domain of use in the invention may be isolated or
may be present in the context of a larger polypeptide molecule
(i.e., one which comprises amino acids other than those of the
natural binding domain), which molecule may be either
naturally-occurring or recombinant and, in the case of the latter,
may comprise either natural or non-natural amino acid sequences
outside the binding domain.
[0032] As used herein with regard to phosphorylation or
dephosphoylation of a polypeptide, the term "site" refers to an
amino acid or amino acid sequence of a natural binding domain or a
binding partner which is recognized by (i.e., a signal for) a
kinase or phosphatase for the purpose of phosphorylation or
dephosphorylation (i.e., addition or removal of a phosphate moiety)
of the polypeptide or a portion thereof. A "site" additionally
refers to the single amino acid which is phosphorylated or
dephosphorylated. It is contemplated that a site comprises a small
number of amino acids, as few as one but typically from 2 to 10,
less often up to 30 amino acids, and further that a site comprises
fewer than the total number of amino acids present in the
polypeptide.
[0033] In an enzymatic assay of the invention, a "site", for
post-translational phosphorylation or dephosphorylation may be
present on either or both of the isolated natural binding domain or
the binding partner therefor. If such sites are present on both the
isolated natural binding domain and its binding partner, binding
between the natural binding domain and the binding partner, or
between two natural binding domains, may be dependent upon the
phosphorylation or dephosphorylation state of either one or both
sites. If a single polypeptide chain comprises the natural binding
domain and the binding partner (or two natural binding domains),
the state of phosphorylation or dephosphorylation of one or both
sites will determine whether binding occurs.
[0034] A site suitable for addition or removal of a phosphate
moiety is present within an isolated natural binding domain or
binding partner thereof of the invention at a position such that
formation of a complex between the isolated natural binding domain
and its binding partner is dependent upon the presence or absence
of the phosphate moiety; and preferably does not overlap with an
amino acid which is part of a fluorescent tag or other detectable
label (including, but not limited to, a radioactive label) or
quencher.
[0035] Similarly, the amino acid that includes a phosphate moiety
may be positioned anywhere within the isolated natural binding
domain such that binding of the isolated natural binding domain and
its binding partner is dependent upon the presence or absence of
the phosphate moiety.
[0036] As used herein, "phosphorylation" and "dephosphorylation"
refer to the addition or removal of a phosphate moiety to/from a
polypeptide, respectively. As used herein, the term
"post-translational modification" refers to the addition or removal
of a phosphate moiety and does not refer to other
post-translational events which do not involve addition or removal
of a phosphate moiety, and thus does not include simple cleavage of
the reporter molecule polypeptide backbone by hydrolysis of a
peptide bond.
[0037] As used herein, the term "moiety" refers to a
post-translationally added or removed phosphate (PO.sub.4) group;
the terms "moiety" and "group" are used interchangeably.
[0038] As used herein, the term "binding partner" refers to a
polypeptide or fragment thereof (a peptide) that binds to a binding
domain, sequence or polypeptide, as defined herein, in a manner
which is dependent upon the state of phosphorylation of a site for
phosphorylation or dephosphorylation which is, at a minimum,
present upon the binding domain, sequence or polypeptide; the
binding partner itself may, optionally, comprise such a site and
binding between the binding domain, fragment or polypeptide with
its corresponding binding partner may, optionally, depend upon
modification of that site. A binding partner does not necessarily
have to contain a site for phosphorylation or dephosphorylation if
such an site is not required to be present on it for
modification-dependent association between it and a binding domain,
sequence or polypeptide. Binding partners of use in the invention
are those which are found in nature and exhibit natural
phosphorylation-dependent binding to a natural binding domain,
sequence or polypeptide of the invention as defined herein. In one
embodiment of the invention, a binding partner is shorter (i.e., by
at least one N-terminal or C-terminal amino acid) than the natural
full-length polypeptide.
[0039] As used herein, the term "associates" or "binds" refers to a
natural binding domain as described herein and its binding partner,
having a binding constant sufficiently strong to allow detection of
binding by FRET or other detection means, which are in physical
contact with each other and have a dissociation constant (Kd) of
about 10 .mu.M or lower. The contact region may include all or
parts of the two molecules. Therefore, the terms "substantially
dissociated" and "dissociated" or "substantially unbound" or
"unbound" refer to the absence or loss of contact between such
regions, such that the binding constant is reduced by an amount
which produces a discernable change in a signal compared to the
bound state, including a total absence or loss of contact, such
that the proteins are completely separated, as well as a partial
absence or loss of contact, so that the body of the proteins are no
longer in close proximity to each other but may still be tethered
together or otherwise loosely attached, and thus have a
dissociation constant greater than 10 .mu.M (Kd). In many cases,
the Kd will be in the mM range. The terms "complex", "dimer",
"multimer" and "oligomer" as used herein, refer to the natural
binding domain and its binding partner in the associated or bound
state. More than one molecule of each of the two or more proteins
may be present in a complex, dimer, multimer or oligomer according
to the methods of the invention.
[0040] As used herein in reference to a natural binding domain or
other polypeptide, the term "isolated" refers to a molecule or
population of molecules that is substantially pure (i.e., free of
contaminating molecules of unlike amino acid sequence).
[0041] As used herein in reference to the purity of a molecule or
population thereof, the term "substantially" refers to that which
is at least 50%, preferably 60-75%, more preferably from 80-95%
and, most preferably, from 98-100% pure.
[0042] "Naturally-occurring" as used herein, as applied to a
polypeptide or polynucleotide, refers to the fact that the
polypeptide or polynucleotide can be found in nature. One such
example is a polypeptide or polynucleotide sequence that is present
in an organism (including a virus) that can be isolated form a
source in nature.
[0043] The term "synthetic", as used herein, is defined as any
amino- or nucleic acid sequence which is produced via chemical
synthesis.
[0044] In an assay of the invention, post-translational
phosphorylation is reversible, such that repeating cycles of
addition and removal of a phosphate moiety may be observed,
although such cycles may not occur in a living cell found in
nature.
[0045] An advantage of assays of the invention is that they may, if
desired, be performed in "real time". As used herein in reference
to monitoring, measurements or observations in assays of the
invention, the term "real time" refers to that which is performed
contemporaneously with the monitored, measured or observed events
and which yields a result of the monitoring, measurement or
observation to one who performs it simultaneously, or effectively
so, with the occurrence of a monitored, measured or observed event.
Thus, a "real time" assay or measurement contains not only the
measured and quantitated result, such as fluorescence, but
expresses this in real time, that is, in hours, minutes, seconds,
milliseconds, nanoseconds, picoseconds, etc. Shorter times exceed
the instrumentation capability; further, resolution is also limited
by the folding and binding kinetics of polypeptides.
[0046] As used herein, the term "binding sequence" refers to that
portion of a polypeptide comprising at least 1, preferably at least
2, more preferably at least 4, and up to 8, 10, 100 or even 1000
contiguous (i.e., covalently linked by peptide bonds) amino acid
residues, that are sufficient for phosphorylation-dependent binding
to a binding partner. A binding sequence will not include a natural
full-length polypeptide, but will include a subset of the amino
acids of a full-length polypeptide, wherein the subset may include
a number of amino acids as high as one fewer than the length of a
given natural full-length polypeptide.
[0047] As used herein in reference to those binding sequences that
are of use in the invention, the term "natural binding sequence"
refers to a binding sequence, as defined above, which consists of
an amino acid sequence which is found in nature and which is
naturally dependent upon the phosphorylation state of a site for
post-translational phosphorylation found within it for binding to a
binding partner. A "natural binding sequence" may be present either
in isolation or in the context of a larger polypeptide molecule,
which molecule may be naturally-occurring or recombinant. If
present, amino acids outside of the binding sequence may be either
natural, i.e., from the same polypeptide sequence from which the
fragment is derived, or non-natural, i.e., from another (different)
polypeptide or from a sequence that is not derived from any known
polypeptide. In assays of the invention, a binding sequence and its
binding partner may exist either on two different polypeptide
chains or on a single polypeptide chain.
[0048] As used herein, the term "binding polypeptide" refers to a
molecule comprising multiple binding sequences, as defined above. A
binding polypeptide of use in the invention is a "natural binding
polypeptide", in which the component binding sequences are natural
binding sequences, as defined above (e.g., wherein the binding
sequences are derived from a single, naturally-occurring
polypeptide molecule), and are both necessary and, in combination,
sufficient to permit phosphorylation state-dependent binding of the
binding polypeptide to its binding partner, wherein the sequences
of the binding polypeptide are either contiguous or are
non-contiguous. As used herein in reference to the component
binding sequences of a binding polypeptide, the term
"non-contiguous" refers to binding sequences which are linked by
intervening naturally-occurring, as defined herein, or non-natural
amino acid sequences or other chemical or biological linker
molecules such are known in the art. The amino acids of a
polypeptide that do not significantly contribute to the natural
phosphorylation-state-dependent binding of that polypeptide to its
binding partner may be those amino acids which are naturally
present and link the binding sequences in a binding polypeptide or
they may be derived from a different natural polypeptide or may be
wholly unknown in nature. In assays of the invention, a binding
polypeptide and its binding partner (which may, itself, be a
binding domain, sequence or polypeptide, as defined herein) may
exist on two different polypeptide chains or on a single
polypeptide chain. According to the invention, a natural binding
polypeptide, like a polypeptide as defined above, is not a
full-length natural polypeptide chain, but instead comprises a
subset that encompasses up to one fewer than the total number of
amino acids in a natural polypeptide chain.
[0049] As used herein, the terms "polypeptide" and "peptide" refer
to a polymer in which the monomers are amino acids and are joined
together through peptide or disulfide bonds. The terms subunit and
domain also may refer to polypeptides and peptides having
biological function. A peptide useful in the invention will at
least have a binding capability, i.e, with respect to binding as-
or to a binding partner, and also may have another biological
function that is a biological function of a protein or domain from
which the peptide sequence is derived. "Polypeptide" refers to a
naturally-occurring amino acid chain comprising a subset of the
amino acids of a full-length protein, wherein the subset comprises
at least one fewer amino acid than does the full-length protein, or
a "fragment thereof" or "peptide", such as a selected region of the
polypeptide that is of interest in a binding assay and for which a
binding partner is known or determinable. "Fragment thereof" thus
refers to an amino acid sequence that is a portion of a full-length
polypeptide, between about 8 and about 1000 amino acids in length,
preferably about 8 to about 300, more preferably about 8 to about
200 amino acids, and even more preferably about 10 to about 50 or
100 amino acids in length. "Peptide" refers to a short amino acid
sequence that is 10-40 amino acids long, preferably 10-35 amino
acids. Additionally, unnatural amino acids, for example,
.beta.-alanine, phenyl glycine and homoarginine may be included.
Commonly-encountered amino acids which are not gene-encoded may
also be used in the present invention. All of the amino acids used
in the present invention may be either the D- or L-optical isomer.
The L-isomers are preferred. In addition, other peptidomimetics are
also useful, e.g. in linker sequences of polypeptides of the
present invention (see Spatola, 1983, in Chemistry and Biochemistry
of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel
Dekker, New York, p. 267).
[0050] As used herein, the terms "protein", "subunit" and "domain"
refer to a linear sequence of amino acids which exhibits biological
function. This linear sequence does not include full-length amino
acid sequences (e.g. those encoded by a full-length gene or
polynucleotide), but does include a portion or fragment thereof,
provided the biological function is maintained by that portion or
fragment. The terms "subunit" and "domain" also may refer to
polypeptides and peptides having biological function. A peptide
useful in the invention will at least have a binding capability,
i.e, with respect to binding as or to a binding partner, and also
may have another biological function that is a biological function
of a protein or domain from which the peptide sequence is
derived.
[0051] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length and up to 1,000 bases or even more,
either ribonucleotides or deoxyribonucleotides or a modified form
of either type of nucleotide. The term includes single and double
stranded forms of DNA.
[0052] Preferably, with regard to the natural binding domain and/or
binding partner therefor, phosphorylation or dephosphorylation is
performed by an enzyme which is a kinase or a phosphatase,
respectively.
[0053] It is preferred that phosphorylation of the site prevents
binding of the isolated natural binding domain to the binding
partner.
[0054] As used herein, the term "prevents" refers to a reduction of
at least 10%, preferably 20-40%, more preferably 50-75% and, most
preferably, 80-100% of binding of the isolated natural binding
domain to the binding partner therefor.
[0055] Preferably, phosphorylation of the site promotes binding of
the isolated natural binding domain to the binding partner.
[0056] As used herein with regard to protein:protein binding, the
term "promotes" refers to that which causes an increase in binding
of the natural binding domain and its binding partner of at least
two-fold, preferably 10- to 20-fold, highly preferably 50- to
100-fold, more preferably from 200- to 1000-fold, and, most
preferably, from 200 to 10,000-fold.
[0057] It is preferred that dephosphorylation of the site prevents
binding of the isolated natural binding domain to the binding
partner.
[0058] It is additionally preferred that dephosphorylation of the
site promotes binding of the isolated natural binding domain to the
binding partner.
[0059] In a preferred embodiment, at least one of the isolated
natural binding domain and the binding partner comprises a
detectable label.
[0060] Preferably, the detectable label emits light.
[0061] More preferably, the light is fluorescent.
[0062] It is preferred that one of the isolated natural binding
domain and the binding partner therefor comprises a quencher for
the detectable label. Labels of use in the invention include, but
are not limited to, a radioactive label, a fluorescent label and a
quencher for either.
[0063] A "fluorescent label", "fluorescent tag" or "fluorescent
group" refers to either a fluorophore or a fluorescent protein or
fluorescent fragment thereof.
[0064] "Fluorescent protein" refers to any protein which fluoresces
when excited with appropriate electromagnetic radiation. This
includes a protein whose amino acid sequence is either natural or
engineered. A "fluorescent protein" is a full-length fluorescent
protein or fluorescent fragment thereof . By the same token, the
term "linker" refers to that which is coupled to both the donor and
acceptor protein molecules, such as an amino acid sequence joining
two natural binding domains or a disulfide bond between two
polypeptides.
[0065] It is contemplated that with regard to fluorescent labels
employed in FRET, the reporter labels are chosen such that the
emission wavelength spectrum of one (the "donor") is within the
excitation wavelength spectrum of the other (the "acceptor"). With
regard to a fluorescent label and a quencher employed in a
single-label detection procedure in an assay of the invention, it
is additionally contemplated that the fluorophore and quencher are
chosen such that the emission wavelength spectrum of the
fluorophore is within the absorption spectrum of the quencher, such
that when the fluorophore and the quencher with which it is
employed are brought into close proximity by binding of the natural
binding domain, sequence or polypeptide upon which one is present
with the binding partner comprising the other, detection of the
fluorescent signal emitted by the fluorophore is reduced by at
least 10%, preferably 20-50%, more preferably, 70-90% and, most
preferably, by 95-100%. A typical quencher reduces detection of a
fluorescent signal by approximately 80%.
[0066] Another aspect of the invention is a kit comprising an
isolated natural binding domain and a binding partner therefor,
wherein the isolated natural binding domain includes a site for
post-translational phosphorylation and binds the binding partner in
a manner dependent upon phosphorylation of the site, and packaging
material therefor.
[0067] It is preferred that the kit further comprises a buffer
which permits phosphorylation-dependent binding of the isolated
natural binding domain and the binding partner.
[0068] As used herein, the term "buffer" refers to a medium which
permits activity of the protein kinase or phosphatase used in an
assay of the invention, and is typically a low-ionic-strength
buffer or other biocompatible solution (e.g., water, containing one
or more of physiological salt, such as simple saline, and/or a weak
buffer, such as Tris or phosphate, or others as described
hereinbelow), a cell culture medium, of which many are known in the
art, or a whole or fractionated cell lysate. Such a buffer permits
phosphorylation-dependent binding of a natural binding domain of
the invention and a binding partner therefor and, preferably,
inhibits degradation and maintains biological activity of the
reaction components. Inhibitors of degradation, such as protease
inhibitors (e.g., pepstatin, leupeptin, etc.) and nuclease
inhibitors (e.g., DEPC) are well known in the art. Lastly, an
appropriate buffer may comprise a stabilizing substance such as
glycerol, sucrose or polyethylene glycol.
[0069] As used herein, the term "physiological buffer" refers to a
liquid medium that mimics the salt balance and pH of the cytoplasm
of a cell or of the extracellular milieu, such that
post-translational protein modification reactions and
protein:protein binding are permitted to occur in the buffer as
they would in vivo.
[0070] Preferably, the buffer permits phosphorylation or
dephosphorylation of the site by a kinase or a phosphatase,
respectively.
[0071] In a preferred embodiment, the kit further comprises one or
both of a kinase and a phosphatase.
[0072] It is preferred that the kit further comprises a substrate
for the phosphatase or kinase, the substrate being MGATP.
[0073] It is contemplated that at least a part of a substrate of an
enzyme of use in an assay of the invention is transferred to a
phosphorylation site on an isolated polypeptide of the invention.
As used herein, the term "at least a part of a substrate" refers to
a portion (e.g., a moiety or a group, as defined above) which
comprises less than the whole of the substrate for the enzyme, the
transfer of which portion to a phosphorylation site on an isolated
polypeptide, both as defined above, is catalyzed by the enzyme.
[0074] It is additionally preferred that the kit further comprises
a cofactor for one or both of the kinase or phosphatase. Cofactors
of use in the invention include, but are not limited to, cAMP,
phosphotidylserine, diolein, Mn.sup.2+ and Mg.sup.2+.
[0075] Preferably, at least one of the isolated natural binding
domain and the binding partner comprises a detectable label.
[0076] It is preferred that the detectable label emits light, and
more preferred that the light is fluorescent.
[0077] An enzyme (e.g., a protein kinase or phosphatase) of use in
the invention may be natural or recombinant or, alternatively, may
be chemically synthesized. If either natural or recombinant, it may
be substantially pure (i.e., present in a population of molecules
in which it is at least 50% homogeneous), partially purified (i.e.,
represented by at least 1% of the molecules present in a fraction
of a cellular lysate) or may be present in a crude biological
sample.
[0078] As used herein, the term "sample" refers to a collection of
inorganic, organic or biochemical molecules which is either found
in nature (e.g., in a biological- or other specimen) or in an
artificially-constructed grouping, such as agents which might be
found and/or mixed in a laboratory. Such a sample may be either
heterogeneous or homogeneous.
[0079] As used herein, the interchangeable terms "biological
specimen" and "biological sample" refer to a whole organism or a
subset of its tissues, cells or component parts (e.g. body fluids,
including but not limited to blood, mucus, lymphatic fluid,
synovial fluid, cerebrospinal fluid, saliva, amniotic fluid,
amniotic cord blood, urine, vaginal fluid and semen). "Biological
sample" further refers to a homogenate, lysate or extract prepared
from a whole organism or a subset of its tissues, cells or
component parts, or a fraction or portion thereof. Lastly,
"biological sample" refers to a medium, such as a nutrient broth or
gel in which an organism has been propagated, which contains
cellular components, such as proteins or nucleic acid
molecules.
[0080] As used herein, the term "organism" refers to all cellular
life-forms, such as prokaryotes and eukaryotes, as well as
non-cellular, nucleic acid-containing entities, such as
bacteriophage and viruses.
[0081] In a method as described above, it is preferred that at
least one of the isolated natural binding domain and the binding
partner is labeled with a detectable label.
[0082] Preferably, the label emits light and, more preferably, the
light is fluorescent.
[0083] In another preferred embodiment, the detection step is to
detect a change in signal emission by the detectable label.
[0084] It is preferred that the method further comprises exciting
the detectable label and monitoring fluorescence emission.
[0085] It is additionally preferred that the method further
comprises the step, prior to or after the detection step, of
contacting the isolated natural binding domain and the binding
partner with an agent which modulates the activity of the
enzyme.
[0086] As used herein with regard to a biological or chemical
agent, the term "modulate" refers to enhancing or inhibiting the
activity of a protein kinase or phosphatase in an assay of the
invention; such modulation may be direct (e.g. including, but not
limited to, cleavage of- or competitive binding of another
substance to the enzyme) or indirect (e.g. by blocking the initial
production or, if required, activation of the kinase or
phosphatase).
[0087] "Modulation" refers to the capacity to either increase or
decease a measurable functional property of biological activity or
process (e.g., enzyme activity or receptor binding) by at least
10%, 15%, 20%, 25%, 50%, 100% or more; such increase or decrease
may be contingent on the occurrence of a specific event, such as
activation of a signal transduction pathway, and/or may be manifest
only in particular cell types.
[0088] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g., nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian)
cells or tissues, or even an inorganic element or molecule.
Modulators are evaluated for potential activity as inhibitors or
activators (directly or indirectly) of a biological process or
processes (e.g., agonist, partial antagonist, partial agonist,
antagonist, antineoplastic agents, cytotoxic agents, inhibitors of
neoplastic transformation or cell proliferation, cell
proliferation-promoting agents, and the like) by inclusion in
screening assays described herein. The activities (or activity) of
a modulator may be known, unknown or partially-known. Such
modulators can be screened using the methods described herein.
[0089] The term "candidate modulator" refers to a compound to be
tested by one or more screening method(s) of the invention as a
putative modulator. Usually, various predetermined concentrations
are used for screening such as 0.01 .mu.M, 0.1 .mu.M, 1.0 .mu.M,
and 10.0 .mu.M, as described more fully hereinbelow. Test compound
controls can include the measurement of a signal in the absence of
the test compound or comparison to a compound known to modulate the
target.
[0090] The invention additionally provides a method of screening
for a candidate modulator of enzymatic activity of a kinase or a
phosphatase, the method comprising contacting an isolated natural
binding domain, a binding partner therefor and an enzyme with a
candidate modulator of the kinase or phosphatase, wherein the
natural binding domain includes a site for post-translational
phosphorylation and binds the binding partner in a manner that is
dependent upon phosphorylation or dephosphorylation of the site by
the kinase or phosphatase and wherein at least one of the isolated
natural binding domain and the binding partner comprises a
detectable label, and monitoring the binding of the isolated
natural binding domain to the binding partner, wherein binding or
dissociation of the isolated natural binding domain and the binding
partner as a result of the contacting is indicative of modulation
of enzymatic activity by the candidate modulator of the kinase or
phosphatase.
[0091] Preferably, the detectable label emits light.
[0092] More preferably, the light is fluorescent.
[0093] It is preferred that the monitoring comprises measuring a
change in energy transfer between a detectable label present on the
isolated natural binding domain and a detectable label present on
the binding partner.
[0094] A final aspect of the invention is a method of screening for
a candidate modulator of enzymatic activity of a kinase or a
phosphatase, the method comprising contacting an assay system with
a candidate modulator of enzymatic activity of a kinase or
phosphatase, and monitoring binding of an isolated natural binding
domain and a binding partner therefor in the assay system, wherein
the isolated natural binding domain includes a site for
post-translational phosphorilation and binds the binding partner in
a manner that is dependent upon phosphorylation or
dephosphorylation of the site by a kinase or phosphatase in the
assay system, wherein at least one of the isolated natural binding
domain and the binding partner comprises a detectable label, and
wherein binding or dissociation of the isolated natural binding
domain and the binding partner as a result of the contacting is
indicative of modulation of enzymatic activity by the candidate
modulator of a the kinase or phosphatase.
[0095] It is highly preferred that in any of the above methods, the
method comprises real-time observation of association of an
isolated natural binding domain and its binding partner.
[0096] Further features and advantages of the invention will become
more fully apparent in the following description of the embodiments
and drawings thereof, and from the claims.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0097] FIG. 1 diagrams double- and single-chain enzymatic assay
formats of the invention.
[0098] FIG. 2 presents a schematic overview of FRET in an assay of
the invention.
[0099] FIG. 3 presents monomer:excimer fluorescence.
[0100] FIG. 4 demonstrates the results of FRET between ZAP70-GFP
and a rhodamine labelled TCR.zeta. derived peptide.
[0101] FIG. 5 demonstrates the dependence of YOP activity on
different concentrations of TCR.zeta. peptide.
[0102] FIG. 6 presents the results of a FRET based assay for
measuring inhibition of YOP by sodium orthovanadate.
[0103] FIG. 7 demonstrates detection of binding of Chk1
phosphorylated, fluorescein labelled Chktide to 14-3-3.zeta. by
fluorescence polarisation.
[0104] FIG. 8 demonstrates inhibition of Chk1 phosphorylation of
Chktide peptide by EDTA.
[0105] FIG. 9 presents the results of a real time assay for Chk1
activity monitoring the fluorescence polarisation of fluorescein
labelled Chktide substrate binding to 14-3-3.zeta. protein.
[0106] FIG. 10 demonstrates that Chk1 activity measured by Chktide:
14-3-3 binding is dependent on ATP and the presence of 14-3-3.zeta.
protein.
[0107] FIG. 11 demonstrates inhibition of Chk1 phosphorylation of
fluorescein labelled Chktide by EDTA.
[0108] FIG. 12 presents the results of an assay for Chk1
phosphorylation of Chktide peptide as measured by 14-3-3.di-elect
cons. binding.
[0109] FIG. 13 demonstrates phosphatase .lambda. activity as
measured by dephosphorylation of fluorescein labelled Chktide and
decreased binding to 14-3-3.zeta..
[0110] FIG. 14 demonstrates phosphatase .lambda. activity as
measured by dephosphorylation of fluorescein labelled Chktide and
decreased binding to 14-3-3.di-elect cons..
[0111] FIG. 15 presents a time course of Chk1 and PKA activity
measured using fluorescence polarisation.
[0112] FIG. 16 demonstrates detection of peptide phosphorylation by
Src kinase, by measuring FRET between ZAP-GFP and a rhodamine
labelled substrate peptide.
[0113] FIG. 17 demonstrates detection of SHPS-1 derived peptide
phosphorylation by Src, and binding of SHPS-1 to SHP2-GFP
partner.
[0114] FIG. 18 demonstrates YOP mediated reversal of FRET between
SHP2-GFP and rhodamine labelled, phosphorylated, SHPS-1
peptide.
[0115] FIG. 19 presents detection of Src inhibition by
staurosporine using a FRET-based assay between rhodamine labelled
SHPS-1 and SHP2-GFP.
[0116] FIG. 20 presents the results of a real-time, FRET-based
assay, measuring Src phosphorylation of SHPS-1 peptide.
DESCRIPTION
[0117] The invention is based upon the discovery that a natural
binding domain, sequence or polypeptide, as defined above,
associates with a binding partner to form a complex or dissociates
from a binding partner, in a manner that is dependent upon the
presence or absence of a phosphate moiety, and that is detectable
and measurable in a highly sensitive manner that may be observed in
real time.
[0118] Polypeptides of Use in the Invention
[0119] The invention provides reporter molecules and assays for
measuring the activity of protein kinases and phosphatases. These
reporter molecules are naturally-occurring polypeptides which
include natural binding domains, natural binding sequences and
natural binding polypeptides, each as defined above, which are used
in assays of the invention in combination with polypeptide binding
partners, also as defined above.
[0120] Minimally, such a reporter molecule comprises or consists of
a natural binding domain. The amino acids of a natural binding
domain are those which are necessary for phosphorylation-dependent
binding of the molecule comprising or consisting of the natural
binding domain with a binding partner, whether such a partner is
present on the same or a different polypeptide chain as the natural
binding domain. Such amino acids may include points of direct
contact between the domain and the binding partner, those which are
recognized and/or modified (i.e., phosphorylated or
dephosphorylated) by a kinase or phosphatase and those which
maintain the three-dimensional structure or charge of the binding
domain in a manner which permits phosphorylation and/or
dephosphorylation and the consequent phosphorylation- and/or
dephosphorylation-dependent binding of the domain to the binding
partner. The amino acids of a natural binding domain may be
contiguous or may be separated by non-domain amino acids; such
non-domain residues may be either those which are naturally present
between the amino acids of the natural binding domain or which are
non-natural. In cases in which non-natural amino acids are found
interspersed with those of a natural binding domain, such
non-natural residues will be residues which do not substantially
(that is, measurably) alter the natural phosphorylation-dependent
binding of the natural binding domain to its binding partner.
[0121] A second reporter molecule of use in the invention is that
which comprises or consists of a naturally-occurring stretch of
contiguous amino acids sufficient for phosphorylation-dependent
binding to a binding partner, as defined above, i.e., at least the
minimum number of contiguous amino acids required to encompass a
natural binding domain. The phosphorylation-dependence of such a
molecule, referred to herein as a "natural binding sequence", is,
itself natural. A reporter molecule of the invention may either
consist of or comprise a natural binding sequence. In the latter
case, amino acids outside of the natural binding sequence do not
substantially influence phosphorylation-dependent binding of the
natural binding domain to the binding partner.
[0122] Lastly, a reporter molecule of use in the invention may be a
"natural binding polypeptide", as defined above. Such a polypeptide
molecule comprises or consists of multiple natural binding domains
(above), which domains are, either individually or in concert with
one another, sufficient to permit natural,
phosphorylation-dependent binding of the natural binding
polypeptide to a binding partner.
[0123] By monitoring the association or dissociation of a natural
binding domain, sequence or polypeptide and its binding partner in
the presence of a known or candidate protein kinase or phosphatase,
the activity of such an enzyme can be measured. In such assays, one
or both of the natural binding domain, sequence or polypeptide and
its binding partner comprises a detectable label including, but not
exclusively, a fluorescent or other light-emitting label, which may
be either chemical or proteinaceous. By measuring changes in signal
emission or absorption before and after addition to the mixture
comprising the natural binding domain, sequence or polypeptide and
its binding partner of the enzyme to be assayed, the extent of
phosphorylation can be calculated. An important feature of the
invention is that such measurements (e.g., of a shift in FRET) can
be performed in real-time. This allows for sensitive assessment of
enzyme reaction kinetics based upon the rate of change of the
protein-binding-dependent signal emission or absorption by the
label(s).
[0124] Assays in which the above reporter molecules are used
according to the invention may be performed either in double- or
single-chain format (FIG. 1). In double-chain format, natural
binding domain, sequence or polypeptide is comprised by a different
polypeptide chain from that comprising or consisting of the binding
partner and is not otherwise covalently linked to it. In
single-chain format, the natural binding domain, sequence or
polypeptide is covalently linked to its binding partner, either
through an intervening amino acid sequence or a chemical
linker.
[0125] The binding partner of a natural binding domain, sequence or
polypeptide may, itself, be a natural binding domain, sequence or
polypeptide as defined herein. If so, binding of the two molecules
may depend upon the phosphorylation state of one or both in a
manner that is comparable to that found in nature.
[0126] Methods by which assays of the invention are performed are
described in detail in the following sections and in the
Examples.
[0127] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g, in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated
herein by reference), chemical methods, pharmaceutical formulation
and delivery and treatment of patients.
[0128] Methods by which to Detect Protein:Protein Binding in Assays
of the Invention
[0129] Methods of detecting the phosphorylation-dependent binding
of a natural binding domain, sequence or polypeptide and a binding
partner in an assay of the invention most usefully, although not
exclusively, are those which employ light-emitting labels. Several
such techniques are described below.
[0130] Fluorescence Energy Resonance Transfer (FRET)
[0131] A tool with which to assess the distance between one
molecule and another (whether protein or nucleic acid) or between
two positions on the same molecule is provided by the technique of
fluorescence resonance energy transfer (FRET), which is now widely
known in the art (for a review, see Matyus, 1992, J. Photochem.
Photobiol. B: Biol., 12: 323-337, which is herein incorporated by
reference). FRET is a radiationless process in which energy is
transferred from an excited donor molecule to an acceptor molecule;
the efficiency of this transfer is dependent upon the distance
between the donor an acceptor molecules, as described below. Since
the rate of energy transfer is inversely proportional to the sixth
power of the distance between the donor and acceptor, the energy
transfer efficiency is extremely sensitive to distance changes.
Energy transfer is said to occur with detectable efficiency in the
1-10 nm distance range, but is typically 4-6 nm for favorable pairs
of donor and acceptor.
[0132] Radiationless energy transfer is based on the biophysical
properties of fluorophores. These principles are reviewed elsewhere
(Lakowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum
Press, New York; Jovin and Jovin, 1989, Cell Structure and Function
by Microspectrofluorometry, eds. E. Kohen and J. G. Hirschberg,
Academic Press, both of which are incorporated herein by
reference). Briefly, a fluorophore absorbs light energy at a
characteristic wavelength. This wavelength is also known as the
excitation wavelength. The energy absorbed by a flurochrome is
subsequently released through various pathways, one being emission
of photons to produce fluorescence. The wavelength of light being
emitted is known as the emission wavelength and is an inherent
characteristic of a particular fluorophore. Radiationless energy
transfer is the quantum-mechanical process by which the energy of
the excited state of one fluorophore is transferred without actual
photon emission to a second fluorophore. That energy may then be
subsequently released at the emission wavelength of the second
fluorophore. The first fluorophore is generally termed the donor
(D) and has an excited state of higher energy than that of the
second fluorophore, termed the acceptor (A). The essential features
of the process are that the emission spectrum of the donor overlap
with the excitation spectrum of the acceptor, and that the donor
and acceptor be sufficiently close. The distance over which
radiationless energy transfer is effective depends on many factors
including the fluorescence quantum efficiency of the donor, the
extinction coefficient of the acceptor, the degree of overlap of
their respective spectra, the refractive index of the medium, and
the relative orientation of the transition moments of the two
fluorophores. In addition to having an optimum emission range
overlapping the excitation wavelength of the other fluorophore, the
distance between D and A must be sufficiently small to allow the
radiationless transfer of energy between the fluorophores.
[0133] FRET may be performed either in vivo or in vitro. Proteins
are labeled either in vivo or in vitro by methods known in the art.
According to the invention, a natural binding domain, sequence or
polypeptide and its binding partner, comprised either by the same
or by different polypeptide molecules, are differentially labeled,
one with a donor and the other with an acceptor, and differences in
fluorescence between a test assay, comprising a protein modifying
enzyme, and a control, in which the modifying enzyme is absent, are
measured using a fluorimeter or laser-scanning microscope. It will
be apparent to those skilled in the art that excitation/detection
means can be augmented by the incorporation of photomultiplier
means to enhance detection sensitivity. The differential labels may
comprise either two different fluorescent labels (e.g., fluorescent
proteins as described below or the fluorophores rhodamine,
fluorescein, SPQ, and others as are known in the art) or a
fluorescent label and a molecule known to quench its signal;
differences in the proximity of the natural binding domain,
sequence or polypeptide with its binding partner with and without
the protein-modifying enzyme can be gauged based upon a difference
in the fluorescence spectrum or intensity observed.
[0134] This combination of protein-labeling methods and devices
confers a distinct advantage over prior art methods for determining
the activity of protein-modifying enzymes, as described above, in
that results of all measurements are observed in real time (i.e.,
as a reaction progresses). This is significantly advantageous, as
it allows both for rapid data collection and yields information
regarding reaction kinetics under various conditions.
[0135] A sample, whether in vitro or in vivo, assayed according to
the invention therefore comprises a mixture at equilibrium of the
labeled natural binding domain, sequence or polypeptide and its
binding partner which, when disassociated from one another,
fluoresce at one frequency and, when complexed together, fluoresce
at another frequency or, alternatively, of molecules which either
do or do not fluoresce or show reduced fluorescence, depending upon
whether or not they are associated.
[0136] The natural binding domain, sequence or polypeptide is
modified to allow the attachment of a fluorescent label to the
surface of that molecule or is fused in-frame with a fluorescent
protein, as described below. The choice of fluorescent label will
be such that upon excitation with light, labeled peptides which are
associated will show optimal energy transfer between fluorophores.
In the presence of a protein kinase or phosphatase, the natural
binding domain, sequence or polypeptide and its binding partner
dissociate due to a structural or electrostatic change which occurs
as a consequence of addition or removal of a phosphate to/from the
enzyme recognition site, thereby leading to a decrease in energy
transfer and increased emission of light by the donor fluorophore.
In this way, the state of polypeptide phosphorylation can be
monitored and quantitated in real-time. This scheme, which
represents the broadest embodiment of the invention, is shown in
FIG. 2.
[0137] As used herein, the terms "fluorophore" and "fluorochrome"
refer interchangeably to a molecule which is capable of absorbing
energy at a wavelength range and releasing energy at a wavelength
range other than the absorbance range. The term "excitation
wavelength" refers to the range of wavelengths at which a
fluorophore absorbs energy. The term "emission wavelength" refers
to the range of wavelength that the fluorophore releases energy or
fluoresces.
[0138] A non-limiting list of chemical fluorophores of use in the
invention, along with their excitation and emission wavelengths, is
presented in Table 1.
1TABLE 1 Fluorophore Excitation (nm) Emission (nm) Color PKH2 490
504 green PKH67 490 502 green Fluorescein (FITC) 495 525 green
Hoechst 33258 360 470 blue R-Phycoerythrin (PE) 488 578 orange-red
Rhodamine (TRITC) 552 570 red Quantum Red .TM. 488 670 red PKH26
551 567 red Texas Red 596 620 red Cy3 552 570 red
[0139] Examples of fluorescent proteins which vary among themselves
in excitation and emission maxima are listed in Table 1 of WO
97/28261 (Tsien et al., 1997, supra). These (each followed by
[excitation max./emission max.] wavelengths expressed in
nanometers) include wild-type Green Fluorescent Protein
[395(475)/508] and the cloned mutant of Green Fluorescent Protein
variants P4 [383/447], P4-3 [381/445], W7 [433(453)/475(501)], W2
[432(453)/480], S65T [489/511], P4-1 [504(396)/480], S65A
[471/504], S65C [479/507], S65L [484/510], Y66F [360/442], Y66W
[458/480], I0c [513/527],W1B [432(453)/476(503)], Emerald [487/508]
and Sapphire [395/511]. This list is not exhaustive of fluorescent
proteins known in the art; additional examples are found in the
Genbank and SwissProt public databases.
[0140] A number of parameters of fluorescence output are envisaged
including
[0141] 1) measuring fluoresence emitted at the emission wavelength
of the acceptor (A) and donor (D) and determining the extent of
energy transfer by the ratio of their emission amplitudes;
[0142] 2) measuring the fluoresence lifetime of D;
[0143] 3) measuring the rate of photobleaching of D;
[0144] 4) measuring the anisotropy of D and/or A; or
[0145] 5) measuring the Stokes shift monomer; excimer
fluorescence.
[0146] Certain of these techniques are presented below.
[0147] Alternative Fluorescent Techniques Suitable for Monitoring
Protein:Protein Binding in Assays of the Invention
[0148] One embodiment of the technology can utilize monomer:excimer
fluorescence as the output. The association of a natural binding
domain with a binding partner in this format is shown in FIG.
3.
[0149] The fluorophore pyrene when present as a single copy
displays fluorescent emission of a particular wavelength
significantly shorter than when two copies of pyrene form a planar
dimer (excimer), as depicted. As above, excitation at a single
wavelength (probably 340 nm) is used to review the excimer
fluorescence (.about.470 nm) over monomer fluorescence (.about.375
nm) to quantify assembly:disassembly of the reporter molecule.
[0150] Additional embodiments of the present invention are not
dependent on FRET. For example the invention can make use of
fluorescence correlation spectroscopy (FCS), which relies on the
measurement of the rate of diffusion of a label (see Elson and
Magde, 1974 Biopolymers, 13: 1-27; Rigler et al., 1992, in
Fluorescence Spectroscopy: New Methods and Applications, Springer
Verlag, pp.13-24; Eigen and Rigler, 1994, Proc. Natl. Acad. Sci.
U.S.A., 91: 5740-5747; Kinjo and Rigler, 1995, Nucleic Acids Res.,
23: 1795-1799).
[0151] In FCS, a focused laser beam illuminates a very small volume
of solution, of the order of 10.sup.-15 liter, which at any given
point in time contains only one molecule of the many under
analysis. The diffusion of single molecules through the illuminated
volume, over time, results in bursts of fluorescent light as the
labels of the molecules are excited by the laser. Each individual
burst, resulting from a single molecule, can be registered.
[0152] A labeled polypeptide will diffuse at a slower rate if it is
large than if it is small. Thus, multimerized polypeptides will
display slow diffusion rates, resulting in a lower number of
fluorescent bursts in any given timeframe, while labeled
polypeptides which are not multimerized or which have dissociated
from a multimer will diffuse more rapidly. Binding of polypeptides
according to the invention can be calculated directly from the
diffusion rates through the illuminated volume.
[0153] Where FCS is employed, rather than FRET, it is not necessary
to label more than one polypeptide. Preferably, a single
polypeptide member of the multimer is labeled. The labeled
polypeptide dissociates from the multimer as a result of
modification, thus altering the FCS reading for the fluorescent
label.
[0154] A further detection technique which may be employed in the
method of the present invention is the measurement of
time-dependent decay of fluorescence anisotropy. This is described,
for example, in Lacowicz, 1983, Principles of Fluorescence
Spectroscopy, Plenum Press, New York, incorporated herein by
reference (see, for example, page 167).
[0155] Fluorescence anisotropy relies on the measurement of the
rotation of fluorescent groups. Larger multimers of polypeptides
rotate more slowly than monomers, allowing the formation of
multimers to be monitored.
[0156] Non-Fluorescent Detection Methods for Use in the
Invention
[0157] The invention may be configured to exploit a number of
non-fluorescent labels. In a first embodiment, the natural binding
domain and binding partner therefor form, when bound, an active
enzyme which is capable of participating in an enzyme-substrate
reaction which has a detectable endpoint. The enzyme may comprise
two or more polypeptide chains or regions of a single chain, such
that upon binding of the natural binding domain to the binding
partner, which are present either on two different polypeptide
chains or in two different regions of a single polypeptide, these
components assemble to form a functional enzyme. Enzyme function
may be assessed by a number of methods, including scintillation
counting and photospectroscopy. In a further embodiment, the
invention may be configured such that the label is a redox enzyme,
for example glucose oxidase, and the signal generated by the label
is an electrical signal.
[0158] Phosphorylation of the natural binding domain and,
optionally, its binding partner according to the invention is
required to inhibit binding and, consequently, enzyme component
assembly, thus reducing enzyme activity.
[0159] In another assay format, an enzyme is used together with a
modulator of enzyme activity, such as an inhibitor or a cofactor.
In such an assay, one of the enzyme and the inhibitor or cofactor
is an natural binding domain, the other its binding partner.
Binding of the enzyme to its inhibitor or cofactor results in
modulation of enzymatic activity, which is detectable by
conventional means (such as monitoring for the conversion of
substrate to product for a given enzyme).
[0160] Fluorescent Protein Labels in Assays of the Invention
[0161] In a FRET assay of the invention, the fluorescent protein
labels are chosen such that the excitation spectrum of one of the
labels (the acceptor) overlaps with the emission spectrum of the
excited fluorescent label (the donor). The donor label is excited
by light of appropriate intensity within the donor's excitation
spectrum. The donor then emits some of the absorbed energy as
fluorescent light and dissipates some of the energy by FRET to the
acceptor fluorescent label. The fluorescent energy it produces is
quenched by the acceptor fluorescent protein label. FRET can be
manifested as a reduction in the intensity of the fluorescent
signal from the donor, reduction in the lifetime of its excited
state, and re-emission of fluorescent light at the longer
wavelengths (lower energies) characteristic of the acceptor. When
the donor and acceptor labels become spatially separated, FRET is
diminished or eliminated.
[0162] One can take advantage of the FRET exhibited by a natural
binding domain, sequence or polypeptide and its binding partner
labeled with different fluorescent proteins, wherein one is linked
to a donor and the other to an acceptor fluorescent protein, in
monitoring protein phosphorylation according to the present
invention. A single polypeptide may comprises a blue fluorescent
protein donor and a green fluorescent protein acceptor, wherein
each is fused to a different assay component (i.e., in which one is
fused to the natural binding domain, sequence or polypeptide and
the other to its binding partner); such a construct is herein
referred to as a "tandem" fusion protein. Alternatively, two
distinct polypeptides ("single" fusion proteins) one comprising a
natural binding domain, sequence or polypeptide and the other its
binding partner may be differentially labeled with the donor and
acceptor fluorescent proteins, respectively. The construction and
use of tandem fusion proteins in the invention can reduce
significantly the molar concentration of peptides necessary to
effect an association between differentially-labeled polypeptide
assay components relative to that required when single fusion
proteins are instead used. The labeled natural binding domain,
sequence or polypeptide and/or its binding partner may be produced
via the expression of recombinant nucleic acid molecules comprising
an in-frame fusion of sequences encoding a such a polypeptide and a
fluorescent protein label either in vitro (e.g., using a cell-free
transcription/translation system, as described below, or instead
using cultured cells transformed or transfected using methods well
known in the art) or in vivo, for example in a transgenic animal
including, but not limited to, insects, amphibians and mammals. A
recombinant nucleic acid molecule of use in the invention may be
constructed and expressed by molecular methods well known in the
art, and may additionally comprise sequences including, but not
limited to, those which encode a tag (e.g., a histidine tag) to
enable easy purification, a secretion signal, a nuclear
localization signal or other primary sequence signal capable of
targeting the construct to a particular cellular location, if it is
so desired.
[0163] The means by which a natural binding domain, sequence or
polypeptide and its binding partner are assayed for association
using fluorescent protein labels according to the invention may be
briefly summarized as follows:
[0164] Whether or not the natural binding domain, sequence or
polypeptide and its binding partner are present on a single
polypeptide molecule, one is labeled with a green fluorescent
protein, while the other is preferably labeled with a red or,
alternatively, a blue fluorescent protein. Useful donor:acceptor
pairs of fluorescent proteins (see Tsien et al., 1997, supra)
include, but are not limited to:
[0165] Donor: S72A, K79R, Y145F, M153A and T203I (excitation
.lambda.395 nm; emission .lambda.511)
[0166] Acceptor: S65G, S72A, K79R and T203Y (excitation .lambda.514
nm; emission .lambda.527 nm), or
[0167] T203Y/S65G, V68L, Q69K or S72A (excitation .lambda.515 nm;
emission .lambda.527 nm).
[0168] An example of a blue:green pairing is P4-3 (shown in Table 1
of Tsien et al., 1997, supra) as the donor label and S65C (also of
Table 1 of Tsien et al., 1997, supra) as the acceptor label. The
natural binding domain, sequence or polypeptide and corresponding
binding partner are exposed to light at, for example, 368 nm, a
wavelength that is near the excitation maximum of P4-3. This
wavelength excites S65C only minimally. Upon excitation, some
portion of the energy absorbed by the blue fluorescent protein
donor is transferred to the acceptor through FRET if the natural
binding domain, sequence or polypeptide and its binding partner are
in close association. As a result of this quenching, the blue
fluorescent light emitted by the blue fluorescent protein is less
bright than would be expected if the blue fluorescent protein
existed in isolation. The acceptor (S65C) may re-emit the energy at
longer wavelength, in this case, green fluorescent light.
[0169] After phosphorylation or dephosphorylation of one or both of
the natural binding domain, sequence or polypeptide and its binding
partner by an kinase or phosphatase, respectively, the natural
binding domain, sequence or polypeptide and its binding partner
(and, hence, the green and red or, less preferably, green and blue
fluorescent proteins) physically separate or associate, accordingly
inhibiting or promoting FRET. For example, if activity of the
modifying enzyme results in dissociation of a protein:protein
dimer, the intensity of visible blue fluorescent light emitted by
the blue fluorescent protein increases, while the intensity of
visible green light emitted by the green fluorescent protein as a
result of FRET, decreases.
[0170] Such a system is useful to monitor the activity of enzymes
that phosphorylate or dephosphorylate the phosphorylation site of a
natural binding domain, sequence or polypeptide and, optionally,
its binding partner to which the fluorescent protein labels are
fused, as well as the activity of kinases or phosphatases or
candidate modulators of those enzymes.
[0171] In particular, this invention contemplates assays in which
the amount- or activity of a modifying enzyme in a sample is
determined by contacting the sample with a natural binding domain,
sequence or polypeptide and its binding partner,
differentially-labeled with fluorescent proteins, as described
above, and measuring changes in fluorescence of the donor label,
the acceptor label or the relative fluorescence of both. Fusion
proteins, as described above, which comprise either one or both of
the labeled natural binding domain, sequence or polypeptide and its
binding partner of an assay of the invention can be used for, among
other things, monitoring the activity of a protein kinase or
phosphatase inside the cell that expresses the recombinant tandem
construct or two different recombinant constructs.
[0172] Advantages of single- and tandem fluorescent
protein/polypeptides comprising a natural binding domain, sequence
or polypeptide fused to a fluorescent protein include the potential
to express the natural binding domain, sequence or polypeptide in
the cell (providing a convenient experimental format), the greater
extinction coefficient and quantum yield of many of these proteins
compared with those of the Edans fluorophore. Also, the acceptor in
such a construct or pair of constructs is, itself, a fluorophore
rather than a non-fluorescent quencher like Dabcyl. Alternatively,
in single-label assays of the invention, whether involving use of a
chemical fluorophore or a single fluorescent fusion construct, such
a non-fluorescent quencher may be used. Thus, the enzyme's
substrate (i.e., the natural binding domain and, optionally, the
corresponding binding partner), and reaction products (i.e., the
natural binding domain and, optionally, the corresponding binding
partner after modification) are both fluorescent but with different
fluorescent characteristics.
[0173] In particular, the substrate and modified products exhibit
different ratios between the amount of light emitted by the donor
and acceptor labels. Therefore, the ratio between the two
fluorescences measures the degree of conversion of substrate to
products, independent of the absolute amount of either, the optical
thickness of the sample, the brightness of the excitation lamp, the
sensitivity of the detector, etc. Furthermore, Aequorea-derived or
-related fluorescent protein labels tend to be protease resistant.
Therefore, they are likely to retain their fluorescent properties
throughout the course of an experiment.
[0174] Reporter Polypeptide Fusion Construct According to the
Invention
[0175] As stated above, recombinant nucleic acid constructs of
particular use in the invention are those which comprise in-frame
fusions of sequences encoding a natural binding domain, sequence or
polypeptide or a binding partner therefor and a fluorescent
protein. If a natural binding domain, sequence or polypeptide and
its binding partner are to be expressed as part of a single
polypeptide, the nucleic acid molecule additionally encodes, at a
minimum, a donor fluorescent protein fused to one, an acceptor
fluorescent protein label fused to the other, a linker that couples
the two and is of sufficient length and flexibility to allow for
folding of the polypeptide and pairing of the natural binding
domain, sequence or polypeptide with the binding partner, and gene
regulatory sequences operatively linked to the fusion coding
sequence. If single fusion proteins are instead encoded (whether by
one or more nucleic acid molecules), each nucleic acid molecule
need only encode a natural binding domain, sequence or polypeptide
or a binding partner therefor, fused either to a donor or acceptor
fluorescent protein label and operatively linked to gene regulatory
sequences.
[0176] "Operatively-linked" refers to polynucleotide sequences
which are necessary to effect the expression of coding and
non-coding sequences to which they are ligated. The nature of such
control sequences differs depending upon the host organism; in
prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, components whose presence can
influence expression, and can also include additional components
whose presence is advantageous, for example, leader sequences and
fusion partner sequences.
[0177] As described above, the donor fluorescent protein label is
capable of absorbing a photon and transferring energy to another
fluorescent label. The acceptor fluorescent protein label is
capable of absorbing energy and emitting a photon. If needed, the
linker connects the natural binding domain, sequence or polypeptide
and its binding partner either directly or indirectly, through an
intermediary linkage with one or both of the donor and acceptor
fluorescent protein labels. Regardless of the relative order of the
natural binding domain, sequence or polypeptide, its binding
partner and the donor and acceptor fluorescent protein labels on a
polypeptide molecule, it is essential that sufficient distance be
placed between the donor and acceptor by the linker and/or the
natural binding domain, sequence or polypeptide and its binding
partner to ensure that FRET does not occur unless the natural
binding domain, sequence or polypeptide and its binding partner
bind. It is desirable, as described in greater detail in
WO97/28261, to select a donor fluorescent protein with an emission
spectrum that overlaps with the excitation spectrum of an acceptor
fluorescent protein. In some embodiments of the invention the
overlap in emission and excitation spectra will facilitate FRET.
Such an overlap is not necessary, however, if intrinsic
fluorescence is measured instead of FRET. A fluorescent protein of
use in the invention includes, in addition to those with intrinsic
fluorescent properties, proteins that fluoresce due intramolecular
rearrangements or the addition of cofactors that promote
fluorescence.
[0178] For example, green fluorescent proteins ("GFPs") of
cnidarians, which act as their energy-transfer acceptors in
bioluminescence, can be used in the invention. A green fluorescent
protein, as used herein, is a protein that fluoresces green light,
and a blue fluorescent protein is a protein that fluoresces blue
light. GFPs have been isolated from the Pacific Northwest
jellyfish, Aequorea victoria, from the sea pansy, Renilla
reniformis, and from Phialidium gregarium. (Ward et al., 1982,
Photochem. Photobiol., 35: 803-808; Levine et al., 1982, Comp.
Biochem. Physiol.,72B: 77-85).
[0179] A variety of Aequorea-related GFPs having useful excitation
and emission spectra have been engineered by modifying the amino
acid sequence of a naturally occurring GFP from Aequorea victoria.
(Prasher et al., 1992, Gene, 111: 229-233; Heim et al., 1994, Proc.
Natl. Acad. Sci. U.S.A., 91: 12501-12504; PCT/US95/14692). As used
herein, a fluorescent protein is an Aequorea-related fluorescent
protein if any contiguous sequence of 150 amino acids of the
fluorescent protein has at least 85% sequence identity with an
amino acid sequence, either contiguous or non-contiguous, from the
wild-type Aequorea green fluorescent protein of SwissProt Accession
No. P42212. Similarly, the fluorescent protein may be related to
Renilla or Phialidium wild-type fluorescent proteins using the same
standards.
[0180] Aequorea-related fluorescent proteins include, for example,
wild-type (native) Aequorea victoria GFP, whose nucleotide and
deduced amino acid sequences are presented in Genbank Accession
Nos. L29345, M62654, M62653 and others Aequorea-related, engineered
versions of Green Fluorescent Protein, of which some are listed
above. Several of these, i.e., P4, P4-3, W7 and W2 fluoresce at a
distinctly shorter wavelength than wild type.
[0181] Recombinant nucleic acid molecules encoding single- or
tandem fluorescent protein/polypeptide comprising a natural binding
domain, sequence or polypeptide or a binding partner therefor fused
to a fluorescent protein useful in the invention may be expressed
for in vivo assay of the activity of a modifying enzyme on the
encoded products. Alternatively, the encoded fusion proteins may be
isolated prior to assay, and instead assayed in a cell-free in
vitro assay system, as described elsewhere herein.
[0182] Protein Phoshorylation in Assays of the Invention
[0183] As highlighted in the Background, the phosphorylation of
proteins is a frequent and important post-translational
modification of proteins. There are many examples of situations in
which dysfunction of the kinases and phosphatases mediating the
phosphorylation state of proteins can lead to disease. The methods
currently available to analyze the phosphorylation state each have
drawbacks, as described above. Assay formats of the invention, as
outlined in the following sections and in the Examples, below, will
allow monitoring of the phosphorylation state of a specific target
protein or activity of a specific kinase or phosphatase in real
time in the cell.
[0184] Three systems, presented in Examples 1 through 4, can be
used to exemplify in non-limiting fashion the phosphorylation
assay, each of which involves the interaction between a binding
domain, sequence or polypeptide and a binding partner, of which at
least the former comprises a modification site that serves as a
substrate for the protein kinases and phosphatases involved in the
system. At the present time, good structural information is
available for such interactions.
[0185] Methods for Detection of Protein Phosphorylation in Real
Time
[0186] A. In Vitro Protein Modification and Detection thereof
[0187] Modifying Enzymes
[0188] The invention requires the presence of a modifying enzyme
which catalyzes either the addition or removal of a modifying
group. A range of kinases, phosphatases and other modifying enzymes
are available commercially (e.g. from Sigma, St. Louis, Mo.;
Promega, Madison, Wis.; Boehringer Mannheim Biochemicals,
Indianapolis, Ind.; New England Biolabs, Beverly, Mass.; and
others). Alternatively, such enzymes may be prepared in the
laboratory by methods well known in the art.
[0189] The catalytic sub-unit of protein kinase A (c-PKA) can be
purified from natural sources (e.g. bovine heart) or from
cells/organisms engineered to heterologously express the enzyme.
Other isoforms of this enzyme may be obtained by these procedures.
Purification is performed as previously described from bovine heart
(Peters et al.,1977, Biochemistry, 16: 5691-5697) or from a
heterologous source (Tsien et al., WO92/00388), and is in each case
briefly summarized as follows:
[0190] Bovine ventricular cardiac muscle (2 kg) is homogenized and
then centrifuged. The supernatant is applied to a strong anion
exchange resin (e.g. Q resin, Bio-Rad) equilibrated in a buffer
containing 50 mM Tris-HCl, 10 mM NaCl, 4 mM EDTA pH 7.6 and 0.2 mM
2-mercaptoethanol. The protein is eluted from the resin in a second
buffer containing 50 mM Tris-HCl, 4 mM EDTA pH 7.6, 0.2 mM
2-mercaptoethanol, 0.5M NaCl. Fractions containing PKA are pooled
and ammonium sulphate added to 30% saturation. Proteins
precipitated by this are removed by centrifugation and the ammonium
sulphate concentration of the supernatant was increased to 75%
saturation. Insoluble proteins are collected by centrifugation
(included c-PKA) and are dissolved in 30 mM phosphate buffer pH
7.0, 1 mM EDTA, 0.2 mM 2-mercaptoethanol. These proteins are then
dialysed against the same buffer (500 volume excess) at 4.degree.
C. for two periods of 8 hours each. The pH of the sample is reduced
to 6.1 by addition of phosphoric acid, and the sample is mixed
sequentially with 5 batches of CM-Sepharose (Pharmacia, .about.80
ml resin each) equilibrated in 30 mM phosphate pH 6.1, 1 mM EDTA,
0.2 mM 2-mercaptoethanol. Cyclic AMP (10 .mu.M) is added to the
material which fails to bind to the CM-Sepharose, and the
sample-cAMP mix is incubated with a fresh resin of CM-Sepharose
(.about.100 ml) equilibrated as before. c-PKA is eluted from this
column following extensive washing in equilibration buffer by
addition of 30 mM phosphate pH 6.1, 1 mM EDTA, 1M KCl, 0.2 mM
2-mercaptoethanol. Fractions containing c-PKA are pooled and
concentrated by filtration through a PM-30 membrane (or similar).
The c-PKA sample is then subjected to gel-filtration chromatography
on a resin such as Sephacryl 200HR (Pharmacia).
[0191] The purification of recombinant c-PKA is as described in WO
92/00388. General methods of preparing pure and partially-purified
recombinant proteins, as well as crude cellular extracts comprising
such proteins, are well known in the art. Molecular methods useful
in the production of recombinant proteins, whether such proteins
are the enzymes to be assayed according to the invention or the
labeled reporter polypeptides of the invention (i.e., the natural
binding domain, sequence or polypeptide and its binding partner),
are well known in the art (for methods of cloning, expression of
cloned genes and protein purification, see Sambrook et al., 1989,
Molecular Cloning. A Laboratory Manual., 2nd Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al.,
Current Protocols in Molecular Biology, copyright 1987-1994,
Current Protocols, copyright 1994-1998, John Wiley & Sons,
Inc.). The sequences of the catalytic subunit of several PKA
molecules are found in the Genbank database (see PKA C.alpha.,
bovine, Genbank Accession Nos. X67154 and S49260; PKA C.beta.1,
bovine, Genbank Accession No. J02647; PKA C.beta.2, bovine, M60482,
the form most likely purified from bovine heart by the protocol
described above).
[0192] According to the invention, assays of the activity of
protein kinases or phosphatases may be performed using crude
cellular extracts, whether to test the activity of a recombinant
protein or one which is found in nature, such as in a biological
sample obtained from a test cell line or animal or from a clinical
patient. In the former case, use of a crude cell extract enables
rapid screening of many samples, which potentially finds special
application in high-throughput screening methods, e.g. of candidate
modulators of protein kinase/phosphatase activity. In the latter
case, use of a crude extract with the labeled reporter polypeptide
comprising a natural binding domain, sequence or polypeptide of the
invention facilitates easy and rapid assessment of the activity of
an enzyme of interest in a diagnostic procedure, e.g., one which is
directed at determining whether a protein kinase or phosphatase is
active at an a physiologically-appropriate level, or in a procedure
designed to assess the efficacy of a therapy aimed at modulating
the activity of a particular enzyme.
[0193] Production of a Natural Binding Domain, Sequence or
Polypeptide
[0194] Polypeptides comprising or consisting of a natural binding
domain, sequence or polypeptide or a binding partner thereof may be
synthesized by Fmoc or Tboc chemistry according to methods known in
the art (e.g., see Atherton et al., 1981, J. Chem. Soc. Perkin I,
1981(2): 538-546; Merrifield, 1963, J. Am. Chem. Soc., 85:
2149-2154, respectively). Following deprotection and cleavage from
the resin, peptides are desalted by gel filtration chromatography
and analysed by mass spectroscopy, HPLC, Edman degradation and/or
other methods as are known in the art for protein sequencing using
standard methodologies.
[0195] Alternatively, nucleic acid sequences encoding such peptides
may be expressed either in cells or in an in vitro
transcription/translation system (see below) and, as with enzymes
to be assayed according to the invention, the proteins purified by
methods well known in the art.
[0196] Labelling of Polypeptides with Fluorophores
[0197] Polypeptides comprising or consisting of natural binding
domains, sequences or polypeptides or a binding partner therefor
are labeled with thiol reactive derivatives of fluorescein and
tetramethylrhodamine (isothiocyanate or iodoacetamide derivatives,
Molecular Probes, Eugene, Oreg., USA) or other fluorophores as are
known in the art using procedures described by Hermanson G. T.,
1995, Bioconjugate Techniques, Academic Press, London.
Alternatively, primary-amine-directed conjugation reactions can be
used to label lysine sidechains or the free peptide N-terminus
(Hermason, 1995, supra).
[0198] Purification of Fluorescent Natural Binding Domains and/or
Binding Partners therefor
[0199] Fluorescent peptides are separated from unreacted
fluorophores by gel filtration chromatography or reverse phase
HPLC.
[0200] Phosphorylation of Natural Binding Domains and, Optionally,
Binding Partners therefor In Vitro
[0201] Natural binding domains and, optionally, binding partners
therefor (0.01-100.0 .mu.M) are phosphorylated by purified c-PKA in
50 mM Histidine buffer pH 7.0, 5 mM MgSO.sub.4, 1 mM EGTA, 0.1-10.0
.mu.M c-PKA, and 0.2 mM [.sup.32P] .gamma.-ATP (specific activity
.about.2 Bq/pmol) at 15-40.degree. C. for periods of time ranging
from 0 to 60 minutes. Where the chemistry of the peptide is
appropriate (i.e. having a basic charge) the phosphopeptide is
captured on a cation exchange filter paper (e.g. phosphocellulose
P81 paper; Whatman), and reactants are removed by extensive washing
in 1% phosphoric acid (see Casnellie, 1991, Methods Enzymol., 200:
115-120). Alternatively, phosphorylation of samples is terminated
by the addition of SDS-sample buffer (Laemmli,1970, Nature, 227:
680-685) and the samples analysed by SDS-PAGE electrophoresis,
autoradiography and scintillation counting of gel pieces.
[0202] Dephosphorylation of a Natural Binding Domain or Binding
Partner therefor In Vitro
[0203] The dephosphorylation of natural binding domains and,
optionally, binding partners therefor, phosphorylated as above is
studied by removal of ATP (through the addition of 10 mM glucose
and 30 U/ml hexokinase; Sigma, St. Louis, Mo.) and addition of
protein phosphatase-1 (Sigma). Dephosphorylation is followed at
15-40.degree. C. by quantitation of the remaining phosphopeptide
component at various time points, determined as above.
[0204] Fluorescence Measurements of Protein Modification In Vitro
in Real Time
[0205] Donor and acceptor fluorophore-labeled polypeptides
comprising or consisting of natural binding domains, sequences or
polypeptides (molar equivalents of fluorophore-labeled polypeptide
or molar excess of acceptor-labeled polypeptide) are first mixed
(if the natural binding domains, sequence or polypeptide and its
binding partner are present on separate polypeptides). Samples are
analyzed in a fluorimeter using excitation wavelengths relevant to
the donor fluorescent label and emission wavelengths relevant to
both the donor and acceptor labels. A ratio of emission from the
acceptor over that from the donor following excitation at a single
wavelength is used to determine the efficiency of fluorescence
energy transfer between fluorophores, and hence their spatial
proximity. Typically, measurements are performed at 0-37.degree. C.
as a function of time following the addition of the modifying
enzyme (and, optionally, a modulator or candidate modulator of
function for that enzyme, as described below) to the system in 50
mM histidine pH 7.0, 120 mM KCl, 5 mM MgSO.sub.4, 5 mM NaF, 0.05 mM
EGTA and 0.2 mM ATP. The assay may be performed at a higher
temperature if that temperature is compatible with the enzyme(s)
under study.
[0206] Alternative Cell-Free Assay System of the Invention
[0207] A cell-free assay system according to the invention is
required to permit binding of an unmodified, labeled natural
binding domain, sequence or polypeptide and its binding partner to
occur. As indicated herein, such a system may comprise a
low-ionic-strength buffer (e.g., physiological salt, such as simple
saline or phosphate- and/or Tris-buffered saline or other as
described above), a cell culture medium, of which many are known in
the art, or a whole or fractionated cell lysate. The components of
an assay of protein modification according to the invention may be
added into a buffer, medium or lysate or may have been expressed in
cells from which a lysate is derived. Alternatively, a cell-free
transcription- and/or translation system may be used to deliver one
or more of these components to the assay system. Nucleic acids of
use in cell-free expression systems according to the invention are
as described for in vivo assays, below.
[0208] An assay of the invention may be peformed in a standard in
vitro transcription/translation system under conditions which
permit expression of a recombinant or other gene. The TNT.RTM. T7
Quick Coupled Transcription/Translation System (Cat. # L1170;
Promega) contains all reagents necessary for in vitro
transcription/translation except the DNA of interest and the
detection label; as discussed below, polypeptides comprising
natural binding domains, sequences or polypeptides or their binding
partners may be encoded by expression constructs in which their
coding sequences are fused in-frame to those encoding fluorescent
proteins. The TNT.RTM. Coupled Reticulocyte Lysate Systems
(comprising a rabbit reticulocyte lysate) include: TNT.RTM. T3
Coupled Reticulocyte Lysate System (Cat. # L4950; Promega);
TNT.RTM. T7 Coupled Reticulocyte Lysate System (Cat. # L4610;
Promega); TNT.RTM. SP6 Coupled Reticulocyte Lysate System (Cat. #
L4600; Promega); TNT.RTM. T7/SP6 Coupled Reticulocyte Lysate System
(Cat. # L5020; Promega); TNT.RTM. T7/T3 Coupled Reticulocyte Lysate
System (Cat. # L5010; Promega).
[0209] An assay involving a cell lysate or a whole cell (see below)
may be performed in a cell lysate or whole cell preferably
eukaryotic in nature (such as yeast, fungi, insect, e.g.,
Drosophila), mouse, or human). An assay in which a cell lysate is
used is performed in a standard in vitro system under conditions
which permit gene expression. A rabbit reticulocyte lysate alone is
also available from Promega, either nuclease-treated (Cat. # L4960)
or untreated (Cat. # L4151).
[0210] Candidate Modulators of Protein Kinases and/or Phosphatases
to be Screened According to the Invention
[0211] Whether in vitro or in an in vivo system (see below), the
invention encompasses methods by which to screen compositions which
may enhance, inhibit or not affect (e.g., in a cross-screening
procedure in which the goal is to determine whether an agent
intended for one purpose additionally affects general cellular
functions, of which protein phosphorylation/dephosphorylation is an
example) the activity of a protein kinase or phosphatase.
[0212] Candidate modulator compounds from large libraries of
synthetic or natural compounds can be screened. Numerous means are
currently used for random and directed synthesis of saccharide,
peptide, and nucleic acid based compounds. Synthetic compound
libraries are commercially available from a number of companies
including Maybridge Chemical Co. (Trevillet, Cornwall, UK),
Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.),
and Microsource (New Milford, Conn.). A rare chemical library is
available from Aldrich (Milwaukee, Wis.). Combinatorial libraries
are available and can be prepared. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available from e.g., Pan Laboratories (Bothell,
Wash.) or MycoSearch (NC), or are readily produceable by methods
well known in the art. Additionally, natural and synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical, and biochemical means.
[0213] Useful compounds may be found within numerous chemical
classes, though typically they are organic compounds, including
small organic compounds. Small organic compounds have a molecular
weight of more than 50 yet less than about 2,500 daltons,
preferably less than about 750, more preferably less than about 350
daltons. Exemplary classes include heterocycles, peptides,
saccharides, steroids, and the like. The compounds may be modified
to enhance efficacy, stability, pharmaceutical compatibility, and
the like. Structural identification of an agent may be used to
identify, generate, or screen additional agents. For example, where
peptide agents are identified, they may be modified in a variety of
ways to enhance their stability, such as using an unnatural amino
acid, such as a D-amino acid, particularly D-alanine, by
functionalizing the amino or carboxylic terminus, e.g. for the
amino group, acylation or alkylation, and for the carboxyl group,
esterification or amidification, or the like.
[0214] Candidate modulators which may be screened according to the
methods of the invention include receptors, enzymes, ligands,
regulatory factors, and structural proteins. Candidate modulators
also include nuclear proteins, cytoplasmic proteins, mitochondrial
proteins, secreted proteins, plasmalemma-associated proteins, serum
proteins, viral antigens, bacterial antigens, protozoal antigens
and parasitic antigens. Candidate modulators additionally comprise
proteins, lipoproteins, glycoproteins, phosphoproteins and nucleic
acids (e.g., RNAs such as ribozymes or antisense nucleic acids).
Proteins or polypeptides which can be screened using the methods of
the present invention include hormones, growth factors,
neurotransmitters, enzymes, clotting factors, apolipoproteins,
receptors, drugs, oncogenes, tumor antigens, tumor suppressors,
structural proteins, viral antigens, parasitic antigens, bacterial
antigens and antibodies (see below).
[0215] Candidate modulators which may be screened according to the
invention also include substances for which a test cell or organism
might be deficient or that might be clinically effective in
higher-than-normal concentration as well as those that are designed
to eliminate the translation of unwanted proteins. Nucleic acids of
use according to the invention not only may encode the candidate
modulators described above, but may eliminate or encode products
which eliminate deleterious proteins. Such nucleic acid sequences
are antisense RNA and ribozymes, as well as DNA expression
constructs that encode them. Note that antisense RNA molecules,
ribozymes or genes encoding them may be administered to a test cell
or organism by a method of nucleic acid delivery that is known in
the art, as described below. Inactivating nucleic acid sequences
may encode a ribozyme or antisense RNA specific for the a target
MRNA. Ribozymes of the hammerhead class are the smallest known, and
lend themselves both to in vitro production and delivery to cells
(summarized by Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S;
Usman et al., 1996, Curr. Opin. Struct. Biol., 6: 527-533).
[0216] As stated above, antibodies are of use in the invention as
modulators (specifically, as inhibitors) of protein kinases and/or
phosphatases. Methods for the preparation of antibodies are well
known in the art, and are briefly summarized as follows:
[0217] Either recombinant proteins or those derived from natural
sources can be used to generate antibodies using standard
techniques, well known to those in the field. For example, the
proteins are administered to challenge a mammal such as a monkey,
goat, rabbit or mouse. The resulting antibodies can be collected as
polyclonal sera, or antibody-producing cells from the challenged
animal can be immortalized (e.g. by fusion with an immortalizing
fusion partner) to produce monoclonal antibodies.
[0218] 1. Polyclonal Antibodies.
[0219] The antigen protein may be conjugated to a conventional
carrier in order to increases its immunogenicity, and an antiserum
to the peptide-carrier conjugate is raised. Coupling of a peptide
to a carrier protein and immunizations may be performed as
described (Dymecki et al., 1992, J. Biol. Chem., 267: 4815-4823).
The serum is titered against protein antigen by ELISA (below) or
alternatively by dot or spot blotting (Boersma and Van Leeuwen,
1994, J. Neurosci. Methods, 51: 317). At the same time, the
antiserum may be used in tissue sections prepared as described
below. The serum is shown to react strongly with the appropriate
peptides by ELISA, for example, following the procedures of Green
et al., 1982, Cell, 28: 477-487.
[0220] 2. Monoclonal Antibodies.
[0221] Techniques for preparing monoclonal antibodies are well
known, and monoclonal antibodies may be prepared using a candidate
antigen whose level is to be measured or which is to be either
inactivated or affinity-purified, preferably bound to a carrier, as
described by Arnheiter et al., Nature, 294, 278-280 (1981).
[0222] Monoclonal antibodies are typically obtained from hybridoma
tissue cultures or from ascites fluid obtained from animals into
which the hybridoma tissue is introduced. Nevertheless, monoclonal
antibodies may be described as being "raised to" or "induced by" a
protein.
[0223] Monoclonal antibody-producing hybridomas (or polyclonal
sera) can be screened for antibody binding to the target protein.
By antibody, we include constructions using the binding (variable)
region of such an antibody, and other antibody modifications. Thus,
an antibody useful in the invention may comprise a whole antibody,
an antibody fragment, a polyfunctional antibody aggregate, or in
general a substance comprising one or more specific binding sites
from an antibody. The antibody fragment may be a fragment such as
an Fv, Fab or F(ab').sub.2 fragment or a derivative thereof, such
as a single chain Fv fragment. The antibody or antibody fragment
may be non-recombinant, recombinant or humanized. The antibody may
be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth. In
addition, an aggregate, polymer, derivative and conjugate of an
immunoglobulin or a fragment thereof can be used where
appropriate.
[0224] Determination of Activity of Candidate Modulator of a
Protein Kinase or Phosphatase
[0225] A candidate modulator of the activity of a protein kinase or
phosphatase may be assayed according to the invention as described
herein, is determined to be effective if its use results in a
difference of about 10% or greater relative to controls in which it
is not present (see below) in FRET resulting from the association
of a labeled natural binding domain, sequence or polypeptide and
its binding partner in the presence of a protein-modifying
enzyme.
[0226] The level of activity of a candidate modulator may be
quantified using any acceptable limits, for example, via the
following formula: 1 Percent Modulation = ( Index Control - Index
Sample ) ( Index Control ) .times. 100
[0227] where Index.sub.control is the quantitative result (e.g.,
amount of- or rate of change in fluorescence at a given frequency,
rate of molecular rotation, FRET, rate of change in FRET or other
index of modification, including, but not limited to, enzyme
inhibition or activation) obtained in assays that lack the
candidate modulator (in other words, untreated controls), and
Index.sub.sample represents the result of the same measurement in
assays containing the candidate modulator. As described below,
control measurements are made with differentially labeled natural
binding domains, sequences or polypeptides and their binding
partners only, and then with these molecules plus a protein kinase
or phosphatase which recognizes a phosphorylation site present on
them.
[0228] Such a calculation is used in either in vitro or in vivo
assays performed according to the invention.
[0229] B. In Vivo Assays of Enzymatic Activity According to the
Invention
[0230] Reporter Group Protein Modification in Living Cells
[0231] Differentially-labeled natural binding domains, sequences or
polypeptides and their corresponding binding partners of the
invention are delivered (e.g., by microinjection) to cells, such as
smooth muscle cells (DDT1) or ventricular cardiac myocytes as
previously described (Riabowol et al., 1988, Cold Spring Harbor
Symposia on Quantitative Biology, 53: 85-90). The ratio of emission
from the labeled molecule(s) is measured as described above via a
photomultiplier tube focused on a single cell. Activation of a
kinase (e.g., PKA by the addition of dibutyryl cAMP or
.beta.-adrenergic agonists) is performed, subsequent inhibition is
performed by removal of stimulus and by addition of a suitable
antagonist (e.g., cAMP antagonist Rp-cAMPS).
[0232] Heterologous Expression of Peptides
[0233] Natural binding domains, sequences or polypeptides and/or
their binding partners can be produced from the heterologous
expression of DNA sequences that encode them or by chemical
synthesis of the same. Expression can be in procaryotic or
eukaryotic cells using a variety of plasmid vectors capable of
instructing heterologous expression. Purification of these products
is achieved by destruction of the cells (e.g. French Press) and
chromatographic purification of the products. This latter procedure
can be simplified by the inclusion of an affinity purification tag
at one extreme of the peptide, separated from the peptide by a
protease cleavage site if necessary.
[0234] The Use of Cells or Whole Organisms in Assays of the
Invention
[0235] When performed using cells, the assays of the invention are
broadly applicable to a host cell susceptible to transfection or
transformation including, but not limited to, bacteria (both
gram-positive and gram-negative), cultured- or explanted plant
(including, but not limited to, tobacco, arabidopsis, carnation,
rice and lentil cells or protoplasts), insect (e.g., cultured
Drosophila or moth cell lines) or vertebrate cells (e.g., mammalian
cells) and yeast.
[0236] Organisms are currently being developed for the expression
of agents including DNA, RNA, proteins, non-proteinaceous
compounds, and viruses. Such vector microorganisms include bacteria
such as Clostridium (Parker et al., 1947, Proc. Soc. Exp. Biol.
Med., 66: 461-465; Fox et al., 1996, Gene Therapy, 3: 173-178;
Minton et al., 1995, FEMS Microbiol. Rev., 17: 357-364), Salmonella
(Pawelek et al., 1997, Cancer Res., 57: 4537-4544; Saltzman et al.,
1996, Cancer Biother. Radiopharm., 11: 145-153; Carrier et al.,
1992, J. immunol., 148: 1176-1181; Su et al., 1992, Microbiol.
Pathol., 13: 465-476; Chabalgoity et al., 1996, Infect. Immunol.,
65: 2402-2412), Listeria (Schafer et al., 1992, J. Immunol., 149:
53-59; Pan et al., 1995, Nature Med., 1: 471-477) and Shigella
(Sizemore et al., 1995, Science, 270: 299-302), as well as yeast,
mycobacteria, slime molds (members of the taxa Dictyosteliida--such
as of the genera Polysphondylium and Dictystelium, e.g.
Dictyostelium discoideum--and Myxomycetes--e.g. of the genera
Physarum and Didymium) and members of the Domain Arachaea
(including, but not limited to, archaebacteria), which have begun
to be used in recombinant nucleic acid work, members of the phylum
Protista, or other cell of the algae, fungi, or any cell of the
animal or plant kingdoms.
[0237] Plant cells useful in expressing polypeptides of use in
assays of the invention include, but are not limited to, tobacco
(Nicotiana plumbaginifolia and Nicotiana tabacum), arabidopsis
(Arabidopsis thaliana), Aspergillus niger, Brassica napus, Brassica
nigra, Datura innoxia, Vicia narbonensis, Vicia faba, pea (Pisum
sativum), cauliflower, carnation and lentil (Lens culinaris).
Either whole plants, cells or protoplasts may be transfected with a
nucleic acid of choice. Methods for plant cell transfection or
stable transformation include inoculation with Agrobacterium
tumefaciens cells carrying the construct of interest (see, among
others, Turpen et al., 1993, J. Virol. Methods, 42: 227-239),
administration of liposome-associated nucleic acid molecules
(Maccarrone et al., 1992, Biochem. Biophys. Res. Commun., 186:
1417-1422) and microparticle injection (Johnston and Tang, 1993,
Genet. Eng. (NY), 15: 225-236), among other methods. A generally
useful plant transcriptional control element is the cauliflower
mosaic virus (CaMV) 35S promoter (see, for example, Saalbach et
al., 1994, Mol. Gen. Genet., 242: 226-236). Non-limiting examples
of nucleic acid vectors useful in plants include pGSGLUC1 (Saalbach
et al., 1994, supra), pGA492 (Perez et al., 1989, Plant Mol. Biol.,
13: 365-373), pOCA18 (Olszewski et al., 1988, Nucleic Acids Res.,
16: 10765-10782), the Ti plasmid (Roussell et al., 1988, Mol. Gen.
Genet., 211: 202-209) and pKR612B1 (Balazs et al., 1985, Gene, 40:
343-348).
[0238] Mammalian cells are of use in the invention. Such cells
include, but are not limited to, neuronal cells (those of both
primary explants and of established cell culture lines) cells of
the immune system (such as T-cells, B-cells and macrophages),
fibroblasts, hematopoietic cells and dendritic cells. Using
established technologies, stem cells (e.g. hematopoietic stem
cells) may be used for gene transfer after enrichment procedures.
Alternatively, unseparated hematopoietic cells and stem cell
populations may be made susceptible to DNA uptake. Transfection of
hematopoietic stem cells is described in Mannion-Henderson et al.,
1995, Exp. Hematol., 23: 1628; Schiffmann et al., 1995, Blood, 86:
1218; Williams, 1990, Bone Marrow Transplant, 5: 141; Boggs, 1990,
Int. J. Cell Cloning, 8: 80; Martensson et al., 1987, Eur. J.
Immunol., 17: 1499; Okabe et al., 1992, Eur. J. Immunol., 22:
37-43; and Banerji et al., 1983, Cell, 33: 729. Such methods may
advantageously be used according to the present invention.
[0239] Nucleic Acid Vectors for the Expression of Assay Components
of the Invention in Cells or Multicellular Organisms
[0240] A nucleic acid of use according to the methods of the
invention may be either double- or single stranded and either naked
or associated with protein, carbohydrate, proteoglycan and/or lipid
or other molecules. Such vectors may contain modified and/or
unmodified nucleotides or ribonucleotides. In the event that the
gene to be transfected may be without its native transcriptional
regulatory sequences, the vector must provide such sequences to the
gene, so that it can be expressed once inside the target cell. Such
sequences may direct transcription in a tissue-specific manner,
thereby limiting expression of the gene to its target cell
population, even if it is taken up by other surrounding cells.
Alternatively, such sequences may be general regulators of
transcription, such as those that regulate housekeeping genes,
which will allow for expression of the transfected gene in more
than one cell type; this assumes that the majority of vector
molecules will associate preferentially with the cells of the
tissue into which they were injected, and that leakage of the
vector into other cell types will not be significantly deleterious
to the recipient organism. It is also possible to design a vector
that will express the gene of choice in the target cells at a
specific time, by using an inducible promoter, which will not
direct transcription unless a specific stimulus, such as heat
shock, is applied.
[0241] A gene encoding a component of the assay system of the
invention or a candidate modulator of protein kinase or phosphatase
activity may be transfected into a cell or organism using a viral
or non-viral DNA or RNA vector, where non-viral vectors include,
but are not limited to, plasmids, linear nucleic acid molecules,
artificial chromomosomes and episomal vectors. Expression of
heterologous genes in mammals has been observed after injection of
plasmid DNA into muscle (Wolff J. A. et al., 1990, Science, 247:
1465-1468; Carson D. A. et al., U.S. Pat. No. 5,580,859), thyroid
(Sykes et al., 1994, Human Gene Ther., 5: 837-844), melanoma (Vile
et al., 1993, Cancer Res., 53: 962-967), skin (Hengge et al., 1995,
Nature Genet., 10: 161-166), liver (Hickman et al., 1994, Human
Gene Therapy, 5: 1477-1483) and after exposure of airway epithelium
(Meyer et al., 1995, Gene Therapy, 2: 450-460).
[0242] In addition to vectors of the broad classes described above
and fusion gene expression construct encoding a natural binding
domain, sequence or polypeptide fused in-frame to a fluorescent
protein, as described above (see "Fluorescent resonance energy
transfer"), microbial plasmids, such as those of bacteria and
yeast, are of use in the invention.
[0243] Bacterial plasmids:
[0244] Of the frequently used origins of replication, pBR322 is
useful according to the invention, and pUC is preferred. Although
not preferred, other plasmids which are useful according to the
invention are those which require the presence of plasmid encoded
proteins for replication, for example, those comprising pT181, FII,
and FI origins of replication.
[0245] Examples of origins of replication which are useful in
assays of the invention in E. coli and S. typhimurium include but
are not limited to, pHETK (Garapin et al., 1981, Proc. Natl. Acad.
Sci. U.S.A., 78: 815-819), p279 (Talmadge et al., 1980, Proc. Natl.
Acad. Sci. U.S.A., 77: 3369-3373), p5-3 and p21A-2 (both from
Pawalek et al., 1997, Cancer Res., 57: 4537-4544), pMB1 (Bolivar et
al., 1977, Gene, 2: 95-113), ColE1 (Kahn et al., 1979, Methods
Enzymol., 68: 268-280), p15A (Chang et al., 1978, J. Bacteriol.,
134: 1141-1156); pSC101 (Stoker et al., 1982, Gene, 18: 335-341);
R6K (Kahn et al., 1979, supra); R1 (temperature dependent origin of
replication, Uhlin et al., 1983, Gene, 22: 255-265); lambda dv
(Jackson et al., 1972, Proc. Nat. Aca. Sci. U.S.A., 69: 2904-2909);
pYA (Nakayama et al., 1988, infra). An example of an origin of
replication that is useful in Staphylococcus is pT181 (Scott, 1984,
Microbial Reviews 48: 1-23). Of the above-described origins of
replication, pMB1, p15A and ColE1 are preferred because these
origins do not require plasmid-encoded proteins for
replication.
[0246] Yeast plasmids:
[0247] Three systems are used for recombinant plasmid expression
and replication in yeasts:
[0248] 1. Integrating. An example of such a plasmid is YIp, which
is maintained at one copy per haploid genome, and is inherited in
Mendelian fashion. Such a plasmid, containing a gene of interest, a
bacterial origin of replication and a selectable gene (typically an
antibiotic-resistance marker), is produced in bacteria. The
purified vector is linearized within the selectable gene and used
to transform competent yeast cells. Regardless of the type of
plasmid used, yeast cells are typically transformed by chemical
methods (e.g. as described by Rose et al., 1990, Methods in Yeast
Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.). The cells are treated with lithium acetate to achieve
transformation efficiencies of approximately 10.sup.4
colony-forming units (transformed cells)/.mu.g of DNA. Yeast
perform homologous recombination such that the cut, selectable
marker recombines with the mutated (usually a point mutation or a
small deletion) host gene to restore function. Transformed cells
are then isolated on selective media.
[0249] 2. Low copy-number ARS-CEN, of which YCp is an example. Such
a plasmid contains the autonomous replicating sequence (ARS1), a
sequence of approximately 700 bp which, when carried on a plasmid,
permits its replication in yeast, and a centromeric sequence
(CEN4), the latter of which allows mitotic stability. These are
usually present at 1-2 copies per cell. Removal of the CEN sequence
yields a YRp plasmid, which is typically present in 100-200 copes
per cell; however, this plasmid is both mitotically and meiotically
unstable.
[0250] 3. High-copy-number 2.mu. circles. These plasmids contain a
sequence approximately 1 kb in length, the 2.mu. sequence, which
acts as a yeast replicon giving rise to higher plasmid copy number;
however, these plasmids are unstable and require selection for
maintenance. Copy number is increased by having on the plasmid a
selection gene operatively linked to a crippled promoter. This is
usually the LEU2 gene with a truncated promoter (LEU2-d), such that
low levels of the Leu2p protein are produced; therefore, selection
on a leucine-depleted medium forces an increase in copy number in
order to make an amount of Leu2p sufficient for cell growth.
[0251] As suggested above, examples of yeast plasmids useful in the
invention include the YRp plasmids (based on
autonomously-replicating sequences, or ARS) and the YEp plasmids
(based on the 2.mu. circle), of which examples are YEp24 and the
YEplac series of plasmids (Gietz and Sugino, 1988, Gene, 74:
527-534). (See Sikorski, "Extrachromosomal cloning vectors of
Saccharomyces cerevisiae", in Plasmids, A Practical Approach, Ed.
K. G. Hardy, IRL Press, 1993; and Yeast Cloning Vectors and Genes,
Current Protocols in Molecular Biology, Section II, Unit 13.4,
Eds., Ausubel et al., 1994).
[0252] In addition to a yeast origin of replication, yeast plasmid
sequences typically comprise an antibiotic resistance gene, a
bacterial origin of replication (for propagation in bacterial
cells) and a yeast nutritional gene for maintenance in yeast cells.
The nutritional gene (or "auxotrophic marker") is most often one of
the following (with the gene product listed in parentheses and the
sizes quoted encompassing the coding sequence, together with the
promoter and terminator elements required for correct
expression):
[0253] TRP1 (PhosphoADP-ribosylanthranilate isomerase, which is a
component of the tryptophan biosynthetic pathway).
[0254] URA3 (Orotidine-5'-phosphate decarboxylase, which takes part
in the uracil biosynthetic pathway).
[0255] LEU2 (3-Isopropylmalate dehydrogenase, which is involved
with the leucine biosynthetic pathway).
[0256] HIS3 (Imidazoleglycerolphosphate dehydratase, or IGP
dehydratase).
[0257] LYS2 (.alpha.-aminoadipate-semialdehyde dehydrogenase, part
of the lysine biosynthetic pathway).
[0258] Alternatively, the screening system may operate in an
intact, living multicellular organism, such as an insect or a
mammal. Methods of generating transgenic Drosophila, mice and other
organisms, both transiently and stably, are well known in the art;
detection of fluorescence resulting from the expression of Green
Fluorescent Protein in live Drosophila is well known in the art.
One or more gene expression constructs encoding one or more of a
labeled natural binding domain, sequence or polypeptide, a binding
partner, a protein kinase or phosphatase and, optionally, a
candidate modulator thereof are introduced into the test organism
by methods well known in the art (see also below). Sufficient time
is allowed to pass after administration of the nucleic acid
molecule to allow for gene expression, for binding of a natural
binding domain, sequence or polypeptide to its binding partner and
for chromophore maturation, if necessary (e.g., Green Fluorescent
Protein matures over a period of approximately 2 hours prior to
fluorescence) before FRET is measured. A reaction component
(particularly a candidate modulator of enzyme function) which is
not administered as a nucleic acid molecule may be delivered by a
method selected from those described below.
[0259] Dosage and Administration of a Labeled Natural Binding
Domain Sequence or Polypeptide, Binding Partner Therefor, Protein
Kinase or Phosphatase or Candidate Modulator thereof for Use in an
In Vivo Assay of the Invention
[0260] Dosage
[0261] For example, the amount of each labeled natural binding
domain or binding partner therefor must fall within the detection
limits of the fluorescence-measuring device employed. The amount of
an enzmye or candidate modulator thereof will typically be in the
range of about 1 .mu.g-100 mg/kg body weight. Where the candidate
modulator is a peptide or polypeptide, it is typically administered
in the range of about 100-500 .mu.g/ml per dose. A single dose of a
candidate modulator, or multiple doses of such a substance, daily,
weekly, or intermittently, is contemplated according to the
invention.
[0262] A candidate modulator is tested in a concentration range
that depends upon the molecular weight of the molecule and the type
of assay. For example, for inhibition of protein/protein or
protein/DNA complex formation or transcription initiation
(depending upon the level at which the candidate modulator is
thought or intended to modulate the activity of a protein kinase or
phosphatase according to the invention), small molecules (as
defined above) may be tested in a concentration range of 1 pg-100
.mu.g/ml, preferably at about 100 pg-10 ng/ml; large molecules,
e.g., peptides, may be tested in the range of 10 ng-100 .mu.g/ml,
preferably 100 ng-10 .mu.g/ml.
[0263] Administration
[0264] Generally, nucleic acid molecules are administered in a
manner compatible with the dosage formulation, and in such amount
as will be effective. In the case of a recombinant nucleic acid
encoding a natural binding domain and/or binding partner therefor,
such an amount should be sufficient to result in production of a
detectable amount of the labeled protein or peptide, as discussed
above. In the case of a protein kinase or phosphatase, the amount
produced by expression of a nucleic acid molecule should be
sufficient to ensure that at least 10% of natural binding domains
or binding partners therefor will undergo modification if they
comprise a target site recognized by the enzyme being assayed.
Lastly, the amount of a nucleic acid encoding a candidate modulator
of a protein kinase or phosphatase of the invention must be
sufficient to ensure production of an amount of the candidate
modulator which can, if effective, produce a change of at least 10%
in the effect of the target protein kinase or phosphatase on FRET
or other label emission resulting from binding of a natural binding
domain to its binding partner or, if administered to a patient, an
amount which is prophylactically and/or therapeutically
effective.
[0265] When the end product (e.g. an antisense RNA molecule or
ribozyme) is administered directly, the dosage to be administered
is directly proportional to the amount needed per cell and the
number of cells to be transfected, with a correction factor for the
efficiency of uptake of the molecules. In cases in which a gene
must be expressed from the nucleic acid molecules, the strength of
the associated transcriptional regulatory sequences also must be
considered in calculating the number of nucleic acid molecules per
target cell that will result in adequate levels of the encoded
product. Suitable dosage ranges are on the order of, where a gene
expression construct is administered, 0.5-to 1 .mu.g, or 1-10
.mu.g, or optionally 10-100 .mu.g of nucleic acid in a single dose.
It is conceivable that dosages of up to 1 mg may be advantageously
used. Note that the number of molar equivalents per cell vary with
the size of the construct, and that absolute amounts of DNA used
should be adjusted accordingly to ensure adequate gene copy number
when large constructs are injected.
[0266] If no effect (e.g., of a protein kinase or phosphatase or an
inhibitor thereof) is seen within four orders of magnitude in
either direction of the starting dosage, it is likely that a
protein kinase or phosphatase does not recognize the target site of
the natural binding domain (and, optionally, its binding partner)
according to the invention, or that the candidate modulator thereof
is not of use according to the invention. It is critical to note
that when high dosages are used, the concentration must be kept
below harmful levels, which may be known if an enzyme or candidate
modulator is a drug that is approved for clinical use. Such a
dosage should be one (or, preferably, two or more) orders of
magnitude below the LD.sub.50 value that is known for a laboratory
mammal, and preferably below concentrations that are documented as
producing serious, if non-lethal, side effects.
[0267] Components of screening assays of the invention may be
formulated in a physiologically acceptable diluent such as water,
phosphate buffered saline, or saline, and further may include an
adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum
phosphate, aluminum hydroxide, or alum are materials well known in
the art. Administration of labeled polypeptides comprising a
natural binding domain, sequence, polypeptide or a binding partner
therefor, a protein kinase or phosphatase or a candidate modulator
as described herein may be either localized or systemic.
[0268] Localized administration:
[0269] Localized administration of a component of an assay of the
invention is preferably by via injection or by means of a drip
device, drug pump or drug-saturated solid matrix from which the
labeled natural binding domain, sequence or polypeptide, binding
partner therefor, protein kinase or phosphatase or candidate
modulator therefor, or nucleic acid encoding any of these can
diffuse implanted at the target site. When a tissue that is the
target of delivery according to the invention is on a surface of an
organism, topical administration of a pharmaceutical composition is
possible.
[0270] Compositions comprising a composition of- or of use in the
invention which are suitable for topical administration can take
one of several physical forms, as summarized below:
[0271] (i) A liquid, such as a tincture or lotion, which may be
applied by pouring, dropping or "painting" (i.e. spreading manually
or with a brush or other applicator such as a spatula) or
injection.
[0272] (ii) An ointment or cream, which may be spread either
manually or with a brush or other applicator (e.g. a spatula), or
may be extruded through a nozzle or other small opening from a
container such as a collapsible tube.
[0273] (iii) A dry powder, which may be shaken or sifted onto the
target tissue or, alternatively, applied as a nebulized spray.
[0274] (iv) A liquid-based aerosol, which may be dispensed from a
container selected from the group that comprises pressure-driven
spray bottles (such as are activated by squeezing), natural
atomizers (or "pump-spray" bottles that work without a compressed
propellant) or pressurized canisters.
[0275] (v) A carbowax or glycerin preparation, such as a
suppository, which may be used for rectal or vaginal administration
of a therapeutic composition.
[0276] In a specialized instance, the tissue to which a candidate
modulator of a protein kinase or phosphatase is to be delivered for
assay (or, if found effective, for therapeutic use) is the lung. In
such a case the route of administration is via inhalation, either
of a liquid aerosol or of a nebulized powder of. Drug delivery by
inhalation, whether for topical or systemic distribution, is well
known in the art for the treatment of asthma, bronchitis and
anaphylaxis. In particular, it has been demonstrated that it is
possible to deliver a protein via aerosol inhalation such that it
retains its native activity in vivo (see Hubbard et al., 1989, J.
Clin. Invest., 84: 1349-1354).
[0277] Systemic administration:
[0278] Systemic administration of a protein, nucleic acid or other
agent according to the invention may be performed by methods of
whole-body drug delivery are well known in the art. These include,
but are not limited to, intravenous drip or injection,
subcutaneous, intramuscular, intraperitoneal, intracranial and
spinal injection, ingestion via the oral route, inhalation,
trans-epithelial diffusion (such as via a drug-impregnated,
adhesive patch) or by the use of an implantable, time-release drug
delivery device, which may comprise a reservoir of
exogenously-produced protein, nucleic acid or other material or
may, instead, comprise cells that produce and secrete a natural
binding domain and/or a binding partner therefor, protein kinase or
phosphatase or candidate modulator thereof. Note that injection may
be performed either by conventional means (i.e. using a hypodermic
needle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol.
Rehabil., 14: 47-49). Components of assays of the invention can be
given in a single- or multiple dose.
[0279] Delivery of a nucleic acid may be performed using a delivery
technique selected from the group that includes, but is not limited
to, the use of viral vectors and non-viral vectors, such as
episomal vectors, artificial chromosomes, liposomes, cationic
peptides, tissue-specific cell transfection and transplantation,
administration of genes in general vectors with tissue-specific
promoters, etc.
[0280] Kits According to the Invention
[0281] A Kit for Assaying the Activity of a Protein Kinase or
Phosphatase
[0282] In order to facilitate convenient and widespread use of the
invention, a kit is provided which contains the essential
components for screening the activity of a protein kinase or
phosphatase, as described above. A natural binding domain, sequence
or polypeptide, as defined above, and its corresponding binding
partner are provided, as is a suitable reaction buffer for in vitro
assay or, alternatively, cells or a cell lysate. A reaction buffer
which is "suitable" is one which is permissive of the activity of
the enzyme to be assayed and which permits
phosphorylation-dependent binding of the natural binding domain to
the binding partner. The labeled polypeptide components are
provided as peptide/protein or a nucleic acid comprising a gene
expression construct encoding the one or more of a peptide/protein,
as discussed above. Natural binding domains, sequences and
polypeptides, as well as their corresponding binding partners, are
supplied in a kit of the invention either in solution (preferably
refrigerated or frozen) in a buffer which inhibits degradation and
maintains biological activity, or are provided in dried form, i.e.,
lyophilized. In the latter case, the components are resuspended
prior to use in the reaction buffer or other biocompatible solution
(e.g. water, containing one or more of physiological salts, a weak
buffer, such as phosphate or Tris, and a stabilizing substance such
as glycerol, sucrose or polyethylene glycol); in the latter case,
the resuspension buffer should not inhibit
phosphorylation-dependent binding of the natural binding domain,
sequence or polypeptide with the binding partner when added to the
reaction buffer in an amount necessary to deliver sufficient
protein for an assay reaction. Natural binding domains, sequences
or polypeptides or their binding partners provided as nucleic acids
are supplied- or resuspended in a buffer which permits either
transfection/transformation into a cell or organism or in vitro
transcription/translation, as described above. Each of these
components is supplied separately contained or in admixture with
one or more of the others in a container selected from the group
that includes, but is not limited to, a tube, vial, syringe or
bottle.
[0283] Optionally, the kit includes cells. Eukaryotic or
prokaryotic cells, as described above, are supplied in- or on a
liquid or solid physiological buffer or culture medium (e.g. in
suspension, in a stab culture or on a culture plate, e.g. a Petri
dish). For ease of shipping, the cells are typically refrigerated,
frozen or lyophilized in a bottle, tube or vial. Methods of cell
preservation are widely known in the art; suitable buffers and
media are widely known in the art, and are obtained from commerical
suppliers (e.g., Gibco/LifeTechnologies) or made by standard
methods (see, for example Sambrook et al., 1989, Molecular Cloning.
A Laboratory Manual., 2nd Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).
[0284] An enzyme being assayed according to the invention is added
to the assay system either as a protein (isolated,
partially-purified or present in a crude preparation such as a cell
extract or even a living cell) or a recombinant nucleic acid.
Methods of expressing a nucleic acid comprising an enzyme or other
protein are well known in the art (see again above).
[0285] An assay of the invention is carried out using the kit
according to the methods described above and in the Examples.
[0286] A Kit for Screening a Candidate Modulator of Protein Kinase
or Phosphatase Activity According to the Invention
[0287] A candidate modulator of post-translational phosphorylation
or dephosphorylation may be assayed using a kit of the invention. A
kit as described above is used for this application, with the assay
performed further comprising the addition of a candidate modulator
of the protein kinase or phosphatase which is present to the
reaction system. Optionally, a protein kinase or phosphatase is
supplied with the kit, either as a protein or nucleic acid as
described above.
[0288] Assays of protein activity are performed as described above.
At a minimum, three detections are performed, one in which the
natural binding domain and binding partner are present without the
protein kinase or phosphatase or candidate modulator thereof
(control reaction A), one in which the polypeptides are incubated
with the modifying enzyme under conditions which permit the
phosphorylation or dephosphorylation reaction to occur (control
reaction B) and one in which the protein kinase or phosphatase and
candidate modulator are both incubated with the labeled
polypeptides under conditions which permit the modification
reaction to occur (test reaction). In each case, conditions are
suitable to permit phosphorylation-dependent association of the
natural binding domain, sequence or polypeptide and the binding
partner. The result of the last detection procedure is compared
with those of the two controls; the candidate modulator is judged
to be efficacious if there is a shift in either of the observed
amount of signal (i.e., total amount- or rate of change of
fluorescence, FRET, mass of a protein complex or inhibition or
activation of an enzyme) of at least 10% away from that observed in
control reaction B toward that observed in control reaction A.
EXAMPLE 1
Use of a Polypeptide Comprising a Natural Binding Domain as a
Phosphorylation Reporter According to the Invention: Assay 1
[0289] An assay of this type involves the following components:
[0290] v-Src SH2 domain (amino acids 148-246; Waksman et al., 1993,
Cell, 72: 779-790; OWL database accession no. M33292), and
[0291] Hamster polyomavirus middle T antigen (Ag, below) (321-331,
EPQYEEIPIYL; Waksman et al., 1993, supra; OWL database accession
no. P03079).
[0292] SH2 domains are found in proteins involved in a number of
signalling pathways and their binding to specific phosphorylated
tyrosine residues is key in mediating the transmission of signals
between tyrosine kinases and the proteins in the cell which respond
to tyrosine phosphorylation (Waksman et al., 1993, supra and
references therein). Individual SH2 domains recognize specific
sequences, and the sequence specificity of a number of SH2 domains
has been determined (Songyang et al., 1993, supra) using a
phosphopeptide library. These data provide a number of possible
domain/peptide pairs which are useful in assays of enzymatic
activity according to the invention. The crystal structure of the
Src SH2 domain complexed with a peptide containing its specific
recognition motif from the hamster middle-T antigen (target
tyrosine for phosphorylation shown in bold above) has been
determined by Waksman et. al.(Cell 72, 779-790).
[0293] Thus, the assay is:
2 1
[0294] F1 is the donor fluorophore, F2 the acceptor fluorophore and
P denotes the addition of a phosphate group to the target tyrosine
residue.
[0295] The peptide as used in the crystallization described above
does not contain suitable residues for convenient labelling, and a
label within this short sequence is too close to the
phosphorylation site. A short linker (e.g., Gly-Gly) is, therefore,
added to either the C- or N-terminus of the peptide with a residue
such as Lys for labelling on the end. The location of this linker
will depend upon the location of F1 in the SH2 domain.
[0296] A number of potential locations for the fluorophore in the
SH2 domain have been identified based upon crystal structure:
3 SH2 Domain Middle T Ag. peptide K 232 C-terminal extension (G-G-K
or similar) R 217 C-terminal extension K 181 N-terminal extension R
156** N-terminal extension **this site is close to the site of
peptide interaction
[0297] If a fluorescent protein (e.g., Green Fluorescent Protein,
GFP) is used instead of a chemical fluorophore, it is placed at the
N-termini of both the SH2 domain and the peptide
EXAMPLE 2
Use of a Polypeptide Comprising a Natural Binding Domain as a
Phosphorylation Reporter According to the Invention: Assay 2
[0298] This assay involves the following components:
[0299] PTB domain of IRS-1 (amino acids 157-267) (Zhou et al.,
1996, Nature Structural Biology, 3: 388-393; OWL accession no.
P35568), and
[0300] Interleukin 4 Receptor (IL-4R) (amino acids 489-499,
LVIAGNPAYRS; Zhou et. al., 1996, supra; OWL accession no.
P24394)
[0301] Phosphotyrosine binding (PTB) domains are found in a number
of proteins involved in signalling pathways utilizing tyrosine
phosphorylation. The PTB domain has functional similarities to the
SH2 domain but differs in its mechanism of action and structure, as
well as in sequence recognition (Laminet et al., 1996, J. Biol.
Chem., 271: 264-269; Zhou et. al., 1996, supra and references
therein). These two classes of domain have little sequence
identity. NMR structural analysis of the PTB domain of IRS-1
complexed with the IL-4 receptor peptide has been performed (Zhou
et al., 1996, supra).
[0302] The assay format is as follows:
4 2
[0303] F1 is the donor fluorophore, F2 the acceptor fluorophore,
and P denotes the addition of a phosphate group to the target
tyrosine residue.
[0304] The peptide of the NMR study described above does not
contain suitable residues for convenient labelling except the
arginine next to the phosphorylation site, and a label within this
short sequence may be too close to the target site for
phosphorylation. A short linker may be added to either the C- or
N-terminus of the peptide with a residue for labelling on the end.
The location of such a linker depends upon the location of F1 in
the PTB domain.
[0305] Several potential locations for the fluorophore in the PTB
sequence have been identified from the NMR structure:
5 PTB domain IL-4R peptide K161 N-terminal extension (G-G-K or
similar) K190 N-terminal extension N-terminal extension N-terminal
extension C-terminal extension C-terminal extension
[0306] Again, if GFP is used in lieu of a chemical fluorophore, it
can be fused in-frame to either the N- or C-terminus of both the
PTB sequence and the binding partner.
EXAMPLE 3
[0307] An assay analogous to that in Example 2 can be configured
according to the invention using the PTB domain of the
proto-oncogene product Cb1 and a peptide derived from the Zap-70
tyrosine kinase. The Cb1 phosphotyrosine-binding domain selects a
D(N/D)XpY motif and binds to the Tyr.sub.292 negative regulatory
phosphorylation site of ZAP-70 (Lupher et al., 1997, J. Biol.
Chem., 272: 33140-33144).
[0308] The components of the assay are:
[0309] The Cb1 N-terminal domain (amino acids 1-357; Lupher et al.,
1996, J. Biol Chem., 271: 24063-24068; OWL accession no. P22681),
and
[0310] Zap-70 (amino acids 284-299, NH.sub.3-IDTLNSDGYTPEPARI-COOH;
Lupher et. al., 1996, supra; OWL accession no. P43403)
EXAMPLE 4
Use of a Polypeptide Comprising a Natural Binding Domain as a
Phosphorylation Reporter According to the Invention: Assay 4
[0311] This assay involves the following component--
[0312] c-Src (residues 86-536; Xu et al., 1997, Nature, 385:
595-602; GenBank Accession No. K03218).
[0313] As stated above, Src is a member of a family of non-receptor
tyrosine kinases involved in the regulation of responses to
extracellular signals. Association of src with both the plasma
membrane and intracellular membranes is mediated by myristoylation
at the N-terminus. The enzyme has four regions which are conserved
throughout the family, the SH2 domain, the SH3 domain, the kinase
or SH1 domain and the C-terminal tail. In addition there is a
unique region which does not have homology between family members
(Brown and Cooper, 1996, Biochim. Biophys. Acta, 1287:
121-149).
[0314] The SH2 domain binds tightly to specific tyrosine
phosphorylated sequences. This affinity plays a role in the
interaction between src and other cellular proteins and also in the
regulation of the kinase by phosphorylation. The C-terminal tail of
src can be phosphorylated on Tyr.sub.530, which phosphorylation
leads to almost complete inhibition of kinase activity. There is
strong evidence that this inhibition is achieved by the interaction
of the C-terminal tail with the SH2 domain. This interaction is
thought to promote a conformational change to the `closed`
conformation which is further stabilized by the participation of
the SH3 and kinase domains in intramolecular contacts.
[0315] The assay is diagramed as follows:
6 3
[0316] where F1 is the donor fluorophore, F2 is the acceptor
fluorophore and P denotes the addition of a phosphate group to the
target tyrosine residue.
[0317] There are several potential sites for labelling in this
structure. Some examples of target residues are shown below:
7 C-Terminal tail SH2 domain E527 D195, K198 C-Terminal extension
(eg. Gly-Gly-Lys) R220, K235,
[0318] When a fluorescent protein is used in an assay such as this,
using an intramolecular interaction to follow chemical
modification, it is appropriate to place GFP between domains using
a flexible linker to preserve protein domain interactions. This
allows the GFP variants to approach more closely and increase the
efficiency of the FRET achieved, but must be balanced by the need
to achieve a good distance between variants in the `No FRET` state.
If sufficient spacing between donor and acceptor fluorophores or,
alternatively, between a fluorophore or other label and a quencher
therefor, is not achieved in this manner, other candidate locations
for fluorescent protein fusion include, but are not limited to, the
C-terminus and the region between the SH2 domain and the SH2-kinase
linker.
EXAMPLE 5
Solution FRET Assay for Yersinia Tyrosine Phosphatase (YOP) Using a
Natural Binding Partner Labelled with a Fluorescent Protein and a
Synthetic Peptide Labelled with a Chemical Fluorophore
[0319] The following solution based assay was performed to detect
YOP activity by measuring disruption of a complex between a
fluorescently labelled SH2 domain of ZAP-70 and a synthetic peptide
based on the TCR.zeta. chain labelled with a chemical
fluorophore.
[0320] A FRET partnership is formed between the SH2 domain of
ZAP-70 and a phosphorylated peptide based on the TCR.zeta. chain,
providing both partners are labelled with suitable fluorophores.
Formation of FRET is followed in real-time by adding the two
binding partners together. Disruption of FRET can be achieved by
the addition of a phosphatase, which removes the phosphate required
for the interaction of the partners.
[0321] Methods:
[0322] TCR Peptide Sequence
[0323] Peptide 1. Phosphorylated TCR.zeta. chain:
[0324] RCKFSRSAEPPAYQQGQNQLY.sub.(p)NELNLGRREEY.sub.(p)DVLD
[0325] Peptide Labelling
[0326] Peptide 1 was labelled with rhodamine under mild conditions
using thiol directed chemistry. 230 .mu.M peptide was labelled in
20 mM TES pH 7 in the presence of a three-fold excess of
rhodamine-6-maleimide (Molecular Probes). Dialysis was utilised to
remove excess dye from the peptide. Labelling was verified by
MALDI-TOF MS.
[0327] ZAP-GFP Cloning and Purification
[0328] DNA Constructs
[0329] ZAP-GFP: Primers were designed based on the published ZAP-70
DNA sequence (Genbank accession number L05148). The SH2 domain
(amino acids 1-259) of ZAP70 was cloned by PCR using the following
oligo-nucleotides:
8 Forward primer GGGATCCATATGCCAGACCCCGCGGCGCACCTG Reverse Primer
GGAATTCGGGCACTGCTGTTGGGGCAGGCCTCC
[0330] The resultant PCR fragment was digested with BamHI and EcoRI
and inserted into pET28a (Novagen) to generate vector pFS45. DNA
encoding GFP in the vector pQBI25-FNI (Quantum) was digested with
MluI and the resultant 5' overhang was "filled in" using T4 DNA
polymerase (NEB). After the polymerase was denatured by heat
treatment the DNA was further digested with EcoRI and the resultant
850 bp band was gel purified. The vector pFS45 was digested with
HindIII and the resultant 5' overhang was "filled in" with T4 DNA
polymerase and then further digested with EcoRI. After the digested
vector was gel purified it was ligated with the purified DNA
encoding GFP to generate pFS46 which was designed to express a
ZAP70-GFP fusion protein in bacteria.
[0331] Expression and Purification Procedure
[0332] Fresh transformants of ZAP-GFP pET-28a in BRL(DE3) were used
to inoculate 3 ml LB/kanamycin (100 .mu.g/ml). The starter cultures
were incubated overnight at 37.degree. C. with constant shaking.
From these starter cultures 1 ml was used to inoculate 400 ml
Terrific Broth/kanamycin (100 .mu.g/ml) in a 2L, baffled flask.
Cultures were incubated at 37.degree. C. at 200 rpm for
approximately 5 hrs until the OD600 nm reached 0.5 Abs units. At
this point cultures were induced by adding IPTG to a concentration
of 1 mM. The cultures were then left incubating at room temperature
overnight with gentle shaking on a benchtop rotator. Bacteria were
harvested by centrifugation at 3000 rpm for 20 mins. The bacterial
pellet was resuspended in 25 ml lysis buffer (50 mM phosphate
buffer pH 7.0, 300 mM NaCl, 2% Proteinase inhibitor cocktail
(Sigma), 0.75 mg/ml Lysozyme). Lysis of the resuspended cells was
initiated by gentle stirring for 1 hr at room temperature. The
partially lysed mixture was subjected to 2 cycles of freeze thawing
in liquid nitrogen. Finally the cells were sonicated on ice using a
10 mm probe at high power. Sonication was performed on a pulse
setting for a period of 3 min. The crude lysate was then
centrifuged at 15,000. rpm for 30 mins to remove cell debris.
Hexa-His tagged proteins were purified from the clear lysate using
TALON.RTM. resin (Clontech). Proteins were bound to the resin in a
batchwise manner by gentle shaking at room temperature for 30 min.
Non-His tagged proteins were removed by washing the resin at least
twice with 10.times. bed volume of wash buffer (50 mM sodium
phosphate pH 7.0, 30 mM NaCl, 5 mM fluorescence-blank imidazole ).
The washed resin was loaded into a 2 ml column and the bound
proteins were released with elution buffer (50 mM sodium phosphate
pH 7.0, 300 mM NaCl, 150 mM fluorescence-blank Imidazole). Elution
was normally achieved after the first 0.5 ml and within 2-3 ml in
total. Proteins were stored at -80.degree. C. after snap freezing
in liquid nitrogen in the presence of 10% glycerol.
[0333] Formation of the FRET Partnership
[0334] ZAP-GFP was diluted to 0.5 .mu.M in YOP assay buffer (50 mM
Tris-HCl pH 7.2, 10 mM .beta.-mercaptoethanol, 0.5 mg/ml BSA,
0.015% Brij 35). 98 .mu.l of this solution was used per assay.
Initial readings of the fluorescence of the ZAP-GFP construct were
made using 485 nm excitation wavelength and 520 nm emission
wavelength. Rhodamine labelled peptide (2 .mu.l of a 115 .mu.M
peptide solution) was added to the ZAP-GFP and formation of FRET
followed in real-time by measuring the decrease in fluorescence
emission of ZAP-GFP at 520 nm.
[0335] YOP Source and Assay Conditions
[0336] Yersinia protein tyrosine phosphatase (YOP) was purchased
from Upstate Biotechnology. YOP, 3 units, was added to the
ZAP-GFP/peptide FRET mixture and the increase in fluorescence
emission at 520 nm, was followed in real-time as the partnership
was disrupted (FIG. 4). Dependence of YOP activity on TCR peptide
concentration and Km determination was measured using different
concentrations of rhodamine labelled TCR.zeta. peptide (FIG.
5).
[0337] The solution FRET assay for YOP was also used to determine
the IC.sub.50 of ortho vanadate, a general protein phosphatase
inhibitor. The YOP assay was performed as above, using 3 units of
YOP and incubating the enzyme with the ZAP-GFP/peptide FRET mixture
at 30.degree. C. Dephosphorylation was followed in real-time by
measuring the increase in fluorescence at 520 nm. Sodium
orthovanadate was added to the reaction mix prior to enzyme
addivity to give final concentrations of (0.03-30 .mu.M). Results
indicate an IC.sub.50 value for vanadate of 0.25 .mu.M and are
shown in FIG. 6.
EXAMPLE6
Assay of Chk1 Kinase Using a Solution Phase FP Assay with
Fluorescein Labelled CDC25 Derived Peptide Substrate and
14-3-3.zeta. Binding Partner
[0338] This assay was performed to measure Chk1 activity by
measuring the fluorescence polarisation change that occurs as a
result of the Chk1 protein kinase-mediated binding of fluorescein
labelled Chktide to 14-3-3 protein.
[0339] Chk1 protein kinase modifies the activity of CDC25
phosphatase via serine phosphorylation. Phosphorylation of CDC25
results in the binding of different isoforms of the 14-3-3 protein
and subsequent inhibition of CDC25 phosphatase activity. A peptide
derived from CDC25 (Chktide) and labelled with a chemical
fluorophore binds to 14-3-3 isoforms zeta and epsilon when
phosphorylated by Chk1 kinase. The activity of Chk1 is monitored by
following the fluorescence polarisation change when fluorescein
labelled Chktide binds to 14-3-3 protein.
[0340] Methods
[0341] Reagent Source
[0342] 14-3-3 .zeta. protein, Chk1 enzyme and Ckhtide peptide are
from Upstate Biotechnology.
[0343] Chktide , Chk1 substrate peptide sequence:
[0344] KKKVSRSGLYRSPS.sup.216MPENLNRPR
[0345] Chktide Labelling with Fluorescein
[0346] Chktide was labelled by incubating the peptide for 2 hours
at a concentration of 0.185 mM in 100 mM NaHCO.sub.3, pH 8.3, and
0.37 mM fluorescein-5EX (Molecular Probes) at room temperature. The
labelled peptide was then dialysed against 3 changes of 50 mM Tris
HCL pH 7.4, 150 mM NaCl (200 ml) for a total of 18 hours.
[0347] Phosphoryation of Chktide by Chk1 Protein Kinase.
[0348] A peptide substrate for Chk1 (Chktide) was labelled with
fluorescein. The labelled peptides were then phosphorylated by
incubating them at a concentration of 30 .mu.M for 30 min at
30.degree. C. in 20 mM MOPS pH 7.2, 10 mM MgCl.sub.2, 25 mM
.beta.-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM
DTT, 0.1 mM ATP, and 62 mU Chk1. Non phosphorylated control samples
were prepared by incubation of the labelled peptides under
identical conditions in the absence of Chk1. The peptides were then
incubated at a concentration of 5 .mu.M in 20 mM MOPS pH 7.2 at
30.degree. C., in a total volume of 30 .mu.l on a half area 96 well
plate. The fluorescence polarisation of the samples was measured at
520 nm (excitation 485 nm) following a 5 min equilibration. 14-3-3
.zeta. (5 .mu.M) was added to the peptide samples, and the
fluorescence polarisation at 520 nm (excitation 485 nm) was
monitored over time as shown in FIG. 7. Inhibition of Chk1 activity
by EDTA was measured by following the above procedure and adding
EDTA (10 mM or 20 mM) prior to enzyme addition, as shown in FIG.
8.
[0349] Activity of Chk1 was monitored in real time as follows.
Fluorescein labelled Chktide was incubated at 30.degree. C. in a
total volume of 30 .mu.l (on a half area 96 well plate) in 20 mM
MOPS pH 7.2, 10 mM MgCl.sub.2, 25 mM .beta.-glycerophosphate, 5 mM
EGTA, 1 mM sodium orthovanadate, 1 mM DTT, and 0.1 mM ATP in the
presence of 5 .mu.M 14-3-3 .zeta.. The samples were allowed to
equilibrate for 5 min, then Chk1 was added (or an equal volume of
H.sub.2O for control samples) and the fluorescence polarisation at
520 nm (excitation 485 nm) was monitored over time as shown in FIG.
9. Dependence of the increased fluorescence polarisation signal on
Chk1 activity and 14-3-3.zeta. binding was shown by the lack of
binding when ATP or 14-3-3.zeta. protein was omitted from the real
time enzyme reaction as shown in FIG. 10. Inhibition of Chk1
activity by EDTA was measured by following the above procedure and
adding EDTA (1 mM, 5 mM or 20 mM) prior to the addition of 21 mU of
Chk1 to start the reaction, as shown in FIG. 11. The fluorescence
polarisation for each sample was determined at the end of the
linear portion of the reaction.
[0350] Chk1 activity was also measured by monitoring the binding of
phosphorylated Chktide peptide to an alternate isoform of the
14-3-3 protein, 14-3-3.di-elect cons..
[0351] Production of 14-3-3 .di-elect cons.
[0352] Under the control of the T7 promoter, the vector FS121
contains DNA encoding the 14-3-3.di-elect cons. (Genbank accession
number U54778) protein fused in-frame to DNA encoding an amino
terminal hexa-His tag. Fresh transformants of pFS121 in BRL(DE3)
pLysS were used to inoculate 3 ml LB/ampicillin (100 .mu.g/ml). The
starter cultures was incubated overnight at 37.degree. C. with
shaking. From these starter cultures 1 ml was used to inoculate 400
ml Terrific Broth/ampicillin (100 .mu.g/ml) in a 2L, baffled flask.
Cultures were incubated at 37.degree. C. at 200 rpm for
approximately 4 hr until the OD.sub.600nm reached 0.5 Abs units. At
this point cultures were induced by adding IPTG to a concentration
of 1 mM and further incubated at 37.degree. C. for 4 hrs.
[0353] Bacteria were harvested by centrifugation at 3000 rpm for 20
min. The bacterial pellet was resuspended in 25 ml lysis buffer (50
mM Phosphate pH 7.0, 300 mM NaCl, 2% Proteinase inhibitor cocktail
(Sigrna), 0.75 mg/ml Lysozyme). Lysis of the resuspended cells was
initiated by gentle stirring for 30 min at room temperature.
Nonidet P-40 was added to a final concentration of 1% and lysis was
continued for an additional 20 min at room temperature. The
partially lysed mixture was subjected to 3 cycles of freeze thawing
in liquid nitrogen. Finally the cells were sonicated on ice using a
10 mm probe at high power. Sonication was performed on a pulse
setting for a period of 4 min. The crude lysate was centrifuged at
15,000 rpm for 30 min to remove cell debris. Hexa-His tagged
proteins were purified from the cleared lysate using TALON.RTM.
resin (Clontech). Proteins were bound to the resin in a batchwise
manner by gentle shaking at room temperature for 30 min. Non-His
tagged proteins were removed by washing the resin at least twice
with a 10.times. bed volume of wash buffer (50 mM sodium phosphate
pH 7.0, 300 mM NaCl, 5 mM fluorescence-blank Imidazole ). The
washed resin was loaded into a 2 ml column and the bound proteins
were released with elution buffer (50 mM sodium phosphate, pH 7.0,
300 mM NaCl, 150 mM fluorescence-blank Inidazole). Elution was
normally achieved within 5 ml. Purified proteins were stored at
-80.degree. C. after snap freezing in liquid nitrogen in the
presence of 10% glycerol.
[0354] End Point Assay of Chk1 Kinase by 14-3-3.di-elect cons.
Binding to Phosphorylated Chktide
[0355] A peptide substrate for Chk1 (Chktide) was labelled with
fluorescein. The labelled peptides were then phosphorylated by
incubating them at a concentration of 30 .mu.M for 30 min at
30.degree. C. in 100 .mu.l of 20 mM MOPS pH 7.2, 10 mM MgCl.sub.2,
25 mM .beta.-glycerophosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, 1 mM DTT, 0.1 mM ATP, and 62 mU Chk1.
Non-phosphorylated control samples were prepared by incubation of
the labelled peptides under identical conditions in the absence of
Chk1. The peptides were then incubated at a concentration of 7.5
.mu.M in 20 mM MOPS pH 7.2 at 30.degree. C., in a total volume of
30 .mu.l on a half area 96 well plate. The fluorescence
polarisation of the samples was measured at 520 nm (excitation 485
nm) following a 5 min equilibration. 14-3-3 .di-elect cons. (4
.mu.l) was added to the peptide samples, and the fluorescence
polarisation at 520 nm (excitation 485 nm) was monitored over time
as shown in FIG. 12.
EXAMPLE 7
Solution Phase FP Assay for the Detection of Phosphatase .lambda.
Activity Using a Fluorescein Labelled CDC25 Derived Peptide
Substrate and 14-3-3.zeta. Binding Partner
[0356] These assays were performed to demonstrate measurement of
phosphatase .lambda. activity as detected by a change in
fluorescence polarisation due to decreased binding of Chktide to
14-3-3 .zeta. or 14-3-3 .di-elect cons.. Binding is decreased as a
result of dephosphorylation of Chktide by phosphatase .lambda..
[0357] Reagents were obtained and prepared as in example 6
above.
[0358] Fluorescein labelled Chktide was phosphorylated by
incubation at a concentration of 30 .mu.M for 30 min at 30.degree.
C. in 20 mM MOPS pH 7.2, 10 mM MgCl.sub.2, 25 mM
.beta.-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM
DTT, 0.1 mM ATP, and 62 mU Chk1. Non-phosphorylated control samples
were prepared by incubation of the labelled peptides under
identical conditions in the absence of Chk1. The peptides were then
incubated at a concentration of 7.5 .mu.M in 20 mM MOPS pH 7.2, 2
mM MnCl.sub.2, 1 mM DTT at 30.degree. C., in a total volume of 30
.mu.l on a half area 96 well plate. The fluorescence polarisation
of the samples was measured at 520 nm (excitation 485 nm) following
a 5 min equilibration. 14-3-3 .zeta. (5 .mu.M) was added to the
peptide samples, and the fluorescence polarisation at 520 nm
(excitation 485 nm) was monitored over time as shown in FIG. 13.
Alternatively 14-3-3 .di-elect cons. (4 .mu.l of purified protein
prepared as in Example 6) was added to the peptide samples, and
after the fluorescence polarisation was stabilised, phosphatase
.lambda.(200U) was added. The fluorescence polarisation at 520 nm
(excitation 485 nm) was monitored over time as shown in FIG.
14.
EXAMPLE 8
Simultaneous Assay of Two Serine Threonine Kinases, Chk1 and cAMP
Dependent Protein Kinase (PKA) by FP
[0359] This assay was performed to simultaneously measure the
activity of Chk1 and PKA by monitoring a change in fluorescence
polarisation due to association or dissociation of peptides and/or
proteins capable of associating in a manner that is dependent upon
their phosphorylation state. Following phosphorylation by PKA,
coiled coil dimers can no longer form. Conversely, phosphorylation
of Chktide by Chk1 results in binding of Chktide to a 14-3-3
protein.
[0360] Serine threonine kinases PKA and Chk1 can be assayed
simultaneously using the natural binding partner reporters
described in example 5 for Chk1 and a peptide based binding partner
assay for PKA (described in WO99/11774).
[0361] Methods
[0362] PKA Peptide sequences:
[0363] Peptide 1. ERE IKALERE IRRLRRA SQALERE IAQLERE
[0364] Peptide 2. LRQR IQCLRYR IRRLRRA SQALRQR IAQLKQR
[0365] PKA peptides form coiled-coil dimers when they are
non-phosphorylated. Each monomer has a PKA phosphorylation site.
Following phosphorylation by PKA, the dimers can no longer form.
The association/disassociation can be measured using a fluorescence
polarisation assay, where PKA peptide 1 is labelled with coumarin,
and peptide 2 is labelled with biotin and bound to streptavidin.
The tumbling rate of coumarin labelled peptide 1 is higher after
PKA phosphorylation and dissociation from the dimer/streptavidin
complex. At the same time, the activity of Chk1 is measured by the
decrease in tumbling rate of phosphorylated fluorescein labelled
Chktide substrate binding to 14-3-3.di-elect cons. protein.
[0366] The labelled PKA peptides (both at a concentration of 2.5
.mu.M) were incubated at 30.degree. C. in a total volume of 50
.mu.l (on a half area 96 well plate) in 20 mM MOPS pH 7.2, 10 mM
MgCl.sub.2, 25 mM .beta.-glycerophosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, 1 mM DTT, and 0.1 mM ATP, 7.5 .mu.M Chktide, 3 .mu.L
14-3-3 .di-elect cons. and 0.06U streptavidin. The samples were
allowed to equilibrate for 5 min, then CHK1 (62 mU) and PKA (0.5
pmoles) were added (or an equal volume of H.sub.2O for control
samples). The fluorescence polarisation at 520 nm (excitation 485
nm) and at 450nm (excitation 340 nm) was monitored over time as
shown in FIG. 15.
EXAMPLE 9
FRET Assay of Src Using ZAP70-GFP Binding Partner and Synthetic
Rhodamine Labelled TCR.zeta. Substrate
[0367] These FRET-based assays were performed to detect Src
activity by measuring the Src dependent formation of a complex
between an SH2 domain of ZAP-70 labelled with a fluorophore and a
peptide based on the TCR.zeta. chain labelled with a
fluorophore.
[0368] A FRET partnership can be formed in a phosphorylation
dependent manner between the SH2 domain of ZAP-70 and a peptide
based on the TCR.zeta. chain, providing both binding partners are
labelled with suitable fluorophores. Src is used to phosphorylate
the TCR.zeta. chain derived substrate.
[0369] Methods:
[0370] TCR Pepide Sequence
[0371] Peptide 1. Phosphorylated TCR.zeta. chain:
[0372] RCKFSRSAEPPAYQQGQNQLY.sub.(p)NELNLGRREEY.sub.(p)DVLD
[0373] Peptide 2. Unphosphorylated TCR.zeta. chain:
[0374] RCKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLD
[0375] Peptide Labelling
[0376] Peptides 1 and 2 were labelled with rhodamine under mild
conditions using thiol directed chemistry. 230 .mu.M peptide was
labelled in 20 mM TES pH 7 in the presence of a three-fold excess
of rhodamine-6-maleimide (Molecular Probes). Dialysis was utilised
to remove excess dye from the peptide. Labelling was verified by
MALDI-TOF MS.
[0377] ZAP-GFP Cloning and Purification
[0378] ZAP-GFP was cloned and purified as described in Example
5.
[0379] Phosphorylation of Peptide 2
[0380] Peptide 2 labelled with rhodamine was phosphorylated using 3
units of Src kinase (Upstate Biotechnology) in a 40 .mu.l reaction
containing 115 .mu.M peptide, 50 mM Tris-HCl pH 7.2, 1 mM ATP, 10
mM MgCl.sub.2, 10 mM .beta.-mercaptoethanol, 0.1 mg/ml BSA and
0.015% (v/v) Brij 35, over a period of 2 hours at 37.degree. C.
Non-phosphorylated control samples were prepared under identical
conditions in the absence of the kinase.
[0381] Formation of the FRET Partnership
[0382] ZAP-GFP was diluted to 0.5 .mu.M in assay buffer (50 mM Tris
pH 7.2, 10 mM .beta.-mercaptoethanol, 0.5 mg/ml BSA, 0.015% Brij
35). 98 .mu.l of this solution was used per assay. Initial readings
of the fluorescence of the ZAP-GFP construct were made using 485 nm
excitation wavelength and 520 nm emission wavelength. Rhodamine
labelled peptide (2 .mu.l of above phosphorylation reactions) was
added to the ZAP-GFP solution and formation of FRET was followed in
real-time by measuring the decrease in fluorescence emission of
ZAP-GFP at 520 nm as shown in FIG. 16.
EXAMPLE 10
Solution Phase FRET Assay of Src Kinase Activity Using a Natural
Binding Partner SHP-2 Labelled with a Fluorescent Protein
[0383] These FRET-based assays are performed to measure Src kinase
activity as determined by the Src kinase dependent formation of a
complex between the SH2 domain of SHP2 labelled with a fluorophore
and an synthetic peptide based on SHPS-1, also labelled with a
fluorophore.
[0384] Interaction between the tandem SH2 domain of SHP2 and a
synthetic peptide based on SHPS-1 is mediated by the state of
phosphorylation of the peptide. A FRET partnership can be
established between phosphorylated peptide and the SH2 domain
providing both moieties are labelled with suitable fluorophores
which are brought into close proximity upon interaction of binding
partners.
[0385] Methods:
[0386] SHP2 Peptide Sequence and Fluorescent Labelling
[0387] SHPS-1 peptide sequence.
[0388] BiotinKQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSC
[0389] The SHPS-1 was labelled using thiol directed chemistry. 200
.mu.M peptide was reacted with 600 .mu.M rhodamine-6-maleimide
(Molecular Probes) in 20 mM TES pH 7 over a period of at least two
hours at room temperature. Excess label was removed using dialysis
and the labelling was verified by MALDI-TOF MS.
[0390] SHP2-GFP Cloning and Purification
[0391] Primers were based on the published SHP-2 DNA sequence
(Genbank accession number L03535. The SH2 domain (amino acids
1-225) of SHP-2 was cloned by PCR using the following
oligo-nucleotides:
9 5' primer- GGGGATCCTCTAGAATGACATCGCGGAGATGGTTTCACCC 3' primer-
GGGGAATTCTTTCAGCAGCATTTATACGAGTCG
[0392] The resultant PCR fragment was digested with XbaI and EcoRI,
gel purified and ligated into pET28a (Novagen) to generate vector
pFS 114. The validity of the construct was confirmed by sequence
analysis. DNA encoding GFP in the vector pFS46 was isolated by
digestion with the restriction enzymes EcoRI and XhoI and the
resultant 860 bp band was gel purified and ligated into pFS114 to
generate a bacterial expression vector for production of the fusion
protein SHP2-GFP (pFS115).
[0393] The hexa-His tagged SHP2-GFP fusion protein was expressed
and purified using TALON.RTM. resin according to standard
procedures.
[0394] Phosphorylation of Peptide SHPS-1 Peptide
[0395] SHPS-1 peptide labelled with rhodamine was phosphorylated
using 3 units of Src kinase (Upstate Biotechnology) in a 40 .mu.l
reaction containing 100 .mu.M peptide, 50 mM Tris-HCl pH 7.2, 1 mM
ATP, 10 mM MgCl.sub.2, 10 mM .beta.-mercaptoethanol, 0.1 mg/ml BSA
and 0.015% (v/v) Brij 35, over a period of 2 hours at 37.degree. C.
Non-phosphorylated control samples were prepared under identical
conditions in the absence of the kinase.
[0396] Formation of the FRET Partnership
[0397] SHP2-GFP was diluted to 0.5 .mu.M in assay buffer (50 mM
Tris HCL pH 7.2, 10 mM .beta.-mercaptoethanol, 0.5 mg/ml BSA,
0.015% Brij 35). 98 .mu.l of this solution was used per assay.
Initial readings of the fluorescence of the SHP2-GFP construct were
made using 485 nm excitation wavelength and 520 nm emission
wavelength. Rhodamine labelled peptide (2 .mu.l of the above
phosphorylation reactions) was added to the SHP2-GFP solution and
formation of FRET was monitored in real-time by measuring the
decrease in fluorescence emission of SHP2-GFP at 520 nm as shown in
FIG. 17.
[0398] Disruption of the FRET Partnership
[0399] The FRET partnership described in the above section was
disrupted easily using the tyrosine phosphatase enzyme, YOP
(Upstate Biotechnology). FRET partnership was formed as in the
above example, using 4 .mu.l of phosphorylated or control
non-phosphorylated SHPS-1 peptide. Addition of 3 units of YOP to
the FRET partnership resulted in disruption of the FRET partnership
as the phosphotyrosines required for the formation of the
partnership were removed (shown in FIG. 18).
[0400] Inhibition by Staurosporine
[0401] Phosphorylation of the peptide was prevented by inhibiting
the enzyme, Src, with the potent kinase inhibitor staurosporine.
Phosphorylation of the rhodamine labelled SHPS-1 peptide was
performed as described above in the presence or absence of 10 .mu.M
staurosporine added to the reaction prior to the addition of the
Src enzyme. The SH2 domain of SHP2 and the SHPS-1 peptide failed to
form a FRET partnership in the presence of the inhibitor, as shown
in FIG. 19.
[0402] Assay of Src Phosphorylation of SHPS-1 Peptide in
Real-Time
[0403] Phosphorylation of the rhodamine labelled SHPS-1 peptide and
formation of the FRET partnership with SHP2-GFP was followed in
real-time by measuring the decrease in fluorescence emission at 520
nm. Reactions containing 0.5 .mu.M SHP2-GFP, 50 mM Tris-HCl pH 7.2,
1 mM ATP, 10 mM MgCl.sub.2, 10 mM .beta.-mercaptoethanol, 0.5 mg/ml
BSA, 0.015% Brij 35 and 40 .mu.M peptide were set up in a black
microtitre plate. An initial equilibrium measurement was made
before adding 6 units of Src kinase, or buffer to control wells.
The decrease in fluorescence emission at 520 nm was followed in
real-time at 37.degree. C. as shown in FIG. 20.
EXAMPLE 11
In Vivo Measurement of Kinase or Phosphatase Activity
[0404] The enzymatic activity of a kinase or phosphatase enzyme is
measured in an in vivo assay performed as follows.
[0405] In vivo assays are carried out by transfecting cells with a
first expression construct encoding a fusion protein comprising a
polypeptide comprising a natural binding domain and further
comprising a site for phosphorylation fused in frame to a
fluorescent protein and a second expression construct comprising a
polypeptide comprising a binding partner for the natural binding
domain fused in frame to a second fluorescent protein.
Alternatively, cells are transfected with a tandem construct
encoding a fusion protein comprising a natural binding domain that
includes a site for phosphorylation and a binding partner for the
natural binding domain, and two different fluorescent proteins. For
all experiments, binding of a natural binding domain to its binding
partner is dependent on phosphorylation or dephosphorylation.
[0406] Plasmids encoding the autofluorescent proteins (AFPs) red
shifted green fluorescent protein (rsGFP) and blue fluorescent
protein (BFP) are purchased from Quantum Biotechnologies, Inc. BFP
is a mutated version of the 28 kDa rsGFP. BFP has an excitation
peak of 387 nm and an emission peak of 450 nm. GFP has an
excitation peak of 473 nm and an emission peak of 509 nm.
[0407] Constructs
[0408] DNA primers are designed encoding a first polypeptide
comprising a natural binding and a phosphorylation site domain or a
second polypeptide comprising a binding partner for the first
polypeptide, a stop codon and unique restriction sites (e.g. BamHI
and EcoRI) at each end to facilitate cloning. Codon usage is
selected in order to allow both mammalian and bacterial expression.
Alternatively, DNA primers are designed to encode a polypeptide
comprising both a natural binding domain that includes a
phosphorylation site and a binding partner for the natural binding
domain.
[0409] Experiments are performed using the following pair of
polypeptides:
[0410] 1. v-SRC SH2 domain (amino acids 148-246; Waksman et al.,
supra; OWL database accession no. M33292 and hamster polyomavirus
middle T antigen (Ag) (321-331, EPQYEEIPIYL), Waksman et al.,
supra; OWL database accession no. P03079,
[0411] 2. Phosphotyrosine binding domain (PTB) of IRS-1 (amino
acids 157-267) Zhou et al., supra; OWL accession no. P35568 and
interleukin 4 receptor (I1-4R) (amino acids 489-499, LVIAGNPAYRS;
Zhou et al., supra; OWL database accession no. P24394, and
[0412] 3. The PTB domain of the proto-oncogene product Cb1 (the Cb1
N-terminal binding domain) (amino acids 1-357); Lupher et al.,
supra; OWL accession no. P22681 and a peptide derived from the
Zap-70 tyrosine kinase (amino acids 284-299,
NH.sub.3-IDTLNSDGYtpepARI-COOH); Lupher et al., supra;.OWL
accession no. P43403.
[0413] 4. SH2 domain of ZAP70 (residues 1-259), GenBank accession
No. L05148. Tandem phosphorylation motif of TCRseta chain (residues
52-163) GenBank accession No. J04132.
[0414] Experiments are also performed using a construct encoding a
polypeptide comprising a natural binding domain including a site
for phosphorylation and further comprising a natural binding
partner for the natural binding domain.
[0415] These experiments are performed using the polypeptide c-SRC
(residues 86-536); Xu et al., supra; GenBank Accession No.
K03218.
[0416] AFP-Polypetide Construction
[0417] The purified DNA fragment, isolated by PCR is digested with
the appropriate enzymes that cleave at the unique restriction sites
located at each end and purified as above prior to ligation into
the mammalian expression vectors pQBI25-fc1 and pQBI50-fc1.
[0418] The v-SRC-SH2 domain and the polyomavirus middle T-antigen
peptide are cloned such that the AFP is placed at the N-termini.
The AFPs can be fused either to the N or C-termini of the PTB
domain of IRS-1 and the IL-4R peptide.
[0419] In the case of the tandem construct expressing the c-SRC
polypeptide which includes both a natural binding domain, including
a site of phosphorylation and a natural binding partner for the
natural binding domain, chemical modification by the kinase or
phosphatase enzyme being assayed is monitored by measuring a change
in an intramolecular reaction. A nucleic acid encoding a natural
binding domain including a site of phosphorylation and its binding
partner to be expressed as part of a single-polypeptide,
additionally encodes, at a minimum, a donor AFP fused to the
natural binding domain and an acceptor AFP fused to its binding
partner, a linker that couples the two AFPs and is of sufficient
length and flexibility to allow for folding of the polypeptide and
pairing of the natural binding domain, sequence or polypeptide with
the binding partner, and gene regulatory sequences operatively
linked to the fusion coding sequence.
[0420] To prepare a construct encoding a polypeptide comprising a
natural binding domain and further comprising a natural binding
partner for the natural binding domain and two different AFP
proteins, the purified DNA (prepared as above) is ligated into a
vector encoding an AFP. A fragment encoding the polypeptide plus an
AFP protein is excised from the vector and ligated into a second
vector encoding a linker and a second AFP. Alternatively, the
purified DNA (prepare as above) is ligated into a vector encoding
an AFP. A DNA fragment encoding a linker and a second AFP is
ligated into the above construct (by digestion with appropriate
restriction enzymes) resulting in a construct encoding a
polypeptide comprising a natural binding domain, a natural binding
partner for the natural binding domain and two AFPs separated by a
linker.
[0421] FRFT in Mammalian Cells
[0422] Experiments are performed using pairs of vectors expressing
the following proteins: a polypeptide comprising a natural binding
domain including a phosphorylation site and an AFP and a
polypeptide comprising a natural binding partner of the natural
binding domain and a second AFP.
[0423] Vectors capable of expressing these proteins are transfected
into COS-7 cells (a well-established cell-line derived from monkey
kidney cells) individually and in combination. Transfections are
performed using Lipofectamine 2000 (GibcoBRL) and the transfected
cells are incubated at 37.degree. C. for 48 hr (to allow the
expressed proteins to accumulate to a detectable level) in the
presence or absence of a kinase activator, a candidate modulator of
kinase activity or both a kinase activator and a candidate
modulator of kinase activity. Alternatively, the transfected cells
are incubated in the presence or absence of a phosphatase
activator, a candidate modulator of phosphatase activity or both a
phosphatase activator and a candidate modulator of phosphatase
activity.
[0424] Additional experiments are performed (using the transfection
protocol described above) using a tandem vector expressing a
polypeptide comprising a natural binding domain including a
phosphorylation site, a natural binding partner of the natural
binding domain and two different AFPs separated by an appropriate
linker. As above, transfected cells are incubated for 48 hrs in the
presence or absence of a kinase, a candidate modulator of kinase
activity, both a kinase and a candidate modulator of kinase
activity, a phosphatase, a candidate modulator of phosphatase
activity or both a phosphatase and a candidate modulator of
phosphatase activity.
[0425] Following the 48 hr incubation period the amount of FRET is
determined by analysis in a BMG Galaxy fluorescent plate reader
using the following regime: excitation at 370 nm (excitation for
BFP) and emission at 520 nm (emission for GFP). USE
[0426] The invention is useful in monitoring the activity of a
protein kinase or phosphatase, whether the protein is isolated,
partially-purified, present in a crude preparation or present in a
living cell. The invention is further useful in assaying a cell or
cell extract for the presence- or level of activity of a protein
kinase or phosphatase. The invention is additionally useful in
assaying the activity of naturally-occurring (mutant) or
non-natural (engineered) isoforms of known protein kinases and/or
phosphatases or, instead, that of novel (natural or non-natural)
enzymes. The invention is of use in assaying the efficacy of
candidate modulators of the activity of a protein kinase or
phosphatase in inhibiting or enhancing the activity of that enzyme;
moreover, is useful to screen potential therapeutic drugs for
activity against cloned and/or purified enzymes that may have
important clinical pathogenicities when mutated. The invention is
further of use in the screening of a candidate bioactive agent
(e.g., drugs) for side effects, whereby the ability of such an
agent to modulate the activity of a protein kinase or phosphatase
may be indicative a propensity toward provoking unintended
side-effects to a therapeutic or other regimen in which that agent
might be employed.
OTHER EMBODIMENTS
[0427] Other embodiments will be evident to those of skill in the
art. It should be understood that the foregoing description is
provided for clarity only and is merely exemplary. The spirit and
scope of the present invention are not limited to the above
examples, but are encompassed by the following claims.
* * * * *