U.S. patent application number 09/884681 was filed with the patent office on 2002-05-23 for assays for protein kinases using fluorescent protein substrates.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Cubitt, Andrew B., Tsien, Roger Y..
Application Number | 20020061546 09/884681 |
Document ID | / |
Family ID | 24728690 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061546 |
Kind Code |
A1 |
Tsien, Roger Y. ; et
al. |
May 23, 2002 |
Assays for protein kinases using fluorescent protein substrates
Abstract
This invention provides assays for protein kinase activity using
fluorescent proteins engineered to include sequences that can be
phosphorylated by protein kinases. The proteins exhibit different
fluorescent properties in the non-phosphorylated and phosphorylated
states.
Inventors: |
Tsien, Roger Y.; (La Jolla,
CA) ; Cubitt, Andrew B.; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
24728690 |
Appl. No.: |
09/884681 |
Filed: |
June 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09884681 |
Jun 19, 2001 |
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09263975 |
Mar 5, 1999 |
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6248550 |
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09263975 |
Mar 5, 1999 |
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08679865 |
Jul 16, 1996 |
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5912137 |
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Current U.S.
Class: |
435/15 ;
435/194 |
Current CPC
Class: |
C07K 14/43595 20130101;
C12Q 1/48 20130101; G01N 2333/91205 20130101; G01N 33/582
20130101 |
Class at
Publication: |
435/15 ;
435/194 |
International
Class: |
C12Q 001/48; C12N
009/12 |
Claims
What is claimed:
1. A method for determining whether a sample contains protein
kinase activity comprising; contacting the sample with a phosphate
donor and a fluorescent protein substrate for a protein kinase, the
fluorescent protein substrate comprising a fluorescent protein
moiety and a phosphorylation site for a protein kinase, wherein the
fluorescent protein substrate exhibits a different fluorescent
property in the phosphorylated state than in the un-phosphorylated
state wherein the fluorescent protein moiety is a green fluorescent
protein (SEQ. ID. NO: 2), and wherein the phosphorylation site is
within ten amino acids of the terminus of the fluorescent protein
moiety; exciting the protein substrate; and measuring the amount of
a fluorescent property of the fluorescent protein substrate that
differs in the unphosphorylated state and phosphorylated state,
whereby an amount that is consistent with the presence of the
fluorescent protein substrate in its phosphorylated state indicates
the presence of protein kinase activity.
2. The method of claim 1, wherein the fluorescent protein is an
Aequorea-related fluorescent protein
3. The method of claim 2, wherein said Aequorea-related fluorescent
protein comprises at least one mutation selected from the group
consisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T,
V163A, 1167T, S175G, S205T and N212K.
4. The method of claim 2, wherein said Aequorea-related fluorescent
protein comprises a phosphorylation site within ten amino acids of
the terminus of said Aequorea-related fluorescent protein.
5. The method of claim 2, wherein said Aequorea-related fluorescent
protein comprises a phosphorylation site within twenty amino acids
of the terminus of said Aequorea-related fluorescent protein.
6. A method for determining whether a cell exhibits protein kinase
activity, comprising the steps of: providing a transfected host
cell comprising a recombinant nucleic acid molecule comprising
expression control sequences operatively linked to a nucleic acid
sequence coding for the expression of a fluorescent protein
substrate for a protein kinase, the fluorescent protein substrate
comprising a fluorescent protein moiety containing a
phosphorylation site for a protein kinase, wherein the fluorescent
protein substrate exhibits a different fluorescent property in the
phosphorylated state than in the un-phosphorylated state, the cell
expressing the fluorescent protein substrate; wherein the
fluorescent protein moiety is a green fluorescent protein (SEQ. ID.
NO: 2), and wherein the phosphorylation site is within ten amino
acids of the terminus of the fluorescent protein moiety; exciting
the protein substrate in the cell; and measuring the amount of a
fluorescent property of the fluorescent protein substrate that
differs in the un-phosphorylated and phosphorylated states, wherein
the presence of the fluorescent property associated with the
fluorescent state indicates the presence of protein kinase activity
in the cell.
7. The method of claim 6 wherein the fluorescent protein is an
Aequorea-related fluorescent protein.
8. The method of claim 7, wherein said Aequorea-related fluorescent
protein comprises at least one selected from the group consisting
of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T, V163A, 1167T,
S175G, S205T and N212K.
9. The method of claim 7, wherein said Aequorea-related fluorescent
protein comprises a phosphorylation site within ten amino acids of
the terminus of said Aequorea-related fluorescent protein.
10. The method of claim 7, wherein said Aequorea-related
fluorescent protein comprises a phosphorylation site within twenty
amino acids of the terminus of said Aequorea-related fluorescent
protein.
11. A method for determining whether a compound alters the activity
of a protein kinase, comprising the steps of; 1) contacting a
sample comprising a protein kinase activity with the compound, a
phosphate donor for the protein kinase and a fluorescent protein
substrate, wherein the fluorescent protein substrate comprises a
fluorescent protein moiety and a phosphorylation site for a protein
kinase, and wherein the fluorescent protein substrate exhibits a
different fluorescent property in the phosphorylated state than in
the un-phosphorylated state; wherein the fluorescent protein moiety
is a green fluorescent protein (SEQ. ID. NO: 2), and wherein the
phosphorylation site is within ten amino acids of the terminus of
the fluorescent protein moiety; 2) exciting the protein substrate;
3) measuring the amount of protein kinase activity in the sample as
a function of the quantity of change or rate of change of a
fluorescent property of the fluorescent protein substrate that
differs in the un-phosphorylated and phosphorylated states; and
comparing the amount of activity in the sample with a standard
activity for the same amount of said protein kinase, whereby a
difference between the amount of protein kinase activity in the
sample and the standard activity indicates that said compound has
an effect on the activity of the protein kinase.
12. The method of claim 11 wherein the fluorescent protein is an
Aequorea-related fluorescent protein.
13. The method of claim 11, wherein said Aequorea-related
fluorescent protein comprises at least one selected from the group
consisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T,
V163A, 1167T, S175G, S205T and N212K.
14. The method of claim 11, wherein said Aequorea-related
fluorescent protein comprises a phosphorylation site within ten
amino acids of the terminus of said Aequorea-related fluorescent
protein.
15. The method of claim 11, wherein said Aequorea-related
fluorescent protein comprises a phosphorylation site within twenty
amino acids of the terminus of said Aequorea-related fluorescent
protein.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to the field of enzymatic assays and,
in particular, assays for protein kinase activity involving
modified fluorescent proteins.
[0002] Protein phosphorylation is one of the most important general
mechanisms of cellular regulation. Protein phosphorylation commonly
occurs on three major amino acids, tyrosine, serine or threonine,
and changes in the phosphorylation state of these amino acids
within proteins can regulate many aspects of cellular metabolism,
regulation, growth and differentiation. Changes in the
phosphorylation state of proteins, mediated through phosphorylation
by kinases, or dephosphorylation by phosphatases, is a common
mechanism through which cell surface signaling pathways transmit
and integrate information into the nucleus. Given their key role in
cellular regulation, it is not surprising that defects in protein
kinases and phosphatases have been implicated in many disease
states and conditions. For example, the over-expression of cellular
tyrosine kinases such as the EGF or PDGF receptors, or the mutation
of tyrosine kinases to produce constitutively active forms
(oncogenes) occurs in many cancer cells. Drucker et al. (1996)
Nature Medicine 2: 561-56. Protein tyrosine kinases are also
implicated in inflammatory signals. Defective Thr/Ser kinase genes
have been demonstrated to be implicated in several diseases such as
myotonic dystrophy as well as cancer, and Alzheimer's disease
(Sanpei et al. (1995) Biochem. Biophys. Res. Commun. 212: 341-6;
Sperber et al (1995) Neurosci. Lett. 197: 149-153; Grammas et al
(1995) Neurobiology of Aging 16: 563-569; Govoni et al. (1996) Ann.
N.Y. Acad. Sci. 777: 332-337).
[0003] The involvement of protein kinases and phosphatases in
disease states makes them attractive targets for the therapeutic
intervention of drugs, and in fact many clinically useful drugs act
on protein kinases or phosphatases. Examples include cyclosporin A
which is a potent immunosuppressant that binds to cyclophilin. This
complex binds to the Ca/calmodulin-dependent protein phosphatase
type 2B (calcineurin) inhibiting its activity, and hence the
activation of T-cells. (Sigal and Dumont (1992), Schreiber and
Crabtree (1992)). Inhibitors of protein kinase C are in clinical
trails as therapeutic agents for the treatment of cancer. (Clin.
Cancer Res. (1995) 1:113-122) as are inhibitors of cyclin dependent
kinase. (J. Mol. Med. (1995) 73:(10):509-14.)
[0004] The number of known kinases and phosphatases are growing
rapidly as the influence of genomic programs to identify the
molecular basis for diseases have increased in size and scope.
These studies are likely to implicate many more kinase and
phosphatase genes in the development and propagation of diseases in
the future, thereby making them attractive targets for drug
discovery. However current methods of measuring protein
phosphorylation have many disadvantages which prevents or limits
the ability to rapidly screen using miniaturized automated formats
of many thousands of compounds. This is because current methods
rely on the incorporation and measurement of .sup.32P into the
protein substrates of interest. In whole cells this necessitates
the use of high levels of radioactivity to efficiently label the
cellular ATP pool and to ensure that the target protein is
efficiently labeled with radioactivity. After incubation with test
drugs, the cells must be lysed and the protein of interest purified
to determine its relative degree of phosphorylation. This method
requires high numbers of cells, long preincubation times, careful
manipulation and washing steps (to avoid artifactual
phosphorylation or dephosphorylation), as well as a method of
purification of the target protein. Furthermore, final radioactive
incorporation into target proteins is usually very low, giving the
assay poor sensitivity. Alternative assay methods, for example
based on phosphorylation-specific antibodies using ELISA-type
approaches, involve the difficulty of producing antibodies that
distinguish between phosphorylated and non-phosphorylated proteins,
and the requirement for cell lysis, multiple incubation and washing
stages which are time consuming, complex to automate and
potentially susceptible to artifacts.
[0005] Kinase assays based on purified enzymes require large
amounts of purified kinases, high levels of radioactivity, and
methods of purification of the substrate protein away from
incorporated .sup.32P-labelled ATP. They also suffer from the
disadvantage of lacking the physiological context of the cell,
preventing a direct assessment of a drugs toxicity and ability to
cross the cells plasma membrane.
[0006] Fluorescent molecules are attractive as reporter molecules
in many assay systems because of their high sensitivity and ease of
quantification. Recently, fluorescent proteins have been the focus
of much attention because they can be produced in vivo by
biological systems, and can be used to trace intracellular events
without the need to be introduced into the cell through
microinjection or permeabilization. The green fluorescent protein
of Aequorea victoria is particularly interesting as a fluorescent
indicator protein. A cDNA for the protein has been cloned. (D. C.
Prasher et al., "Primary structure of the Aequorea victoria
green-fluorescent protein," Gene (1992) 111:229-33.) Not only can
the primary amino acid sequence of the protein be expressed from
the cDNA, but the expressed protein can fluoresce. This indicates
that the protein can undergo the cyclization and oxidation believed
to be necessary for fluorescence. The fluorescence of green
fluorescent protein is generated from residues S65-Y66-G67.
[0007] Fluorescent proteins have been used as markers of gene
expression, tracers of cell lineage and as fusion tags to monitor
protein localization within living cells. (M. Chalfie et al.,
"Green fluorescent protein as a marker for gene expression,"
Science 263:802-805; A. B. Cubitt et al., "Understanding, improving
and using green fluorescent proteins," TIBS 20, November 1995, pp.
448-455. U.S. Pat. No. 5,491,084, M. Chalfie and D. Prasher.
Furthermore, mutant versions of green fluorescent protein have been
identified that exhibit altered fluorescence characteristics,
including altered excitation and emission maxima, as well as
excitation and emission spectra of different shapes. (R. Heim et
al., "Wavelength mutations and posttranslational autoxidation of
green fluorescent protein," Proc. Natl. Acad. Sci. USA, (1994)
91:12501-04; R. Heim et al., "Improved green fluorescence," Nature
(1995) 373:663-665.) These properties add variety and utility to
the arsenal of biologically based fluorescent indicators.
[0008] There is a need for assays of protein phosphorylation that
are simple, sensitive, non-invasive, applicable to living cells and
tissues and that avoid the use of any radioactivity.
SUMMARY OF THE INVENTION
[0009] When fluorescent proteins are modified to incorporate a
phosphorylation site recognized by a protein kinase, the
fluorescent proteins not only can become phosphorylated by the
protein kinase, but they also can exhibit different fluorescent
characteristics in their un-phosphorylated and phosphorylated forms
when irradiated with light having a wavelength within their
excitation spectrum. This characteristic makes fluorescent protein
substrates particularly useful for assaying protein kinase activity
in a sample.
[0010] This invention provides methods for determining whether a
sample contains protein kinase activity. The methods involve
contacting the sample with a phosphate donor, usually ATP, and a
fluorescent protein substrate of the invention; exciting the
fluorescent protein substrate with light of an appropriate
wavelength; and measuring the amount of a fluorescent property that
differs in the un-phosphorylated state and phosphorylated state. An
amount that is consistent with the presence of the fluorescent
protein substrate in its phosphorylated state indicates the
presence of protein kinase activity, and an amount that is
consistent with the presence of the protein substrate in its
un-phosphorylated state indicates the absence of protein kinase
activity.
[0011] One embodiment of the above method is for determining the
amount of protein kinase activity in a sample. In this method,
measuring the amount of a fluorescent property in the sample
comprises measuring the amount at two or more time points after
contacting the sample with a phosphate donor and a fluorescent
protein substrate of the invention, and determining the quantity of
change or rate of change of the measured amount. The quantity or
rate of change of the measured amount reflects the amount of
protein kinase activity in the sample.
[0012] In another aspect, the invention provides methods for
determining whether a cell exhibits protein kinase activity. The
methods involve the steps of providing a transfected host cell of
the invention that produces a fluorescent protein substrate of the
invention; exciting the protein substrate in the cell with light of
an appropriate wavelength; and measuring the amount of a
fluorescent property that differs in the un-phosphorylated and
phosphorylated states. An amount that is consistent with the
presence of the protein substrate in its phosphorylated state
indicates the presence of protein kinase activity, and an amount
that is consistent with the presence of the protein substrate in
its un-phosphorylated state indicates the absence of protein kinase
activity or the presence of phosphatase activity.
[0013] In another aspect, the invention provides methods for
determining the amount of activity of a protein kinase in one or
more cells from an organism. The methods involve providing a
transfected host cell comprising a recombinant nucleic acid
molecule comprising expression control sequences operatively linked
to a nucleic acid sequence coding for the expression of a
fluorescent protein substrate of the invention, the cell expressing
the fluorescent protein substrate; exciting the protein substrate
in the cell with light; and measuring the amount of a fluorescent
property that differs in the un-phosphorylated and phosphorylated
states at two or more time points after contacting the sample with
a phosphate donor and a fluorescent protein substrate, and
determining the quantity or rate of change of the measured amount.
The quantity or rate of change of the measured amount reflects the
amount of protein kinase activity in the sample.
[0014] This invention also provides screening methods for
determining whether a compound alters the activity of a protein
kinase. The methods involve contacting a sample containing a known
amount of protein kinase activity with the compound, a phosphate
donor for the protein kinase and a fluorescent protein substrate of
the invention; exciting the protein substrate; measuring the amount
of protein kinase activity in the sample as a function of the
quantity or rate of change of a fluorescent property that differs
in the un-phosphorylated and phosphorylated states; and comparing
the amount of activity in the sample with a standard activity for
the same amount of the protein kinase. A difference between the
amount of protein kinase activity in the sample and the standard
activity indicates that the compound alters the activity of the
protein kinase.
[0015] Another aspect of the drug screening methods involve
determining whether a compound alters the protein kinase activity
in a cell. The methods involve providing first and second
transfected host cells exhibiting protein kinase activity and
expressing a fluorescent protein substrate of the invention;
contacting the first cell with an amount of the compound;
contacting the second cell with a different amount of the compound;
exciting the protein substrate in the first and second cells;
measuring the amount of protein kinase activity as a function of
the quantity of change or rate of change of a fluorescent property
that differs in the un-phosphorylated and phosphorylated states in
the first and second cells; and comparing the amount in the first
and second cells. A difference in the amount indicates that the
compound alters protein kinase activity in the cell.
[0016] This invention also provides fluorescent protein substrates
for a protein kinase. Fluorescent protein substrates for a protein
kinase comprise a fluorescent protein moiety and a phosphorylation
site for a protein kinase. The protein substrate exhibits a
different fluorescent property in the phosphorylated state than in
the unphosphorylated state. In a preferred embodiment, the
fluorescent protein is an Aequorea-related fluorescent protein. In
another embodiment, the phosphorylation site is located within
about 5, 10, 15 or 20 amino acids of a terminus, e.g., the
amino-terminus, of the fluorescent protein moiety. In another
embodiment, the protein substrate comprises the phosphorylation
site more than 20 amino acids from a terminal of the fluorescent
protein moiety and within the fluorescent protein moiety. The
phosphorylation site can be one recognized by, for example, protein
kinase A, a cGMP-dependent protein kinase, protein kinase C,
Ca.sup.2+/calmodulin-dependent protein kinase I,
Ca.sup.2+/calmodulin-dependent protein kinase II or MAP kinase
activated protein kinase type 1.
[0017] This invention also provides nucleic acid molecules coding
for the expression of a fluorescent protein substrate for a protein
kinase of the invention. In one aspect, the nucleic acid molecule
is a recombinant nucleic acid molecule comprising expression
control sequences operatively linked to a nucleic acid sequence
coding for the expression of a fluorescent protein substrate for a
protein kinase of the invention. In another aspect, the invention
provides transfected host cells transfected with a recombinant
nucleic acid molecule comprising expression control sequences
operatively linked to a nucleic acid sequence coding for the
expression of a fluorescent protein substrate for a protein kinase
of the invention.
[0018] In another aspect, this invention provides collections of
fluorescent protein candidate substrates comprising at least 10
different members, each member comprising a fluorescent protein
moiety and a variable peptide moiety around the terminus of the
fluorescent protein moiety.
[0019] In another embodiment, the invention provides collections of
recombinant nucleic acid molecules comprising at least 10 different
recombinant nucleic acid molecule members, each member comprising
expression control sequences operatively linked to nucleic acid
sequences coding for the expression of a different fluorescent
protein candidate substrate of the invention. The invention also
provides collections of host cells comprising at least 10 different
host cell members, each member comprising the above recombinant
nucleic acid molecules.
[0020] The collections of cells are useful in determining the
specificity of cellular kinases, from either diseased or normal
tissues. The screening methods involve providing a collection of
transfected host cells of the invention; culturing the collection
of host cells under conditions for the expression of the
fluorescent protein candidate substrate; and determining for each
of a plurality of members from the collection whether the member
contains a fluorescent protein candidate substrate that exhibits a
fluorescent property different than the fluorescent property
exhibited by the non-phosphorylated candidate substrate. The
presence of fluorescent protein candidate substrate that exhibits a
fluorescent property different than the fluorescent property
exhibited by the candidate substrate indicates that the candidate
substrate possesses a peptide moiety that can be phosphorylated by
the kinase present in the host cells.
[0021] This invention also provides kits comprising a fluorescent
protein substrate and a phosphate donor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a flow chart showing the steps in an assay method
for protein kinase activity.
[0023] FIG. 2 depicts molecular events in a cell in altering and
detecting fluorescent properties of a fluorescent protein substrate
for a protein kinase.
[0024] FIGS. 3A and 3B depict the nucleotide sequence (SEQ ID NO:
1) and deduced amino acid sequence (SEQ ID NO:2) of a wild-type
Aequorea green fluorescent protein.
[0025] FIGS. 4A and 4B provide a list of the positions and amino
acid changes made for phosphorylation mutants made more than
fifteen amino acids in the primary sequence from the N-terminus
(nucleotide=SEQ ID NO:36 amino acid=SEQ ID NO:37), as compared to
FIG. 3. Amino acids underlined represent the phosphorylation motif,
amino acids in brackets represent wild type sequence at those
positions.
[0026] FIG. 5 depicts plasmid pRSET containing a region encoding
GFP that is fused in frame with nucleotides encoding an N-terminal
polyhistidine tag (nucleotide SEQ ID NO:38 and SEQ ID NO:40; amino
acid=SEQ ID NO:39).
[0027] FIGS. 6A-6E show the fluorescence excitation spectra before
and after phosphorylation of N-terminal phospborylation mutants by
protein kinase A using standard phosphorylation conditions. 6 A:
1MSRRRRSI (SEQ ID NO:31). 6B: 1MRRRRSII (SEQ ID NO:32). 6C:
-1MRRRRSIII (SEQ ID NO:33). 6D: -2MRRRRSIIIF (SEQ ID NO:34). 6E:
-3MRRRRSIIIIF (SEQ ID NO:35). In all cases the spectrum after
phosphorylation has higher amplitude than the spectrum before
phosphorylation.
[0028] FIG. 7 depicts an expression vector having expression
control sequences operably linked to sequences coding for the
expression of protein kinase A catalytic subunit (PKA cat) upstream
from sequences coding for the expression of a fluorescent protein
substrate (nucleotide=SEQ ID NO:41 and SEQ ID NO:42).
[0029] FIG. 8 depicts the fluorescence excitation spectrum of
1MRRRRSII (SEQ ID NO:33): S65A, N149K, V163A and 1167T before and
after phosphorylation by protein kinase A using standard
phosphorylation conditions. The spectrum after phosphorylation has
higher amplitude than the spectrum before phosphorylation.
DETAILED DESCRIPTION OF THE INVENTION
[0030] I. Methods for Assaying Samples for Protein Kinases
[0031] Protein kinases add a phosphate residue to the
phosphorylation site of a protein, generally through the hydrolysis
of ATP to ADP. Fluorescent protein substrates for protein kinases
are useful in assays to determine the amount of protein kinase
activity in a sample. The assays of this invention take advantage
of the fact that phosphorylation of the protein substrate results
in a change in a fluorescent property of the fluorescent protein.
Methods for determining whether a sample has kinase activity
involve contacting the sample with a fluorescent protein substrate
having a phosphorylation site recognized by the protein kinase to
be assayed and with a phosphate donor under selected test
conditions. A phosphate donor is a compound containing a phosphate
moiety which the kinase is able to use to phosphorylate the protein
substrate. ATP (adenosine-5'-triphosphate) is by far the most
common phosphate donor. In certain instances, the sample will
contain enough of a phosphate donor to make this step unnecessary.
Then the fluorescent protein substrate is excited with light in its
excitation spectrum. If the protein substrate has been
phosphorylated, the substrate will exhibit different fluorescent
properties, indicating that the sample contains protein kinase
activity. For example, if the phosphorylated form of the protein
substrate has higher fluorescence than the un-phosphorylated form,
the amount of fluorescence in the sample will increase as a
function of the amount of substrate that has been phosphorylated.
If the fluorescent property is a change in the wavelength maximum
of emission, the change will be detected as a decrease in
fluorescence at the wavelength maximum of the un-phosphorylated
substrate and an increase in fluorescence at the wavelength maximum
of the phosphorylated substrate.
[0032] The amount of kinase activity in a sample can be determined
by measuring the amount of a fluorescent property in the sample at
a first time and a second time after contact between the sample,
the fluorescent protein substrate and a phosphate donor, and
determining the degree of change or the rate of change in a
fluorescent property. For example, if phosphorylation results in an
increase in fluorescence at the excitation wavelength maximum, the
fluorescence of the substrate at this wavelength can be determined
at two times. The amount of enzyme activity in the sample can be
calculated as a function of the difference in the determined amount
of the property at the two times. For example, the absolute amount
of activity can be calibrated using standards of enzyme activity
determined for certain amounts of enzyme after certain amounts of
time. The faster or larger the difference in the amount, the more
enzyme activity must have been present in the sample. The amount of
a fluorescent property can be determined from any spectral or
fluorescence lifetime characteristic of the excited substrate, for
example, by determining the intensity of the fluorescent signal
from the protein substrate or the excited state lifetime of the
protein substrate, the ratio of the fluorescences at two different
excitation wavelengths, the ratio of the intensities at two
different emission wavelengths, or the excited lifetime of the
protein substrate.
[0033] Fluorescence in a sample is measured using a fluorimeter. In
general, excitation radiation from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics cause the excitation radiation to excite the sample. In
response, fluorescent proteins in the sample emit radiation which
has a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage moves a microtiter
plate holding a plurality of samples in order to position different
wells to be exposed. The multi-axis translation stage, temperature
controller, auto-focusing feature, and electronics associated with
imaging and data collection can be managed by an appropriately
programmed digital computer. The computer also can transform the
data collected during the assay into another format for
presentation. This process can be miniaturized and automated to
enable screening many thousands of compounds.
[0034] Methods of performing assays on fluorescent materials are
well known in the art and are described in, e.g., Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York:Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y. -L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern
Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing
Col, Inc. (1978), pp. 296-361.
[0035] Enzymatic assays also can be performed on isolated living
cells in vivo, or from samples derived from organisms transfected
to express the substrate. Because fluorescent protein substrates
can be expressed recombinantly inside a cell, the amount of enzyme
activity in the cell or organism of which it is a part can be
determined by determining a fluorescent property or changes in a
fluorescent property of cells or samples from the organism.
[0036] In one embodiment, shown in FIG. 2, a cell is transiently or
stably transfected with an expression vector 200 encoding a
fluorescent protein substrate containing a phosphorylation site for
the enzyme to be assayed. This expression vector optionally
includes controlling nucleotide sequences such as promotor or
enhancing elements. The expression vector expresses the fluorescent
protein substrate 210 that contains the phosphorylation site 211
for the kinase to be detected. The enzyme to be assayed may either
be intrinsic to the cell or may be introduced by stable
transfection or transient co-transfection with another expression
vector encoding the enzyme and optionally including controlling
nucleotide sequences such as promoter or enhancer elements. The
fluorescent protein substrate and the enzyme preferably are located
in the same cellular compartment so that they have more opportunity
to come into contact.
[0037] If the cell possesses a high degree of enzyme activity
(K="kinase" 220), the fluorescent protein substrate will be
phosphorylated 230 (PO.sub.4), usually through the hydrolysis of
ATP. If the cell does not possess kinase activity, or possesses
very little, the cell contains-substantial amounts of
un-phosphorylated substrate 240. Upon excitation with light of the
appropriate wavelength (hv.sub.1) the phosphorylated substrate will
fluoresce light (hv.sub.2). Un-phosphorylated substrate exhibits
different fluorescent characteristics upon excitation at the same
wavelength, and may, for example, not fluoresce at all, or
fluoresce weakly. The amount of the fluorescent property is
measured generally using the optics 250 and detector 260 of a
fluorimeter.
[0038] If the cell contains phosphatases that compete with the
protein kinases, removing the phosphate from the protein substrate,
the level of enzyme activity in the cell can reach an equilibrium
between phosphorylated and un-phosphorylated states of the protein
substrate, and the fluorescence characteristics will reflect this
equilibrium level. In one aspect, this method can be used to
compare mutant cells to identify which ones possess greater or
lesser ratio of kinase to phosphatase activity. Such cells can be
sorted by a fluorescent cell sorter based on fluorescence.
[0039] A contemplated variation of the above assay is to use the
controlling nucleotide sequences to produce a sudden increase in
the expression of either the fluorescent protein substrate or the
enzyme being assayed, e.g., by inducing expression of the
construct. A fluorescent property is monitored at one or more time
intervals after the onset of increased expression. A high amount of
the property associated with phosphorylated state reflects a large
amount or high efficiency of the kinase. This kinetic determination
has the advantage of minimizing any dependency of the assay on the
rates of degradation or loss of the fluorescent protein
moieties.
[0040] In another embodiment, the vector may be incorporated into
an entire organism by standard transgenic or gene replacement
techniques. An expression vector capable of expressing the enzyme
optionally may be incorporated into the entire organism by standard
transgenic or gene replacement techniques. Then, a sample from the
organism containing the protein substrate is tested. For example,
cell or tissue homogenates, individual cells, or samples of body
fluids, such as blood, can be tested.
[0041] II. Screening Assays
[0042] The enzymatic assays of the invention can be used in drug
screening assays to determine whether a compound alters the
activity of a protein kinase. In one embodiment, the assay is
performed on a sample in vitro containing the enzyme. A sample
containing a known amount of enzyme activity is mixed with a
substrate of the invention and with a test compound. The amount of
the enzyme activity in the sample is then determined as above,
e.g., by measuring the amount of a fluorescent property at a first
and second time after contact between the sample, the protein
substrate, a phosphate substrate, and the compound. Then the amount
of activity per mole of enzyme in the presence of the test compound
is compared with the activity per mole of enzyme in the absence of
the test compound. A difference indicates that the test compound
alters the activity of the enzyme.
[0043] In another embodiment, the ability of a compound to alter
kinase activity in vivo is determined. In an in vivo assay, cells
transfected with a expression vector encoding a substrate of the
invention are exposed to different amounts of the test compound,
and the effect on fluorescence in each cell can be determined.
Typically, the difference is calibrated against standard
measurements to yield an absolute amount of kinase activity. A test
compound that inhibits or blocks the activity or expression of the
kinase can be detected by a relative increase in the property
associated with the un-phosphorylated state. The cell can also be
transfected with an expression vector to co-express the kinase or
an upstream signaling component such as a receptor, and fluorescent
substrate. This method is useful for detecting signaling to a
protein kinase of interest from an upstream component of the
signaling pathway. If a signal from an upstream molecule, e.g., a
receptor, is inhibited by a drug activity, then the kinase activity
will not be altered from basal. This provides a method for
screening for compounds which affect cellular events (including
receptor-ligand binding, protein-protein interactions or kinase
activation) which signal to the target kinase.
[0044] This invention also provides kits containing the fluorescent
protein substrate and a phosphate substrate for the protein kinase.
In one embodiment, the kit has a container holding the fluorescent
protein substrate and another container holding the phosphate
substrate. Protein kinases of known activity could be included for
use as positive controls and standards.
[0045] III. Fluorescent Protein Substrates for Protein Kinases
[0046] As used herein, the term "fluorescent property" refers to
the molar extinction coefficient at an appropriate excitation
wavelength, the fluorescence quantum efficiency, the shape of the
excitation spectrum or emission spectrum, the excitation wavelength
maximum and emission wavelength maximum, the ratio of excitation
amplitudes at two different wavelengths, the ratio of emission
amplitudes at two different wavelengths, the excited state
lifetime, or the fluorescence anisotropy. A measurable difference
in any one of these properties between the phosphorylated and
un-phosphorylated states suffices for the utility of the
fluorescent protein substrates of the invention in assays for
kinase activity. A measurable difference can be determined by
determining the amount of any quantitative fluorescent property,
e.g., the amount of fluorescence at a particular wavelength, or the
integral of fluorescence over the emission spectrum. Optimally, the
protein substrates are selected to have fluorescent properties that
are easily distinguishable in the un-phosphorylated and
phosphorylated states.
[0047] Determining ratios of excitation amplitude or emission
amplitude at two different wavelengths ("excitation amplitude
ratioing" and "emission amplitude ratioing", respectively) are
particularly advantageous because the ratioing process provides an
internal reference and cancels out variations in the absolute
brightness of the excitation source, the sensitivity of the
detector, and light scattering or quenching by the sample.
Furthermore, if phosphorylation of the protein substrate changes
its ratio of excitation or emission amplitudes at two different
wavelengths, then such ratios measure the extent of phosphorylation
independent of the absolute quantity of the protein substrate. Some
of the fluorescent protein substrates described herein do exhibit a
phosphorylation-induced change in the ratio of excitation
amplitudes at two different wavelengths. Even if a fluorescent
protein substrate does not exhibit a phosphorylation-induced change
in excitation or emission amplitudes at two wavelengths, cells can
be provided that co-express another fluorescent protein that is not
sensitive to phosphorylation and whose excitation or emission
spectrum is peaked at wavelengths distinct from those of the
phosphorylation substrate. Provided that the expression of the two
proteins are both controlled by the same nucleotide control
sequences, their expression levels should be closely linked.
Therefore ratioing the excitation or emission amplitude of the
phosphorylation substrate at its preferred wavelength to the
corresponding excitation or emission amplitude of the
phosphorylation-insensitive reference protein at its separate
preferred wavelength is an alternative method for canceling out
variations in the absolute quantity of cells or overall level of
protein expression.
[0048] A. Fluorescent Proteins
[0049] As used herein, the term "fluorescent protein" refers to any
protein capable of fluorescence when excited with appropriate
electromagnetic radiation. This includes fluorescent proteins whose
amino acid sequences are either naturally occurring or engineered
(i.e., analogs). Many cnidarians use green fluorescent proteins
("GFPs") as energy-transfer acceptors in bioluminescence. A "green
fluorescent protein," as used herein, is a protein that fluoresces
green light. Similarly, "blue fluorescent proteins" fluoresce blue
light and "red fluorescent proteins" fluoresce red light. GFPs have
been isolated from the Pacific Northwest jellyfish, Aequorea
Victoria, the sea pansy, Renilla reniformis, and Phialidium
gregarium. W. W. Ward et al., Photochem. Photobiol., 35:803-808
(1982); L. D. Levine et al., Comp. Biochem. Physiol., 72B:77-85
(1982).
[0050] A variety of Aequorea-related fluorescent proteins having
useful excitation and emission spectra have been engineered by
modifying the amino acid sequence of a naturally occurring GFP from
Aequorea victoria. (D.C. Prasher et al., Gene, 111:229-233 (1992);
R. Heim et al., Proc. Natl. Acad. Sci., USA, 91:12501-04 (1994);
U.S. patent application Ser. No. 08/337,915, filed Nov. 10, 1994;
International application PCT/US95/14692, filed Nov. 10, 1995.)
[0051] 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 238 amino-acid wild-type Aequorea green
fluorescent protein of FIG. 3 (SEQ ID NO:2). More preferably, a
fluorescent protein is an Aequorea-related fluorescent protein if
any contiguous sequence of 200 amino acids of the fluorescent
protein has at least 95% sequence identity with an amino acid
sequence, either contiguous or non-contiguous, from the wild type
Aequorea green fluorescent protein of FIG. 3 (SEQ ID NO:2).
Similarly, the fluorescent protein may be related to Renilla or
Phialidium wild-type fluorescent proteins using the same
standards.
[0052] Optimal alignment of sequences for aligning a comparison
window may be conducted by the local homology algorithm of Smith
and Waterman (1981) Adv. Appl. Math., 2:482, by the homology
alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.,
48:443, by the search for similarity method of Pearson and Lipman
(1988) Proc. Natl. Acad. Sci., U.S.A., 85:2444, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection. The best alignment (i.e., resulting in the highest
percentage of homology over the comparison window, i.e., 150 or 200
amino acids) generated by the various methods is selected.
[0053] The percentage of sequence identity is calculated by
comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical amino acid occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity.
[0054] Aequorea-related fluorescent proteins include, for example
and without limitation, wild-type (native) Aequorea victoria GFP
(D.C. Prasher et al., "Primary structure of the Aequorea victoria
green fluorescent protein," Gene, (1992) 111:229-33), whose
nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence
(SEQ ID NO:2) are presented in FIG. 3, allelic variants of this
sequence, e.g., Q80R, which has the glutamine residue at position
80 substituted with arginine (M. Chalfie et al., Science, (1994)
263:802-805), those Aequorea-related engineered versions described
in Table I, variants that include one or more folding mutations and
fragments of these proteins that are fluorescent, such as Aequorea
green fluorescent protein from which the two amino-terminal amino
acids have been removed. Several of these contain different
aromatic amino acids within the central chromophore and fluoresce
at a distinctly shorter wavelength than wild type species. For
example, mutants P4 and P4-3 contain (in addition to other
mutations) the substitution Y66H, whereas W2 and W7 contain (in
addition to other mutations) Y66W. Other mutations both close to
the chromophore region of the protein and remote from it in primary
sequence may affect the spectral properties of GFP and are listed
in the first part of the table below.
1TABLE I Extinct. Excitation Emission Coeff. Quantum Clone
Mutation(s) max (nm) max (nm) (M.sup.-1 cm.sup.-1) yield Wild none
395 (475) 508 21,000 0.77 type (7,150) P4 Y66H 383 447 13,500 0.21
P4-3 Y66H 381 445 14,000 0.38 Y145F W7 Y66W 433 (453) 475 (501)
18,000 0.67 N1461 (17,100) M153T V163A N212K W2 Y66W 432 (453) 480
10,000 0.72 I123V (9,600) Y145H H148R M153T V163A N212K S65T S65T
489 511 39,200 0.68 P4-1 S65T 504 (396) 514 14,500 0.53 M153A
(8,600) K238E S65A S65A 471 504 S65C S65C 479 507 S65L S65L 484 510
Y66F Y66F 360 442 Y66W Y66W 458 480
[0055] Additional mutations in Aequorea-related fluorescent
proteins, referred to as "folding mutations," improve the ability
of GFP to fold at higher temperatures, and to be more fluorescent
when expressed in mammalian cells, but have little or no effect on
the peak wavelengths of excitation and emission. It should be noted
that these may be combined with mutations that influence the
spectral properties of GFP to produce proteins with altered
spectral and folding properties. Folding mutations include: T44A,
F64L, V68L, S72A, F99S, Y145F, N1461, M153T or A, V163A, 1167T,
S175G, S205T and N212K.
[0056] This invention contemplates the use of other fluorescent
proteins in fluorescent protein substrates for protein kinases. The
cloning and expression of yellow fluorescent protein from Vibrio
fischeri strain Y-1 has been described by T. O. Baldwin et al.,
Biochemistry (1990) 29:5509-15. This protein requires flavins as
fluorescent co-factors. The cloning of Peridinin-chlorophyll a
binding protein from the dinoflagellate Symbiodinium sp. was
described by B. J. Morris et al., Plant Molecular Biology, (1994)
24:673:77. One useful aspect of this protein is that it fluoresces
red. The cloning of phycobiliproteins from marine cyanobacteria
such as Synechococcus, e.g., phycoerythrin and phycocyanin, is
described in S. M. Wilbanks et al., J. Biol. Chem. (1993)
268:1226-35. These proteins require phycobilins as fluorescent
co-factors, whose insertion into the proteins involves auxiliary
enzymes. The proteins fluoresce at yellow to red wavelengths.
[0057] As used herein, the "fluorescent protein moiety" of a
fluorescent protein substrate is that portion of the amino acid
sequence of a fluorescent protein substrate which, when the amino
acid sequence of the fluorescent protein substrate is optimally
aligned with the amino acid sequence of a naturally occurring
fluorescent protein, lies between the amino terminal and carboxy
terminal amino acids, inclusive, of the amino acid sequence of the
naturally occurring fluorescent protein.
[0058] It has been found that fluorescent proteins can be
genetically fused to other target proteins and used as markers to
identify the location and amount of the target protein produced.
Accordingly, this invention provides fusion proteins comprising a
fluorescent protein moiety and additional amino acid sequences.
Such sequences can be, for example, up to about 15, up to about 50,
up to about 150 or up to about 1000 amino acids long. The fusion
proteins possess the ability to fluoresce when excited by
electromagnetic radiation. In one embodiment, the fusion protein
comprises a polyhistidine tag to aid in purification of the
protein.
[0059] B. Phosphorylation Sites For Protein Kinases
[0060] Fluorescent protein substrates for a protein kinase are the
subset of fluorescent proteins as defined above whose amino acid
sequence includes a phosphorylation site. Fluorescent protein
substrates can be made by modifying the amino acid sequence of an
existing fluorescent protein to include a phosphorylation site for
a protein kinase. Fluorescent protein substrates for protein
kinases are not meant to include naturally occurring fluorescent
proteins or currently known mutant fluorescent proteins. Such
previously known fluorescent proteins or mutants may be substrates
for protein kinases, but do not exhibit any detectable change in
fluorescent properties upon phosphorylation.
[0061] As used herein, the term "phosphorylation site for a protein
kinase" refers to an amino acid sequence which, as part of a
polypeptide, is recognized by a protein kinase for the attachment
of a phosphate moiety. The phosphorylation site can be a site
recognized by, for example, protein kinase A, a cGMP-dependent
protein kinase, protein kinase C, Ca.sup.2+/calmodulin-dependent
protein kinase I, Ca.sup.2+/calmodulin-dependent protein kinase II
or MAP kinase activated protein kinase type 1.
[0062] The preferred consensus sequence for protein kinase A is
RRXSZ (SEQ ID NO:3) or RRXTZ (SEQ ID NO:4), wherein X is any amino
acid and Z is a hydrophobic amino acid, preferably valine, leucine
or isoleucine. Many variations in the above sequence are allowed,
but generally exhibit poorer kinetics. For example, lysine (K) can
be substituted for the second arginine. Many consensus sequences
for other protein kinases have been tabulated, e.g. by Kemp, B. E.
and Pearson, R. B. (1990) Trends Biochem. Sci. 15: 342-346;
Songyang, Z. et al. (1994) Current Biology 4: 973-982.
[0063] For example, a fluorescent protein substrate selective for
phosphorylation by cGMP-dependent protein kinase can include the
following consensus sequence: BKISASEFDR PLR (SEQ ID NO:5), where B
represents either lysine (K) or arginine (R), and the first S is
the site of phosphorylation (Colbran et al. (1992) J. Biol. Chem.
267: 9589-9594). The residues DRPLR (SEQ ID NO:6) are less critical
than the phenylalanine (F) just preceding them for specific
recognition by cGMP-dependent protein kinase in preference to
cAMP-dependent protein kinase.
[0064] Either synthetic or naturally occurring motifs can be used
to create a protein kinase phosphorylation site. For example,
peptides including the motif XRXXSXRX (SEQ ID NO:7), wherein X is
any amino acid, are among the best synthetic substrates (Kemp and
Pearson, supra) for protein kinase C. Alternatively, the
Myristoylated Alanine-Rich Kinase C substrate ("MARCKS") is one of
the best substrates for PKC and is a real target for the kinase in
vivo. The sequence around the phosphorylation site of MARCKS is
KKKKRFSFK (SEQ ID NO:8) (Graff et al. (1991) J. Biol. Chem.
266:14390-14398). Either of these two sequences can be incorporated
into a fluorescent protein to make it a substrate for protein
kinase C.
[0065] A protein substrate for Ca.sup.2+/calmodulin-dependent
protein kinase I is derived from the sequence of synapsin I, a
known optimal substrate for this kinase. The recognition sequence
around the phosphorylation site is LRRLSDSNF (SEQ ID NO:9) (Lee et
al. (1994) Proc. Natl. Acad. Sci. USA 91:6413-6417).
[0066] A protein substrate selective for
Ca.sup.2+/calmodulin-dependent protein kinase II is derived from
the sequence of glycogen synthase, a known optimal substrate for
this kinase. The recognition sequence around the phosphorylation
site is KKLNRTLTVA (SEQ ID NO: 10) (Stokoe et al. (1993) Biochem.
J. 296:843-849). A small change in this sequence to KKANRTLSVA (SEQ
ID NO: 11) makes the latter specific for MAP kinase activated
protein kinase type 1.
[0067] In one embodiment, the fluorescent protein substrate
contains a phosphorylation site around one of the termini, in
particular, the amino-terminus, of the fluorescent protein moiety.
The site preferably is located in a position within five, ten,
fifteen, or twenty amino acids of a position corresponding to the
wild type amino-terminal amino acid of the fluorescent protein
moiety ("within twenty amino acids of the amino-terminus"). This
includes sites engineered into the existing amino acid sequence of
the fluorescent protein moiety and sites produces by extending the
amino terminus of the fluorescent protein moiety.
[0068] One may, for example, modify the existing sequence of wild
type Aequorea GFP or a variant or it as listed above to include a
phosphorylation site within the first ten or twenty amino acids. In
one embodiment, the naturally occurring sequence is modified as
follows:
2 wild type: MSKGEELFTG (SEQ ID NO:43) substrate: MRRRRSIITG (SEQ
ID NO:12).
[0069] One may include modifying the naturally occurring sequence
of Aequorea GFP by introducing a phosphorylation site into an
extended amino acid sequence of such a protein created by adding
flanking sequences to the amino terminus, for example:
3 wild type: MSKGEELFTG (SEQ ID NO:43) substrate: MRRRRSIIIIFTG
(SEQ ID NO:13).
[0070] Fluorescent protein substrates having a phosphorylation site
around a terminus of the fluorescent protein moiety offer the
following advantages. First, it is often desirable to append
additional amino acid residues onto the fluorescent protein moiety
in order to create a specific phosphorylation consensus sequence.
Such a sequence is much less likely to disrupt the folding pattern
of the fluorescent protein when appended onto the terminus than
when inserted into the interior of the protein sequence. Second,
different phosphorylation motifs can be interchanged without
significant disruption of GFP therefore providing a general method
of measuring different kinases. Third, the phosphorylation site is
exposed to the surface of the protein and, therefore, more
accessible to protein kinases. Fourth, we have discovered that
phosphorylation at sites close to the N-terminus of GFP can provide
large changes in fluorescent properties if the site of
phosphorylation is chosen such that the Ser or Thr residue which is
phosphorylated occupies a position which in the wild-type protein
was originally negatively or positively charged. Specifically,
replacement of Glu 6 by a non-charged Ser or Thr residue can
significantly disrupt fluorescence of GFP when made within the
right context of surrounding amino acids. Phosphorylation of the
serine or threonine will restore negative charge to this position
and thereby increases fluorescence.
[0071] In another embodiment, the fluorescent protein substrate
includes a phosphorylation site remote from the terminus, e.g.,
that is separated by more than about twenty amino acids from the
terminus of the fluorescent protein moiety and within the
fluorescent protein moiety. One embodiment of this form includes
the Aequorea-related fluorescent protein substrate comprising the
substitution H217S, creating a consensus protein kinase A
phosphorylation site. Additionally, phosphorylation sites
comprising the following alterations based on the sequence of wild
type Aequorea GFP exhibit fluorescent changes upon phosphorylation:
69RRFSA (SEQ ID NO:14) and 214KRDSM (SEQ ID NO:15).
[0072] The practitioner should consider the following in selecting
amino acids for substitution within the fluorescent protein moiety
remote in primary amino acid sequence from the terminus. First, it
is preferable to select amino acid sequences within the fluorescent
protein moiety that resemble the sequence of the phosphorylation
site. In this way, fewer amino acid substitutions in the native
protein are needed to introduce the phosphorylation site into the
fluorescent protein. For example, protein kinase A recognizes the
sequence RRXSZ (SEQ ID NO:46) or RRXTZ (SEQ ID NO:47), wherein X is
any amino acid and Z is a hydrophobic amino acid. Serine or
threonine is the site of phosphorylation. It is preferable to
introduce this sequence into the fluorescent protein moiety at
sequences already containing Ser or Thr, so that Ser or Thr are not
substituted in the protein. More preferably the phosphorylation
site is created at locations having some existing homology to the
sequence recognized by protein kinase A, e.g., having a proximal
Arg or hydrophobic residues with the same spatial relationship as
in the phosphorylation site.
[0073] Second, locations on the surface of the fluorescent protein
are preferred for phosphorylation sites. This is because surface
locations are more likely to be accessible to protein kinases than
interior locations. Surface locations can be identified by computer
modeling of the fluorescent protein structure or by reference to
the crystal structure of Aequorea GFP. Also, charged amino acids in
the fluorescent protein are more likely to lie on the surface than
inside the fluorescent protein, because such amino acids are more
likely to be exposed to water in the environment.
[0074] In cases where the phosphorylation site is either at the
N-terminus or remote from it, the amino acid context around the
phosphorylation site needs to be optimized in order to maximize the
change in fluorescence. Amino acid substitutions that change large
bulky and or hydrophobic amino acids to smaller and less
hydrophobic replacements are generally helpful. Similarly large
charged amino acids can be replaced by smaller, less charged amino
acids. For example:
[0075] a/Hydrophobic to less hydrophobic
[0076] Phe to Leu
[0077] Leu to Ala
[0078] b/Charged to charged but smaller
[0079] Glu to Asp
[0080] Arg to Lys
[0081] c/Charged to less charged
[0082] Glu to Gln
[0083] Asp to Asn
[0084] d/Charged to polar
[0085] Glu to Thr
[0086] Asp to Ser
[0087] e/Charged to non-polar
[0088] Glu to Leu
[0089] Asp to Ala
[0090] These changes can be accomplished by directed means or using
random iterative approaches where changes are made randomly and the
best ones selected based upon their change in fluorescent
properties after phosphorylation by an appropriate kinase.
[0091] Third, amino acids at distant locations from the actual site
of phosphorylation can be varied to enhance fluorescence changes
upon phosphorylation. These mutations can be created through site
directed mutagenesis, or through random mutagenesis, for example by
error-prone PCR, to identify mutations that enhance either absolute
fluorescence or the change in fluorescence upon phosphorylation.
The identification of mutants remote in primary sequence from the
N-terminus identifies potentially interacting sequences which may
provide additional areas in which further mutagenesis could be used
to refine the change in fluorescence upon phosphorylation. For
example, it has been determined that mutations around the amino
terminus phosphorylation site interact (either transiently during
folding, or in a stable fashion) with amino acids at positions 171
and 172, and that point mutations that significantly disrupt
fluorescence of GFP by changing negative to positive charges near
the amino terminus can be rescued by changing a positive to a
negative charge at position 171.
[0092] In the phosphorylation mutant 50 the sequence is a/ and for
reference the wild type sequence b/ is listed below.
4 a/ MSKRRDSLT (SEQ ID NO:16) b/ MSKGEELFT (SEQ ID NO:44)
[0093] The phosphorylation mutant has only 7% of the fluorescence
of wild type protein. However, its fluorescence can be restored to
80% of wild type by 2 amino acid changes, E171K and I172V,
positions which are quite remote in linear sequence from the amino
terminus.
[0094] Thus, changes in charge at E171K (negative to positive) can
almost completely restore the fluorescence of the phosphorylation
mutant, strongly suggesting that the original loss of fluorescence
arose primarily through changes in charge caused by the point
mutations. It is clear that the addition and loss of charge at
positions around, and at the phosphorylation site, have a
significant impact on fluorescence formation. The fact that charge
alone can significantly affect the fluorescence properties of GFP
is highly significant within the scope of the present application
since phosphorylation involves the addition of 2 negative charges
associated with the phosphate group (OPO.sub.3.sup.-2) on the
serine residue.
[0095] In the above case the mutations restore fluorescence of the
phosphorylation mutant, without significantly increasing the
magnitude of the change in fluorescence upon phosphorylation.
Nevertheless the identification of these positions in GFP provides
a valuable tool to further optimize changes in fluorescence upon
phosphorylation by creating random mutations at codons around
positions 171, 172 and 173 to identify mutations that enhance
changes in fluorescence upon phosphorylation.
[0096] This can be achieved by co-expressing the kinase of interest
with the fluorescent substrate of the invention containing random
mutations which may enhance the fluorescence changes upon
phosphorylation in bacteria (in the example above these would be
NNK mutations at codons 171, 172 and 173, where N represents a
random choice of any of the four bases and K represents a random
choice of guanine or thymine). The expression vector containing the
mutated fluorescent substrates and the kinase are transformed into
host bacteria and the individual bacterial colonies grown up. Each
colony is derived from a single cell, and hence contains a single
unique mutant fluorescent substrate grown up.
[0097] The individual colonies may then be grown up and screened
for fluorescence either by fluorescence activated cell sorting
(FACS), or by observation under a microscope. Those that exhibit
the greatest fluorescence can then be rescreened under conditions
in which the kinase gene is inactivated. This can be achieved by
appropriate digests of the kinase gene by restriction enzymes that
specifically cut within the kinase but not GFP. Comparison of the
brightness of the mutant first in the presence of kinase then in
its absence indicates the relative effect of phosphorylation on the
mutant GFP.
[0098] C. Production of Fluorescent Protein Substrates for Protein
Kinases
[0099] While certain fluorescent protein substrates for protein
kinases can be prepared chemically, for example, by coupling a
peptide moiety to the amino terminus of a fluorescent protein, it
is preferable produce fluorescent protein substrates
recombinantly.
[0100] Recombinant production of a fluorescent protein substrate
involves expressing a nucleic acid molecule having sequences that
encode the protein. As used herein, the term "nucleic acid
molecule" includes both DNA and RNA molecules. It will be
understood that when a nucleic acid molecule is said to have a DNA
sequence, this also includes RNA molecules having the corresponding
RNA sequence in which "U" replaces "T." The term "recombinant
nucleic acid molecule" refers to a nucleic acid molecule which is
not naturally occurring, and which comprises two nucleotide
sequences which are not naturally joined together. Recombinant
nucleic acid molecules are produced by artificial combination,
e.g., genetic engineering techniques or chemical synthesis.
[0101] In one embodiment, the nucleic acid encodes a fusion protein
in which a single polypeptide includes the fluorescent protein
moiety within a longer polypeptide. In another embodiment the
nucleic acid encodes the amino acid sequence of consisting
essentially of a fluorescent protein modified to include a
phosphorylation site. In either case, nucleic acids that encode
fluorescent proteins are useful as starting materials.
[0102] Nucleic acids encoding fluorescent proteins can be obtained
by methods known in the art. For example, a nucleic acid encoding a
green fluorescent protein can be isolated by polymerase chain
reaction of cDNA from A. victoria using primers based on the DNA
sequence of A. victoria green fluorescent protein, as presented in
FIG. 3. PCR methods are described in, for example, U.S. Pat. No.
4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant.
Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NY,
1989).
[0103] Mutant versions of fluorescent proteins can be made by
site-specific mutagenesis of other nucleic acids encoding
fluorescent proteins, or by random mutagenesis caused by increasing
the error rate of PCR of the original polynucleotide with 0.1 mM
MnCl.sub.2 and unbalanced nucleotide concentrations. See, e.g.,
U.S. patent application Ser. No. 08/337,915, filed Nov. 10, 1994 or
International application PCT/US95/14692, filed Nov. 10, 1995.
[0104] Nucleic acids encoding fluorescent protein substrates which
are fusions between a polypeptide including a phosphorylation site
and a fluorescent protein and can be made by ligating nucleic acids
that encode each of these. Nucleic acids encoding fluorescent
protein substrates which include the amino acid sequence of a
fluorescent protein in which one or more amino acids in the amino
acid sequence of a fluorescent protein are substituted to create a
phosphorylation site can be created by, for example, site specific
mutagenesis of a nucleic acid encoding a fluorescent protein.
[0105] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques also well known in the art. Sambrook et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., (1989) and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc.
[0106] Nucleic acids used to transfect cells with sequences coding
for expression of the polypeptide of interest generally will be in
the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used, the term nucleotide
sequence "coding for expression of" a polypeptide refers to a
sequence that, upon transcription and translation of mRNA, produces
the polypeptide. As any person skilled in the art recognizes, this
includes all degenerate nucleic acid sequences encoding the same
amino acid sequence. This can include sequences containing, e.g.,
introns. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are "operatively linked" to a nucleic acid
sequence when the expression control sequences control and regulate
the transcription and, as appropriate, translation of the nucleic
acid sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the MRNA, and
stop codons.
[0107] The recombinant nucleic acid can be incorporated into an
expression vector comprising expression control sequences
operatively linked to the recombinant nucleic acid. The expression
vector can be adapted for function in prokaryotes or eukaryotes by
inclusion of appropriate promoters, replication sequences, markers,
etc.
[0108] The expression vector can be transfected into a host cell
for expression of the recombinant nucleic acid. Host cells can be
selected for high levels of expression in order to purify the
protein. E. coli is useful for this purpose. Alternatively, the
host cell can be a prokaryotic or eukaryotic cell selected to study
the activity of an enzyme produced by the cell. The cell can be,
e.g., a cultured cell or a cell in vivo.
[0109] Recombinant fluorescent protein substrates can be produced
by expression of nucleic acid encoding for the protein in E. coli.
Aequorea-related fluorescent proteins are best expressed by cells
cultured between about 15.degree. C. and 30.degree. C. but higher
temperatures (e.g. 37.degree. C.) are possible. After synthesis,
these enzymes are stable at higher temperatures (e.g., 37.degree.
C.) and can be used in assays at those temperatures.
[0110] The construct can also contain a tag to simplify isolation
of the substrate. For example, a polyhistidine tag of, e.g., six
histidine residues, can be incorporated at the amino or carboxyl
terminal of the fluorescent protein substrate. The polyhistidine
tag allows convenient isolation of the protein in a single step by
nickel-chelate chromatography.
[0111] Alternatively, the substrates need not be isolated from the
host cells. This method is particularly advantageous for the
assaying for the presence of protein kinase activity in situ.
[0112] IV. Libraries of Candidate Substrates
[0113] The inclusion of a phosphorylation site around the amino
terminus of a fluorescent protein moiety can provide a fluorescent
protein that, when phosphorylated, can alter a fluorescent property
of the protein. Accordingly, this invention provides libraries of
fluorescent protein candidate substrates useful for screening in
the identification and characterization of sequences that can be
recognized and efficiently phosphorylated by a kinase. Libraries of
these proteins can be screened to identify sequences that can be
phosphorylated by kinases of unknown substrate specificity, or to
characterize differences in kinase activity in, or from, diseased
and normal cells or tissues.
[0114] As used herein, a "library" refers to a collection
containing at least 10 different members. Each member of a
fluorescent protein candidate substrate library comprises a
fluorescent protein moiety and a variable peptide moiety, which is
preferably located near the amino-terminus of the fluorescent
protein moiety and preferably has fewer than about 15 amino acids.
The variety of amino acid sequences for the peptide moiety is at
the discretion of the practitioner. For example, the library can
contain a quite diverse collection of variable peptide moieties in
which most or all of the amino acid positions are subjected to a
non-zero but low probability of substitution. Also, the library can
contain variable peptide moieties having an amino acid sequence in
which only a few, e.g., one to ten, amino acid positions are
varied, but the probability of substitution at each position is
relatively high.
[0115] Preferably, libraries of fluorescent protein candidate
substrates are created by expressing protein from libraries of
recombinant nucleic acid molecules having expression control
sequences operatively linked to nucleic acid sequences that code
for the expression of different fluorescent protein candidate
substrates. Methods of making nucleic acid molecules encoding a
diverse collection of peptides are described in, for example, U.S.
Pat. No. 5,432,018 (Dower et al.), U.S. Pat. No. 5,223,409 (Ladner
et al.), U.S. Pat. No. 5,264,563 (Huse), and International patent
publication WO 92/06176 (Huse et al.). For expression of
fluorescent protein candidate substrates, recombinant nucleic acid
molecules are used to transfect cells, such that each cell contains
a member of the library. This produces, in turn, a library of host
cells capable of expressing the library of different fluorescent
protein candidate substrates. The library of host cells is useful
in the screening methods of this invention.
[0116] In one method of creating such a library, a diverse
collection of oligonucleotides having preferably random codon
sequences are combined to create polynucleotides encoding peptides
having a desired number of amino acids. The oligonucleotides
preferably are prepared by chemical synthesis. The polynucleotides
encoding variable peptide moiety can then be coupled to the 5' end
of a nucleic acid coding for the expression of a fluorescent
protein moiety or a carboxy-terminal portion of it. That is, the
fluorescent protein moiety can be cut back to eliminate up to 20
amino acids of the reference fluorescent protein. This creates a
recombinant nucleic acid molecule coding for the expression of a
fluorescent protein candidate substrate having a peptide moiety
fused to the amino terminus of the fluorescent protein. This
recombinant nucleic acid molecule is then inserted into an
expression vector to create a recombinant nucleic acid molecule
comprising expression control sequences operatively linked to the
sequences encoding the candidate substrate.
[0117] To generate the collection of oligonucleotides which forms a
series of codons encoding a random collection of amino acids and
which is ultimately cloned into the vector, a codon motif is used,
such as (NNK).sub.x, where N may be A, C, G, or T (nominally
equimolar), K is G or T (nominally equimolar), and x is the desired
number of amino acids in the peptide moiety, e.g., 15 to produce a
library of 15-mer peptides. The third position may also be G or C,
designated "S". Thus, NNK or NNS (i) code for all the amino acids,
(ii) code for only one stop codon, and (iii) reduce the range of
codon bias from 6:1 to 3:1. The expression of peptides from
randomly generated mixtures of oligonucleotides in appropriate
recombinant vectors is discussed in Oliphant et al., Gene
44:177-183 (1986).
[0118] An exemplified codon motif (NNK).sub.6 (SEQ ID NO: 17)
produces 32 codons, one for each of 12 amino acids, two for each of
five amino acids, three for each of three amino acids and one
(amber) stop codon. Although this motif produces a codon
distribution as equitable as available with standard methods of
oligonucleotide synthesis, it results in a bias against peptides
containing one-codon residues.
[0119] An alternative approach to minimize the bias against
one-codon residues involves the synthesis of 20 activated
tri-nucleotides, each representing the codon for one of the 20
genetically encoded amino acids. These are synthesized by
conventional means, removed from the support but maintaining the
base and 5-HO-protecting groups, and activating by the addition of
3'O-phosphoramidite (and phosphate protection with beta-cyanoethyl
groups) by the method used for the activation of mononucleosides,
as generally described in McBride and Caruthers, Tetrahedron
Letters 22:245 (1983). Degenerate "oligocodons" are prepared using
these trimers as building blocks. The trimers are mixed at the
desired molar ratios and installed in the synthesizer. The ratios
will usually be approximately equimolar, but may be a controlled
unequal ratio to obtain the over- to under-representation of
certain amino acids coded for by the degenerate oligonucleotide
collection. The condensation of the trimers to form the oligocodons
is done essentially as described for conventional synthesis
employing activated mononucleosides as building blocks. See
generally, Atkinson and Smith, Oligonucleotide Synthesis, M. J.
Gait, ed. p35-82 (1984). Thus, this procedure generates a
population of oligonucleotides for cloning that is capable of
encoding an equal distribution (or a controlled unequal
distribution) of the possible peptide sequences.
[0120] Libraries of amino terminal phosphorylation sites may also
be annealed to libraries of randomly mutated GFP sequences to
enable the selection of optimally responding substrates.
[0121] V. Methods for Screening Libraries of Candidate
Substrates
[0122] Libraries of host cells expressing fluorescent protein
candidate substrates are useful in identifying fluorescent proteins
having peptide moieties that alter a fluorescent property of the
fluorescent protein. Several methods of using the libraries are
envisioned. In general, one begins with a library of recombinant
host cells, each of which expresses a different fluorescent protein
candidate substrate. Each cell is expanded into a clonal population
that is genetically homogeneous.
[0123] In a first method, the desired fluorescent property is
measured from each clonal population before and at least one
specified time after a known change in intracellular protein kinase
activity. This change in kinase activity could be produced by
transfection with a gene encoding the kinase, by induction of
kinase gene expression using expression control elements, or by any
condition that post-translationally modulates activity of a kinase
that has already been expressed. Examples of the latter include
cell surface receptor mediated elevation of intracellular cAMP to
activate cAMP-dependent surface receptor mediated increases of
intracellular cGMP to activate cGMP-dependent protein kinase,
cytosolic free calcium to activate Ca.sup.2+/calmodulin-dependent
protein kinase types I, II, or IV, or the production of
diacylglycerol to activate protein kinase C, etc. One then selects
for the clone(s) that show the biggest or fastest change in the
desired fluorescence property. This method detects fluorescent
protein mutants whose folding and maturation was influenced by
phosphorylation as well as those affected by phosphorylation after
maturation.
[0124] One embodiment of this method exploits the fact that the
catalytic subunit of cAMP-dependent protein kinase is
constitutively active in the absence of the regulatory subunit and
is growth-inhibitory in E. coli and most mammalian cells.
Therefore, the cells tend to shed the kinase gene by recombination.
The change in kinase activity is obtained by culturing the cells
for a time sufficient to lose the kinase gene.
[0125] In a second method the host cells do not express the protein
kinase of interest. Each clonal population is separately lysed. ATP
is then added to the lysate. After an incubation period to allow
phosphorylation by background kinases, the fluorescence property is
measured. Then exogenous protein kinase is added to the lysate and
the fluorescent property is re-measured at one or more specified
time points. Again one selects for the clone(s) that show the
biggest or fastest change(s) in the desired fluorescence property.
Because little or no fresh protein synthesis is likely to occur in
the lysate, this method would discriminate against mutants which
are sensitive to phosphorylation only during their folding and
maturation.
[0126] In one embodiment of this method, the lysate is split into
two aliquots, one of which is mixed with kinase and ATP, the other
of which receives only ATP. One selects for the clone(s) that show
the biggest difference in fluorescence property between the two
aliquots.
[0127] The nucleic acids from cells exhibiting the different
properties can be isolated from the cells. Candidate substrates
having different fluorescent properties can be tested further to
identify the source of the difference.
[0128] The host cell also can be transfected with an expression
vector capable of expressing an enzyme, such as a protein kinase,
whose effect on the fluorescent property is to be tested.
EXAMPLES
[0129] A. Phosphorylation Sites Located in the Amino Acid Sequence
of Aeguorea GFP Remote in the Primary Amino Acid Sequence from the
N-terminus
[0130] Potential sites for phosphorylation were chosen at or close
to positions in GFP which had previously been identified to exert
significant effects on fluorescence, or which had a higher
probability of surface exposure based on computer algorithms (FIG.
4). For example, in a mutant called H9, Ser202 and Thr203 are
mutated to F and I respectively, creating a large change in
spectral properties (see also Ehrig et al, 1995). Therefore in one
mutant, 199RRLSI (SEQ ID NO: 18), a potential site of
phosphorylation was created around Ser202, whose phosphorylation
should significantly affect the fluorescent properties. Similarly
the amino acids located at positions 72 and 175 have been
implicated in increased folding efficiency of GFP at higher
temperatures and were made into potential sites of phosphorylation
in separate mutants.
[0131] A complete list of the positions and amino acid changes made
for each phosphorylation mutant in this series is outlined in FIG.
4. GFP was expressed in E. coli using the expression plasmid pRSET
(Invitrogen), in which the region encoding GFP was fused in frame
with nucleotides encoding an N-terminal polyhistidine tag (FIG. 5).
The sequence changes were introduced by site-directed mutagenesis
using the Bio-Rad mutagenesis kit (Kunkel, T. A. (1985) Proc. Natl.
Acad. Sci. 82:488-492, Kunkel,T. A., Roberts, J. D., and Zakour, R.
A. (1987) Meth Enzymol 154:367-382) and confirmed by sequencing.
The recombinant proteins were induced with IPTG and expressed in
bacteria and purified by nickel affinity chromatography. The
sequence changes, relative fluorescence, relative rate of
phosphorylation and % change in fluorescence upon phosphorylation
are listed in Table II. In those cases where the protein exhibited
no fluorescence after insertion of the phosphorylation site no
determinations were made on the effect of phosphorylation on
fluorescence.
5TABLE II Relative fluorescence, rate of phosphorylation and change
in fluorescence upon phosphorylation for mutants incorporating
phosphorylation sites remote from the N-terminus Fluorescence %
Change in SEQ before Relative fluorescence ID phosphorylation rates
of after incubation NO: Sequence (% of wild type) phosphorylation
with kinase 19 25RRFSV 95 1.75 -5 20 68RRFSR 0 n.d n.d 14 68RRFSA 6
0.6 +10 21 94RRSIF 0 n.d n.d 22 131RRGSIL 0 n.d n.d 23 155KRKSGI 86
2.5 0 24 172RRGSV 90 1.57 0 18 199RRLSI 0 n.d n.d 15 214KRDSM 21
1.88 +40
[0132] Bold letters indicate site of phosphorylation. Numbers prior
to the sequence indicate amino acid position in wild type GFP (FIG.
3, SEQ ID NO:2) where phosphorylation site starts. The relative
rates of phosphorylation compare the rate of phosphorylation of the
given phosphorylation site with the endogenous protein kinase A
phosphorylation site in Aequorea GFP (HKFSV (SEQ ID NO:45))
measured by incorporation of .sup.32P after incubation of the
purified substrate and protein kinase A catalytic subunit in the
presence of .sup.32P-labelled ATP using 3 .mu.g GFP, 5 .mu.g
protein kinase A catalytic subunit for 10 minutes at 30.degree. C.
in standard phosphorylation buffer (20 mM MOPS pH 6.5, 100 mM KCl,
100 .mu.M ATP, 3 mM MgCl.sub.2 1 mM DTT and 100 uCi
.sup.32P-labeled ATP. Reactions were terminated by blotting onto
phosphocellulose paper and washing with 10% phosphoric acid. The %
change in fluorescence represents the increase in fluorescence (475
nm excitation, 510 nm emission) observed in each purified protein
resulting from incubation with excess protein kinase A catalytic
subunit for 1 hour at 30.degree. C. using the same phosphorylation
conditions as described above except that no .sup.32P-labeled ATP
was present and that after the reaction time was complete samples
were analyzed in the fluorimeter rather than blotted onto
phosphocellulose paper.
[0133] The greatest changes in fluorescence occurred in mutant
214KRDSM (SEQ ID NO: 15) which exhibited a 40% change in
fluorescence upon phosphorylation. However analysis of the kinetics
of phosphorylation using .gamma.-.sup.32P-labeled ATP demonstrated
that the site is poorly phosphorylated by protein kinase A. Wild
type GFP contains a mediocre consensus phosphorylation site
(25HKFSV (SEQ ID NO:45)) that can be phosphorylated by protein
kinase A in vitro with relatively slow kinetics. While
phosphorylation at this position has no detectable effect on the
fluorescence of GFP, the rate of phosphorylation at this position
is used as an internal control between experiments to determine the
relative rates of phosphorylation at sites engineered into the
protein by site directed mutagenesis.
[0134] B. Phosphorylation Sites Around the Amino Terminus
[0135] Sites at the N-terminus of GFP were engineered into GFP by
PCR. Initial studies attempted to preserve the native sequence as
much as possible. As discussed earlier the positions chosen for
phosphorylation were within the first 5 amino acids of GFP and
encompassed all charged residues within this region. The sequence
changes, relative fluorescence, relative rates of phosphorylation
and % change in fluorescence upon phosphorylation are tabulated in
Table III.
6TABLE III Relative fluorescence, rate of phosphorylation and
change in fluorescence upon phosphorylation for phosphorylation
sites inserted at the N-terminus Relative fluorescence Relative SEQ
ID as a % of rates of % Change in NO: Sequence wild type
phosphorylation fluorescence 48 1MSKGEELF 100 1.0 0 25 1MRKGSCLF 40
5.1 5.7 26 1MRKGSLLF 52 1.6 8.0 27 1MRRESLLF 30 3.0 6.0 28
1MRRDSCLF 27 3.7 17 29 1MSRRDSCF 43 2.1 25 30 1MSKRRDSL 7 5.5
5.1
[0136] Numbers prior to the sequence indicate amino acid position
in wild type GFP where phosphorylation site starts. The relative
rates of phosphorylation compare the rate of phosphorylation of the
given phosphorylation site with the endogenous protein kinase A
phosphorylation site in Aequorea GFP (HKFSV (SEQ ID NO:45))
measured by incorporation of .sup.32p after incubation of the
purified substrate and protein kinase A catalytic subunit in the
presence of .sup.32P-labelled ATP using the standard protocols
described earlier. The % change in fluorescence represents the
change in fluorescence (488 nm excitation, 511 nm emission)
observed in each purified protein as a result of incubation with
excess protein kinase A catalytic subunit for 1 hour at 30.degree.
C. using phosphorylation conditions described earlier.
[0137] These results demonstrated that mutants whose sequence
closely resembles the native protein retain considerable
fluorescence, display good kinetics of phosphorylation, but show
relatively small changes in fluorescence after phosphorylation. To
improve the effect of phosphorylation on fluorescence, amino acids
around the phosphorylation site were mutated to create an optimal
phosphorylation sequence even if it disordered the existing local
tertiary structure. Such disruption was predicted and found to
decrease the basal fluorescence of these constructs in their
non-phosphorylated state (Table IV).
7TABLE IV Relative fluorescence before phosphorylation and change
in fluorescence upon phosphorylation for more drastically altered
phosphorylation sites inserted at the N-terminus Relative % Change
in SEQ fluorescence fluorescence ID as a % of upon NO: Sequence
wild-type phosphorylation 48 1MSKGEELF(=WT) .congruent.100 0 31
1MSRRRRSI 5.8 40 32 1MRRRRSII 5.1 70 33 -1MRRRRSIII n.d. 43 34
-2MRRRRSIIIF 0.7 15 35 -3MRRRRSIIIIF 0.6 70
[0138] Numbers prior to the sequence indicate amino acid position
in wild type GFP where phosphorylation site starts. Negative
numbers indicate extensions onto the wild-type N-terminus. The %
change in fluorescence represents the change in fluorescence (488
excitation, 511 emission) observed in each purified protein
resulting from incubation with excess protein kinase A catalytic
subunit for 1 hour at 30.degree. C. using standard phosphorylation
conditions described earlier.
[0139] Perhaps because of the reduced basal fluorescence,
phosphorylation by protein kinase A produced greater percentage
increases in fluorescence in these constructs than in the more
conservative mutations of Table II. Constructs 1MRRRRSII (SEQ ID
NO:32) and -3MRRRRSIIIIF (SEQ ID NO:35) displayed the greatest
increases, about 70%, in fluorescence upon phosphorylation using
the standard conditions, as shown in FIG. 6. However, these
increased percentage increases were obtained at the cost of a
reduced ability to fold at higher temperatures and relatively poor
fluorescence even after phosphorylation. To improve these
characteristics, these mutants were further optimized by additional
random mutagenesis with a novel selection procedure.
[0140] C. Further Optimization of N-terminal Phosphorylation Sites
by Random Mutagenesis of the Remainder of GFP
[0141] The two best constructs from above (1MRRRRSII (SEQ ID NO:32)
and -3MRRRRSIII IF (SEQ ID NO:35)) were further mutagenized and
screened for variants that are highly fluorescent when
phosphorylated, but weakly fluorescent when non-phosphorylated. The
method involved expression of a randomly mutated fluorescent
substrate with or without simultaneous co-expression of the
constitutively active catalytic subunit of protein kinase A in
bacteria, and screening the individual mutants to determine those
that are highly fluorescent in the presence but not the absence of
the kinase.
[0142] To enable co-expression of the kinase and potential
substrates, a new expression vector with the kinase C subunit
upstream from the fluorescent substrate was constructed (FIG. 7).
Random mutations were introduced into GFP by error-prone PCR and
the resulting population of mutants cloned into the co-expression
vector using the appropriate restriction sites. The expression
vector containing the mutated fluorescent substrates were
transformed into host bacteria and individual bacterial colonies
(each derived from a single cell, and hence containing a single
unique mutant fluorescent substrate) were grown up.
[0143] The colonies were screened for fluorescence either by
fluorescence-activated cell sorting (FIG. 8) or by observation
under a microscope. Those that exhibited the greatest fluorescence
were re-screened under conditions in which the kinase gene was
inactivated. This was achieved in either of two ways. In the first
method the co-expression vector was isolated and treated with
restriction endonucleases and modifying enzymes (EcoR1, klenow
fragment and T4 DNA ligase) to cut the kinase gene, add additional
bases and relegate the DNA, causing a frame shift and hence
inactivating the gene. The treated and non-treated plasmids were
then re-transformed into bacteria and compared in fluorescence.
Alternatively the plasmids were initially grown in a RecA.sup.-
(recombinase A negative) bacterial strain, where the kinase is
stable, to screen for brighter mutants in the presence of the
kinase. The plasmid DNA was then isolated and re-transformed into a
strain of bacteria which is RecA.sup.+, in which the kinase is
unstable and is lost through homologous recombination of the
tandomly repeated ribosome binding sites (rbs). The bacteria have a
strong tendency to eliminate the kinase C subunit because it slows
their multiplication, so cells that splice out the kinase by
recombination have a large growth advantage.
[0144] Comparison of the brightness of the mutant first in the
presence of kinase then in its absence indicates the relative
effect of phosphorylation on the mutant GFP fluorescence (after
normalizing for GFP expression levels). A library of approximately
2.times.10.sup.6 members was screened by this approach.
Approximately 500 displayed higher levels of fluorescence when
screened in the presence of the kinase. After inactivation of the
kinase, one mutant out of the 500 displayed reduced levels of
fluorescence. The increased fluorescence of the remainder of the
500 mutants was independent of the presence of the kinase. This
mutant GFP was isolated and sequenced and found to contain the
following mutations compared to wild-type GFP (FIG. 3, SEQ ID NO:2)
(in addition to the N-terminal phosphorylation site 1MRRRRSII (SEQ
ID NO:32)): S65A, N149K, V163A and I167T.
[0145] To confirm that this mutant was indeed directly sensitive to
protein kinase A phosphorylation and to quantify its responsively,
it was expressed in the absence of kinase. The E. coli were lysed
and the protein purified as described earlier using a nickel
affinity column. The protein exhibited high levels of fluorescence
when induced at 30.degree. C. but displayed reduced fluorescence
when incubated at 37.degree. C. After such preincubation
(37.degree. C. overnight) and separation of the less fluorescent
material by centrifugation, this protein exhibited the largest
change in fluorescence upon phosphorylation yet observed (FIG. 8).
The tolerance of this mutant for 37.degree. C. treatment suggests
that this mutant is suitable for use in mammalian cells.
[0146] The present invention provides novel assays for protein
kinase activity involving novel fluorescent protein substrates.
While specific examples have been provided, the above description
is illustrative and not restrictive. Many variations of the
invention will become apparent to those skilled in the art upon
review of this specification. The scope of the invention should,
therefore, be determined not with reference to the above
description, but instead should be determined with reference to the
appended claims along with their full scope of equivalents.
[0147] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 1
1
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