Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF MEASURING PROTEIN-PROTEIN
INTERACTIONS IN LIVING CELLS
This invention was made with Government support under National Institutes
of Health grants GM-48113 and GM-51457. The Government has certain rights to
this invention.
Field of the Invention
The present invention concerns methods of detecting or determining binding
between two different proteins, or oligomer formation by the same protein, in
living
i 5 cells, along with cells and kits useful for carrying out such methods.
Background of the Invention
The binding of proteins to one another, or the disruption of binding of one
protein to another by a competitive inhibitor, is typically measured in vitro.
Such
binding assays are typically used as a model for in vivo, including
intracellular,
binding events. While such techniques are well established, the in vitro
binding
conditions do not control for the vast number of variables introduced when a
binding
event occurs within a cell. Because of the importance of screening for new
binding
partners, or inhibitors of known binding partners, to the development of new
therapeutic molecules, the development of techniques that measure binding
within a
cell is extremely important.
The "pull down" assay is known, in which the binding of a pair of proteins of
interest is determined by forming a co-precipitate with an antibody in vitro
and then
centrifuging down, or "pulling down" the aggregate so formed. Disadvantages of
this
technique are that it is carried out in vitro, and that substantial
nonspecific binding
occurs.
The yeast "two hybrid" technique employs a pair of transcription factors that
trigger the transcription of a selectable or detectable protein. The technique
has been
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adapted and extended to a number of situations, including examination of
enzyme-
substrate interactions. R. Sikorski and R. Peters, Science 281, 1822-1823 (18
Sept.
1998). In general, a first hybrid is formed of a first protein of interest and
one of the
transcription factors; a second hybrid is formed of a second protein of
interest and
5 another of the transcription factors. If the two proteins of interest
associate, then the
two transcription factors associate and transcription of the detectable or
selectable
protein is initiated. Advantages of this technique are that binding occurs in
a cell, and
it can be readily adapted to the screening of combinatorial libraries by
inserting
various members of the library in different "two hybrid" cells and expressing
the
10 library transcription praducts therein. Disadvantages of this technique are
that it is
limited to the use of transcription factors, the binding reaction must be in a
narrow
range, is typically earned out in yeast, and the binding event must occur in
the cell
nucleus.
Accordingly, there is a need for new methods of screening for protein-protein
15 interactions that can be carried out in living cells, that can be carried
out in a variety
of locations within a living cell, and that are readily adapted to the
screening of
combinatorial libraries.
Summary of the Invention
20 A first aspect of the present invention is a method of detecting a protein-
protein interaction in a living cell. The method comprises (a) providing a
cell that
contains a first heterologous conjugate and a second heterologous conjugate,
wherein
the first heterologous conjugate comprises a first protein of interest
conjugated to a
detectable group, and wherein the second heterologous conjugate comprises a
second
25 protein of interest conjugated to a protein that specifically binds to an
internal
structure within the cell, and then (b) detecting the presence or absence of
binding of
the detectable group to the internal structure, the presence of the binding
indicating
that the first and second proteins of interest specifically bind to one
another.
The proteins of interest may be the same or different; the proteins of
interest
30 may be members of a specific binding pair. Preferably, the detectable group
is a
protein, and the first protein of interest and the detectable group together
comprise a
fusion protein. Preferably, the second heterologous conjugate is also a fusion
protein.
If desired, the cell may contain and express a nucleic acid encoding either,
or both,
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fusion proteins, or the heterologous constructs may be administered
exogenously to
the cells. The cell is preferably a eukaryotic cell.
Additional aspects of the present invention include nucleic acids encoding
fusion proteins as described above, cells containing and expressing such
fusion
5 proteins, kits useful for carrying out the methods described above, and
nucleic acid
libraries useful as screening tools for carrying out the methods described
above.
The invention is useful for screening compounds for the ability to disrupt or
inhibit the binding of known binding pairs and thereby identifying competitive
inhibitors thereof. The invention is useful for screening one known protein of
interest
10 against a library of other proteins of interest to identify compounds that
bind to the
known protein of interest.
The foregoing and other objects and aspects of the present invention are
explained in detail in the drawings herein and the specification below.
15 Brief Description of the Drawings
Figure 1. Cellular localization and mobility of GFP-tagged CaMKIIa and
CaMKIIb
Figure lA. Schematic representation of the domain organization of the GFP-
tagged CaMKII isoforms. The catalytic-domain (C), regulatory-domain (R),
variable-
20 domain (V) and oligomerization-domain (A) are shown.
Figure 1B. Autophosphorylation of GFP-tagged CaMKII isoforms.
Comparison of the baseline (left), calcium/CaM-dependent (middle) and burst
(right)
autophosphorylation activity of CaMKIIa, GFP-CaMKIIa and GFP-CaMKIIb. The
kinase activity of the in. vitro translated constructs are shown. Translated
GFP alone
25 was included as a control.
Figure 1C. Relative kinase activity corrected for the amount of expressed
CaMKII or GFP-CaMKII protein (measured as the ratio of 32P incorporation and
35S-
Met incorporation). The dark bars show CaMKII autophosphorylation after
incubation with 32P-ATP in high Ca2+/CaM for 30 seconds. The light bars show
30 the calcium independent "burst" autophosphorylation after 30 s in high
calcium and
120 seconds in EGTA.
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Figure 1D. Confocal image of GFP-tagged CaMKIIa (left) and CaMKIIb
(right) expressed in living hippocampal CAI-CA3 neurons. Notice the homogenous
distribution of GFP-CaMKIIa throughout the soma and major branches, while
CaMKIIb is enriched in the dendritic branches.
Figure lE. Confbcal image of GFP-tagged CaMKIIa (left) and CaMKIIb
{right) expressed in living RBL-cells. Notice the cortical staining and non-
uniform
internal staining of GFP-CaMKIIb and the homogenous distribution of GFP-
CaMKIIa.
Figure 1F. Comparison of the calculated diffusion coefficients for GFP-
CaMKIIa and GFP-CaMKIIb.
Figure 2. Development of a "Pull-Out" binding assay to study protein-protein
interactions in living cells.
Figure 2A. Principle of the Pull-out binding assay. The binding interaction
between a Protein X and Protein Y can be measured by tagging Protein X with an
I S inducible plasma membrane binding domain (PM-domain) and Protein Y with
GFP.
If a significant fraction of the two proteins bind to each other, drug
addition targets
the GFP to the plasma membrane. In contrast, the cytosolic distribution
remains
unaltered if Protein X and Y do not bind to each other.
Figure 2B. Property of a minimal phorbol ester binding domain used as an
inducible PM-domain in the Pull-Out binding assay. A fusion protein between
GFP
and the phorbol ester binding domain can be pulled from the cytosol to the
plasma
membrane by addition of phorbol ester. Left, distribution of the fusion
protein before
phorbol ester addition. Right, distribution of the fusion protein after
phorbol ester
addition. Bottom, line scans of the fluorescence intensity across the cell
before and
after phorbol ester addition.
Figure 2C. Demonstration that nearly all CaMKIIa molecules are part of
oligomers. Phorbol ester addition to cells with co-expressed PM-CaMKIIa and
GFP-
CaMKIIa leads to the near complete plasma membrane translocation of GFP-
CaMKIIa.
Figure 2D. Control measurements showing that expressed GFP itself is not
affected by phorbol ester addition. Calibration bars are 10~m.
Figure 3. CaMKIIa forms larger oligomers than CaMKIIb.
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Figure 3A. Schematic representation of the assay to measure the size of
CaMKIia and CaMICIIb oligomers in living cells.
Figure 3B. Quantitative comparison of the concentrations of expressed GFP-
CaMKIIa and PM-CaMKIIa measured by in vitro translation. The relative
S concentration of the expressed proteins was compared by 35S-Met
incorporation into
in vitro translated proteins. The same RNA was used for the in vitro
translation and
the RNA transfection of cells.
Figure 3C. Only for dilutions below 10% does PM-CaMKIIa begin to loose
its potency to translocate GFP-CaMKIIa to the plasma membrane. Dilutions were
achieved by mixing the RNA's for PM-CaMKIIa and GFP-CaMKIIa at decreasing
ratios. Top and middle, plasma membrane translocation is still near maximal at
dilutions of 1:1 and 1:5. Bottom, plasma membrane translocation is markedly
reduced at a 1:40 dilutian. Calibration bars are 10~m.
Figure 3D. Line scan profiles of three different dilution after phorbol ester
addition.
Figure 3E. Schematic representations of the quantitative analysis used to
measure the relative plasma membrane translocation. Ipre and DPM are measured
before and after PMA addition, respectively.
Figure 3F. Plot of the relative plasma membrane translocation of CaMKIIa
and CaMKIIb at decreasing ratios of expressed PM-CaMKII and GFP-CaMKII. Each
point is an average of at least 10 experiments. The solid curves are best fits
to the two
set of data and the dashed lines show the confidence interval. Best fits were
obtained
assuming an average of 13.5 subunits for CaMKIIa and 4.2 subunits for CaMKIIb.
Figure 4. Requirement for more than one CaMKIIb subunits for targeting
CaMKIIa/b hetero-oligomers to the actin cytoskeleton.
Figure 4A. Insertion of CaMKIIb into hetero-oligomers of mostly CaMKIIa
is a stochastic process. Plot of the relative plasma membrane translocation of
GFP-
CaMKIIa at decreasing ratios of expressed PM-CaMKIIb. Each point is an average
of
at least 10 experiments. The dashed line shows the predicted curve for a
stochastic
insertion of PM-CaMKIIb into hetero-oligomers ( p = 1 - (R / (R+1))N with R as
the
dilution ratio and N as the number of subunits; since the PM-domain induces a
near
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irreversible membrane binding interaction, it was assumed that one PM-domain
per
oligomer is suffcient for inducing translocation).
Figure 4B. The insertion of CaMKIIa into hetero-oligomers with mostly
CaMKIIb is a stochastic process. Plot of the relative plasma membrane
translocation
of GFP-CaMKIIa at decreasing ratios of expressed PM-CaMKIIb. Each point is an
average of at least 10 experiments. The predicted curve for a stochastic
insertion of
PM-CaMKIIa into CaMKIIb oligomers overlaps with the fit of the data.
Figure 4C. Relative cortical localization of GFP-CaMKIIa plotted as a
function of increasing dilutions of CaMKIIb. Relative cortical localization is
defined
as DpM/Ia", with DpM as the intensity difference between the PM and the
cytosol and
Ia,, as the average fluorescence intensity of a particular cell. Each point is
an average
of at least 10 experiments.
Figure 4D. Measurement of the change in the diffusion coefficient as a
function of an increasing dilution of CaMKIIb to GFP-CaMKIIa. The apparent
diffusion coefficient of GFP-CaMKIIa increased from 0.2 to 1 mm2/s as the
ratio of
CaMKIIb to CaMKIIa was lowered from 1:2 to 1:9. The outermost left and right
data
points show the diffusion coefficients of GFP-CaMKIIb and CaMKIIa,
respectively.
Detailed Description of Preferred Embodiments
"Detectable groups" or "detectable proteins" used to carry out the present
invention include fluorescent proteins, such as green fluorescent protein
(GFP) and
apoaequorin, including analogs and derivatives thereof. Green fluorescent
protein is
obtained from the jellyfish Aequorea victoria and has been expressed in a wide
variety of microbial, plant, insect and mammalian cells. A. Crameri et al.,
Nature
Biotech. 14, 315-319 (1996). Any detectable group may be employed, and other
suitable detectable groups include other fluorophores or fluorescent
indicators, such
as a fusion tag with any binding domain such as avidin, streptavidin and
ligand
binding domains of receptors. Coupling of biotin or other ligands to the
fluorophore
or indicator of interest may be achieved using a dextran matrix or other
linker system.
The detectable protein may be one which specifically binds a fluorophore, as
in
FLASH technology. Fluorescent detectable groups (including both fluorescent
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proteins and proteins that bind a separate fluorophore molecule thereto) are
currently
preferred.
"Internal structure" as used herein refers to a separate, discreet,
identifiable
component contained within a cell. The term "structure" as applied to the
constituent
S parts of a cell is known (see, e.g., R. Dyson, Cell Biology: A Molecular
Approach, pg,
(2d ed. 1978)), and the term "internal structure" is intended to exclude
external
structures such as flagella and pili. Such internal structures are, in
general, anatomical
structures of the cell in which they are contained. Examples of internal
structures
include both structure located in the cytosol or cytoplasm outside of the
nucleus (also
10 called "cytoplasmic structures"), and structures located within the nucleus
(also called
"nuclear structures"). The nucleus itself including the nuclear membrane are
internal
structures. Structures located within the cytoplasm outside of the nucleus are
currently preferred. Thus the term "internal structure" is specifically
intended to
include any non-uniformly distributed cellular component, including proteins,
lipids,
carbohydrates, nucleic acids, etc., and derivatives thereof.
"Library" as used herein refers to a collection of different compounds,
typically organic compounds, assembled or gathered together in a form that
they can
be used together, either simultaneously or serially. The compounds may be
small
organic compounds or biopolymers, including proteins and peptides. The
compounds
20 may be encoded and produced by nucleic acids as intermediates, with the
collection of
nucleic acids also being referred to as a library. Where a nucleic acid
library is used,
it may be a random or partially random library, commonly known as a
"combinatorial
library" or "combinatorial chemistry library", or it may be a library obtained
from a
particular cell or organism, such as a genomic library or a cDNA library.
Small
25 organic molecules can be produced by combinatorial chemistry techniques as
well.
Thus in general, such libraries comprise are organic compounds, including but
not
limited oligomers, non-oligomers, or combinations thereof. Non-oligomers
include a
wide variety of organic molecules, such as heterocyclics, aromatics,
alicyclics,
aliphatics and combinations thereof, comprising steroids, antibiotics, enzyme
30 inhibitors, ligands, hormones, drugs, alkaloids, opioids, benzodiazepenes,
terpenes,
prophyrins, toxins, catalysts, as well as combinations thereof. Oligomers
include
peptides (that is, oligopeptides) and proteins, oligonucleotides (the term
oligonucleotide also referred to simply as "nucleotide, herein) such as DNA
and
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RNA, oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,
polyureas,
polyethers, poly (phosphorus derivatives) such as phosphates, phosphonates,
phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly (sulfur
derivatives) such as sulfones, sulfonates, sulfites, sulfonamides,
sulfenamides, etc.,
where for the phosphorous and sulfur derivatives the indicated heteroatom for
the
most part will be bonded to C, H, N, O or S, and combinations thereof. See,
e.g., U.S.
Patent No. 5,565,324 to Still et al., U.S. Patent No. 5,284,514 to Ellman et
al., U.S.
Patent No. 5,445,934 to Fodor et al. (the disclosures of all United States
patents cited
herein are to be incorporated herein by reference in their entirety).
"Nucleic acid" as used herein refers to both DNA and RNA.
"Protein" as used herein is intended to include protein fragments, or
peptides.
Thus the term "protein" is used synonymously with the phrase "protein or
fragment
thereof' (for the purpose of brevity), particularly with reference to proteins
that are
"proteins of interest" or members of a specific binding pair. Protein
fragments may or
1 S may not assume a secondary or tertiary structure. Protein fragments may be
of any
length, from 2, 3, 5 or 10 peptides in length up to 50, 100, or 200 peptides
in length or
more, up to the full length of the corresponding protein.
"Specifically binds" and "specific binding" as used herein includes but is not
limited to stereospecific binding, electrostatic binding, or hydrophlic
binding
interactions. Thus, specifically binds and specific binding are exhibited by
at least a
two or three fold (or two or three times}, greater apparent binding affinity
between the
binding partners as compared to other proteins or binding partners within the
cell in
which binding is being detected.
"Specific binding pair" refers to a pair of molecules (e.g., a pair of
proteins)
that specifically bind to one another. A pair of molecules that specifically
bind to one
another, which may be the same or different, are referred to as members of a
specific
binding pair. A protein that is a member of a specific binding pair may be a
protein
that has been previously determined to be a member of a specific binding pair
or a
protein that is a putative member of a specific binding pair. Examples of the
latter
include members of a Library, such as the products of a cDNA or combinatorial
library, or a protein for which a binding partner has not yet been identified,
where it is
desired to identify a naturally occuring (e.g., a product of a cDNA or genomic
DNA
library) or non-naturally occuring (e.g., combinatorial) binding partner
therefore.
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"Translocation" as used herein refers to a change in distribution of a protein
or
conjugate (including a fusion protein) from one physical distribution within a
cell to
another, different, physical distribution within a cell. Preferably,
translocation is from
either a uniform or non-uniform distribution to a non-uniform distribution.
Translocation could also be from a non-uniform to a uniform distribution.
As noted above, the present invention provides a method of detecting a
protein-protein interaction. The method comprises first providing a cell that
contains
a first heterologous conjugate and a second heterologous conjugate. The first
heterologous conjugate comprises a first protein of interest conjugated to a
detectable
10 group. The second heterologous conjugate comprises a second protein of
interest
(which may be the same as or different from the first protein of interest)
conjugated to
a protein that specifically binds to an internal structure within the cell.
The binding of
the protein that specifically binds to an internal structure may be immediate,
may be
induced (as discussed below), or may be a prior binding in the case of a
protein that is
15 previously localized to or permanently located at the internal structure of
interest.
The two conjugates are preferably each present in the cell at a total
concentration
between about 1 or 10 nM to about 1 or 10 mM.
The presence or absence of binding of the detectable group to the internal
structure is then detected, the presence of the binding indicating that the
first and
20 second proteins of interest specifically bind to one another. Detection may
be by any
suitable means depending upon the detectable group employed, but preferably
the
detectable group is a fluorescent group and detection is earned out by optical
or visual
reading, which may be done manually, by an automated apparatus, or by
combinations thereof.
25 If desired, the second heterologous conjugate can further comprise a
detectable group, which detectable group is preferably different from the
detectable
group located on the first heterologous conjugate and fluoresces at a
different
wavelength therefrom. For example, both detectable groups could be a green
fluorescent proteins, yet simply different mutants of green fluorescent
protein that
30 fluoresce at different wavelengths.
Either or both of the heterologous conjugates may be introduced directly in
the
cell by any suitable means, such as by electroporation or lipofection. In the
alternative, when the heterologous conjugates are fusion proteins, a nucleic
acid
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(typically a DNA) may be stable introduced into the cell (for example, as a
plasmid),
which nucleic acid includes a suitable promoter segment that controls and
causes the
expression of a nucleic acid encoding the fusion protein. Again, either or
both of the
fusion proteins may be produced in the cell in this matter.
5 ~ Binding events in the instant invention may be direct or indirect binding
events. Indirect binding events are those mediated through an intermediate, or
bridging, molecule or conjugate. Administration of such a bridge molecule can
be a
signal to induce translocation (discussed below). For example, the bridging
molecule
may be a covalent conjugate of FK506 and cyclosporin, to cause the indirect
binding
10 of FKBP12 and cyclophilin (both conventionally cytosolic proteins) to one
another.
Either of the FKBP 12 or the cyclophilin can be modified so that it binds to
the plasma
membreane, such as by lipidating the protein or forming a fusion protein with
the
transmembrane domain of a transmembrane protein.
Cells used to carry out the present invention are typically eukaryotic cells,
15 which may be yeast, plant, or animal cells. Yeast and animal cells,
particularly
mammalian cells, are currently preferred. Example plant cells include, but are
not
limited to, arabidopsis, tobacco, tomato and potato plant cells. Example
animal cells
include, but are not limited to, human, monkey, chimpanzee, rat, cat, dog, and
mouse
cells.
20 Any internal structure as defined above can be used to carry out the
present
invention, as long as the binding of the detectable group to the internal
structure
provides a different detectable signal from the cell than when the detectable
group is
not bound to the internal structure. In one preferred embodiment the internal
structure
is contained in the cell cytoplasm. Examples of internal structures include,
but are not
25 limited to, plasma membrane, cytoskeleton (including but not limited to
actin
cytoskeleton, tubulin cytoskeleton, intermediate filaments, focal adhesions,
etc.),
centromere, nucleus, mitochondria, endoplasmic reticulum, vacuoles, golgi
apparatus,
and chloroplasts. Preferably, the internal structure is either the plasma
membrane or
cortical cytoskeleton.
30 In a preferred embodiment of the invention, the protein that specifically
binds
to an internal structure is a translocatable protein. In this embodiment, the
method
further comprises the step of inducing translocation of the second
heterologous
conjugate prior to the detecting step. Induction of translocation may be
carried out by~
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any suitable means, such as by administration of a physical or chemical signal
(e.g.,
administration of a compound such as a phorbol ester). Such a protein may be
selected from the group consisting of cytosolic protein kinases, protein
phosphatases,
adapter proteins, cytoskeletal proteins, cytoskeleton associated proteins, GTP-
binding
5 proteins, plasma transmernbrane proteins, plasma membrane associated
proteins, (3-
arrestin, and visual arrestin (including fragments thereof that specifically
bind to an
internal structure). Preferably, the protein is a protein kinase C isoform or
a fragment
thereof that specifically binds to an internal structure, such as a C 1 domain
fragment
or a C2 domain fragment, where the induction signal is administration of a
phorbol
10 ester. In addition, induction of translocation may be induced by
stimulation of a
receptor, such as a glutamate receptor, beta-adrenergic receptor, or PAF
receptor, with
a receptor agonist to induce a signaling step which in turn induces
translocation.
Finally, numerous proteins may be modified to make them translocatable by
employing bridging molecules, as discussed above.
15 As noted above, in one embodiment of the invention the first and second
proteins of interest may together comprise members of a specific binding pair.
In this
embodiment, the invention may further include the step of administering a test
compound to the cell prior to the detecting step, wherein the absence of
binding of the
detectable group to the internal structure indicates that the test compound
inhibits the
20 binding of the members of the specific binding pair. Any test compound can
be used,
including peptides, oligonucleotides, expressed proteins, small organic
molecules,
known drugs and derivatives thereof, natural or non-natural compounds, etc.
Administration of the test compound may be by any suitable means, including
direct
administration such as by electroporation or lipofection if the compound is
not
25 otherwise membrane permeable, or (where the test compound is a protein), by
introducing a heterologous nucleic acid that encodes and expresses the test
compound
into the cell. Such methods are useful for screening libraries of compounds
for new
compounds which disrupt the binding of a known binding pair.
The method may be used to quantitatively determine binding affinity by
30 varying the concentration of either construct to measure the binding
affinity of the
constructs at different concentrations, or (where the members of the specific
binding
pair are the same) to establish the size of the oligomers formed.
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As also noted above, in another embodiment of the invention, the method
further comprises the step of: (c) repeating steps (a) and (b) a plurality of
times with a
library of proteins of interest, wherein one of the first and second proteins
of interest
is maintained the same and the other of the first and second proteins of
interest (the
variable protein or the protein being screened) is replaced with a different
member of
the library, so that the library is screened for proteins that specifically
bind to one of
the first or second proteins of interest. Repeating of the steps may be
carried out
serially, simultaneously, or both serially and simultaneously. Administration
of the
protein of interest that is varied may be by any suitable means, including
direct
administration such as by electroporation or lipofection if the compound is
not
otherwise membrane permeable, or (where the test compound is a protein), by
introducing a heterologous nucleic acid that encodes and expresses the
variable
protein of interest into the cell. Such methods are useful for screening
libraries of
compounds for new candidates for binding to a known protein.
The invention provides fusion proteins comprising a protein that specifically
binds to an internal structure within a cell and a protein of interest, such
as a protein
that is a member of a specific binding pair, along with nucleic acids encoding
such
fusion proteins and cells that contain and express such nucleic acids (the
nucleic acid
thus including regulatory sequences operative in the cell and operatively
associated
with the nucleic acid segment that encodes the fusion protein). Likewise the
present
invention provides fusion proteins comprising a protein that is a detectable
group and
a protein of interest, such as a member of a specific binding pair,
In one embodiment, a kit useful for detecting protein-protein interactions
within a living cell, comprises (a) a cell as described above that contains
and
expresses a nucleic acid encoding a first fusion protein, the fusion protein
comprising
a protein that specifically binds to an internal structure within the cell and
a first
protein of interest; together with (b) a vector for the cell, the vector
containing an
expression cassette. The expression cassette comprises a promoter operable in
the
cell and operatively associated with a nucleic acid encoding a detectable
protein, and
has a splice site positioned adjacent the nucleic acid encoding a detectable
protein so
that a heterologous nucleic acid encoding a second protein of interest can be
inserted
therein to produce a nucleic acid segment encoding a second fusion protein.
The
second fusion protein comprising the detectable protein and the second protein
of
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interest. In an alternate embodiment, a kit useful for detecting protein-
protein
interactions within a living cell, comprises (a) a cell as described above
that contains
and expresses a nucleic acid encoding a first fusion protein, the fusion
protein
comprising a detectable protein and a first protein of interest; together with
(b) a
5 vector for the cell, the vector containing an expression cassette, the
expression
cassette comprising a promoter operable in the cell and operatively associated
with a
nucleic acid encoding a protein that specifically binds to an internal
structure within
the cell, as described above. The expression cassette likewise has a splice
site
positioned adjacent the nucleic acid encoding the protein that specifically
binds to an
10 internal structure within the cell (as described above; preferably a
translocatable
protein as described above) so that a heterologous nucleic acid encoding a
second
protein of interest can be inserted therein to produce a nucleic acid segment
encoding
a second fusion protein. The second fusion protein comprises the protein that
specifically binds to an internal structure within the cell and the second
protein of
15 interest. Such kits can be provided in any suitable form, and are typically
packed
together with suitable instructions. Any vector may be employed, but the
vector is
typically a plasmid vector.
In using kits as described above, the vector is used in one embodiment to
create, from a source nucleic acid library as described above, a product
nucleic acid
20 library comprising a plurality of separate nucleic acids, each of the
separate nucleic
acids encoding a fusion protein, the fusion protein comprising a protein of
interest
(encoded by the source library) and a detectable protein, wherein the protein
of
interest encoded by each of the separate nucleic acids is different from the
protein of
interest encoded by the other nucleic acids of the library. The product
library, in the
25 vector, can then be used to transform multiple cells so that the library
constituents can
be screened in the manner described above.
In the alternate embodiment described above, the vector is used to create,
from
a source nucleic acid library as described above, a product nucleic acid
library
comprising a plurality of separate nucleic acids, each of the separate nucleic
acids
30 encoding a fusion protein, the fusion protein comprising a protein of
interest (from the
source library) and a protein that specifically binds to an internal structure
within a
cell as described above (preferably a translocatable protein as described
above),
wherein the protein of interest encoded by each of the separate nucleic acids
is
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different from the protein of interest encoded by the other nucleic acids of
the library.
Again the product library, in the vector, can then be used to transform or
transfect
multiple cells so that the library constituents can be screened in the manner
described
above.
Screening of libraries, in either of the foregoing embodiments, may be carried
out in accordance with canventional techniques. Typically, the screening will
be
carried out by transfecting pools (subsets of the members of the library) into
different
groups of cells to express the protein of interest. This allows one to
identify pools
that contain binding partners that interact with the protein of interest.
Pools with
binding partners can then again be divided into subpools until individual
members
(for example, individual cDNA sequences) of the library are identified that
bind to the
protein of interest.
The present invention is explained in greater detail in the following non-
limiting examples.
Examples
Ca2+/calmodulin dependent protein kinase II (CaMKII) is a ubiquitous kinase
which is expressed at high concentrations in neurons and at lower
concentrations in
most other cell types. Previous studies suggested that CaMKII is an essential
mediator for long term potentiation and other forms of synaptic plasticity
(reviewed in
Braun and Schulman, 1995; Soderling, 1993) . Furthermore, CaMKII activity may
have an important role in stabilizing the dendritic architecture (Wu and
Cline, 1998).
A critical neuronal function of the a-isoform of CaMKII (CaMKIIa) was directly
demonstrated by studying mice which were either lacking CaMKIIa or which
expressed mutated CaMKIIa. CaMKIIa deficient mice as well as transgenic mice
expressing an autonomously active or an autophosphorylation deficient CaMKIIa
showed impaired long term potentiation as well as defects in spatial learning
and
memory (Chapman et al., 1995; Glazewski et al., 1996; Gordon et al., 1996;
Mayford
et al., 1996; Mayford et al., 1995; Silva et al., 1992; Giese et al., 1998).
Since not only CaMKIIa but also CaMKII(3 is a prominent isoform in the
central nervous system, the question arises whether CaMKIIa and CaMKII(3 have
different roles in regulating neuronal functions. Such functional differences
between
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the two isofarms would have a direct impact on our understanding of cell type
specific signaling processes since the relative expression of CaMKIIa and
CaMKII/3
is markedly different in different brain regions and at different
developmental stages.
For example, the ratios of a and (3 subunits are about 3:1 and i :4 in adult
forebrain
and cerebellum, respectively, while in 10-day postnatal mice, the forebrain a
: b ratio
is 1:1 (Miller and Kennedy, 1985). On a structural basis, recombinant CaMKIIa
as
well as purified brain CaMKII has been shown to form oligomers with
approximately
8 to 12 subunits (Kanaseki et al., 1991, Bennett et a1.,1983) . CaMKIIø and
CaMKIIa have a similar overall domain organization and corresponding
autophosphorylation consensus sequences and even though the calmodulin binding
affinity of CaMKII~3 is slightly higher than that of CaMKIIa, the regulation
of
different CaMKII isoforms by Ca2+/CaM and autophosphorylation is overall
similar
(Miller and Kennedy, 1985; De Koninck and Schulman, 1998; GuptaRoy and
Griffith,
1996). Despite these similarities, it has been controversial whether CaMKII(3
forms
1 S oligomers on its own (Yamauchi et al., 1989), whether CaMKIIa and CaMKII(3
form
hetero-oligomers when expressed at the same time (Kanaseki et al., 1991) and
whether the two isoforms are differentially localized within cells (Scholz et
aL, 1988,
Nomura et al., 1997).
Here green fluorescent protein (GFP) - tagged CaMKIIa and CaMKII~i
isoforms are used to explore the subcellular localization and oligomerization
of
CaMKIIa and CaMKII~i. It was found that dendritic spines and filopodia as well
as
the cortical cytoskeleton are the primary docking sites for expressed
CaMKII(3. In
contrast, expressed CaMKIIa was uniformly distributed in the soma and
processes
and was largely absent from spines. However, when expressed in the same cell,
CaMKII~i targeted CaMKIIa to dendritic spines and the cell cortex. In vitro
binding
studies suggested that this targeting results from a direct binding
interaction of
CaMKII(i with F-actin.
A GFP-based protein-protein interaction assay (Pull-Out binding assay) was
then developed to explore the binding interactions between CaMKIIa and
CaMKII~3
isoforms in living cells. When expressed alone, CaMKIIb was found to form homo-
oligomers with an average size that is markedly smaller than the approximately
thirteen subunits measured for CaMKIIa homo-oligomers. When expressed at the
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same time, CaMKIIb isoforms incorporated equally well into either CaMKIIa or
CaMKIIb oligomers (and vice versa). Half maximal targeting of CaMKIIa
oligomers
to the cytoskeleton was achieved if at least 15% of CaMKIIb were present in
the same
cell, suggesting that a small number of CaMKIIb subunits are required to dock
5 CaMKIIa/b hetero-oligomers with approximately thirteen subunits to F-actin.
Our
studies suggests that the synaptic localization of CaMKII activity is
controlled by the
relative expression of CaMKIIb F-actin docking modules.
1. PROCEDURES
a. Cloning of CaMKII fusion constructs.
The cDNA for rat CaMKIIa ,(3 and (3' were generous gifts from Dr. Howard
Schulman. The construction of the GFP-CaMKIIa vector was described previously
(Shen and Meyer, 1998). To obtain the in vitro transcription vector for
CaMKIIa
without GFP, the CaMKIIa cDNA was amplified by PCR and cloned into the in
vitro
I 5 transcription vector dSHiro3. DNA sequencing were performed to exclude PCR
errors. GFP-CaMKIIb and CaMKIIb were also cloned into the SHiro3 and dSHiro3
vectors using a similar PCR strategy. The construction of PM-GFP or Cys-GFP
was
described previously (Oancea et al., 1998). PM-CaMKIIa and (3 were made by
replacing the GFP sequence with CaMKIIa and (3 coding sequence in the same
SHiro3 vector.
b. In vitro translation.
In vitro translation with SP6 RNA polymerise was performed according to the
manufacturer's protocol using a commercial kit (TNT Coupled reticulocyte
lysate
system, Promega). In vitro transcription reactions were performed using mRNAs
is
templates. The relative molar concentration of the different translated
proteins was
calculated by calibration using 35S-methionine incorporation and by counting
the
number of methionines in the respective protein. Non-radioactive methionine
was
used to obtain CaMKIIs and fusion constructs for the autophosphorylation
assay.
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c. Ca2+ dependent and independent autophosphorylation of CaMHII and GFP
fusion proteins.
CaMKII autophosphorylation assay were performed as described previously
(Hanson et al., 1994). Briefly, CaMKII isoforms and fusion constructs were
5 autophosphorylated at 30~C in 25m1 reactions containing 50 mM PIPES (pH
7.0),
IOmM MgCl2~ 500 mM CaCl2, 600 nM calmodulin, 50 mg/mI BSA, and 200 mM [g-
32p~ATp (6000cpm/pmol). The Ca2+ dependent autophosphorylation reaction was
started by adding in vitro translation product into the reaction rnix and
stopped by
addition of EDTA (16.7 mM final concentration) 30 seconds later. To measure
the
10 extent of the Ca2+-independent autophosphorylation, a 30 second reaction at
high
Ca2+ was followed by a 120 second secondary incubation in the presence of
added
EGTA (3.3 mM final concentration). In control experiments, 3.3 mM EGTA were
included in the initial reaction mix. The reaction mix was then resolved on a
SDS-
PAGE and subjected to Phosphorimager analysis. The densitometry of bands were
1 S measured and corrected by the amount of kinase which was determined in a
separate
in vitro translation reaction with 35S-methionine as described above.
d. In vitro transcription and RNA processing.
In vitro transcription and RNA processing were performed as described before
20 (Yokoe and Meyer, 1996; Shen and Meyer, 1998). Briefly, in vitro
transcription with
SP6 RNA polymerase was performed according to the manufacturer's protocol
using a
commercial kit (mMESSAGE mMACHINETM, Ambion). 10 mM EDTA was used to
terminate the reaction. RNA was purified by column chromatography (RNeasy
column, Qiagen) followed by the addition of a polyA tail. Poly adenylation was
25 carried out for 30 minutes at 37oC in a 50 pl reaction mixture containing
40 mM Tris-
HCl (pH 8.0), 10 mM MgCl2, 2.5 mM MnCl2, 250 mM NaCI, 0.25 mg / ul RNA, 250
mM ATP, 5 units poly(A) ~polymerase (Life Technologies). The reaction was
terminated by addition of 20 mM EDTA. Unincorporated ATP and salts were
removed by applying the mRNA to a RNeasy column. The eluent was dried and
30 mRNA was dissolved at 1 ug / pl in the electroporation buffer (5 mM KCI,
125 mM
NaCI, 20 mM HEPES pH 7.4 and 10 mM glucose).
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e. Cell culturing and electroporation
RBL 2H3 cells were maintained in Dulbecco's Minimum Essential Medium
(DMEM) supplemented with 20% fetal bovine serum (Life Technologies,
Gaithersburg, MD) at 37oC with 5% C02. The cells were plated at 5 X 104 cells
/
5 cm2 on glass cover slips and were allowed to attach to the coverslip for a
minimum of
3 hours. Hippocampal neurons obtained from 2 to 4 days postnatal rats were
cultured
as described in Ryan and Smith ( 1995) and used 10 days to three weeks after
plating.
A self built small volume electroporation device for adherent cells was used
for
electroporation (Teruel and Meyer, 1997). For the transfection of neurons,
modified
versions of the device and buffer conditions were used. After transfection,
the
electroporation buffers were replaced with the same culture medium.
f. Functional labeling of presynaptic terminals with FM 4-64
Functional presynaptic terminals were visualized with FM 4-64 (N-(3-
15 triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)
pyridinium,dibromide; Molecular Probes). FM 4-64 (Henkel and Betz, 1995 ) is
similar in structure and properties to FM 1-43 but its longer wavelength
emission
spectra make it more suitable for dual-channel fluorescence microscopy in
conjunction with green fluorescent protein (Ziv and Smith, 1996). Cells were
loaded
with FM 4-64 by replacing the saline in imaging chamber with high potassium
solution (100mM KCL, 20 mM HEPES, l.SmM CaCl2, 30mM NaCL,
l.SmMMgCL2, pH 7.4 and 6 pM of FM 4-64) for 20 seconds and switch back to a
saline solution for 5-10 min. After collecting a digital image of the labeled
field, the
cells were stimulated again by switching to the same high potassium solution.
The
spatial distribution of the active presynaptic terminals could then be
determined from
a difference image.
g. Diffusion analysis
The diffusion coefficients were determined using an analysis by which a
photobleached area is produced by a focused laser pulse and the fluorescence
recovery is fit to sequential 2-dimensional Gaussian distributions. Ratio
images of the
fluorescence distribution after the bleach pulse to the distribution before
the pulse
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were used for the analysis. This analysis follows at the same time the
decrease in the
bleach amplitude and the increase in the bleach radius. Assuming mass
conservation,
the resulting fluorescence distributions were fit by:
Fn( x ~ Y ) = 1 - (Fo * ao~ / ant) * exp ( - ( (x - xo)2 + (Y - Yo)2 ) / ant
)~ With x and y
as the pixel coordinates, an as the radius of the bleach diameter in the n-th
image, and
Fn(x, y) as the local relative fluorescence intensity. A least square fit
routine was
used to fit at the same time Gaussian profiles to all images in the time
series. An
approximate diffusion coefficient was then determined from a graph of the
square of
the radius, ant, versus time. The diffusion coefficient can be directly
obtained from
the slope of this graph (Dy / D x = 4 * D; with D as the diffusion coe~cient,
(Shen
and Meyer, 1998)).
h. Model calculations of a stochastic insertion of CaMKIIb subunits into
hetero-
oligomers
15 The probability of having one or more subunits randomly inserting into a
hetero-oligomer is [1-(probability to have no subunit inserted)]. The
probability of
having none inserted is (R/(R+I))N with R as the ratio of GFP-CaMKIIa to
CaMKIIb and N as the number of subunits.
i.Immunofluorescence
NIH-3T3, RBL cells, and hippocampal neurons were cultured on glass
coverslips and transfected with mRNA encoding GFP-CaMKIIb fusion construct.
Seven to eight hours after transfection, the cells were fixed for 10 minutes
at 4 oC
with 4% paraformaldehyde in PBS (1.2 mM KH2P04, 8.1 mM Na2HP04, 138 mM
NaCI and 2.7 mM KCl [pH 7.4]). NIH-3T3 cells and RBL cells were permeabilized
for S minutes at 4 oC with 0.1% Triton in PBS. Hippocampal neurons were
permeabilized for 10 minutes at 4 oC with 0.1% Triton. For F-actin staining,
rhodamine phalloidin (Molecular Probes) was incubated with the cells for 30
min at
room temperature at a dilution of 1:300. in PBS. For the staining of post-
synaptic
densities, hippocampal neurons were incubated with an monolonal PSD-95
antibody
(Cat.# OS-428, Upstate Biotechnology, Lake Placid, N~ overnight at 4 oC at a
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dilution of 1:200 and then in secondary Cy3 labeled anti-mouse antibody (Cat.#
11 S-
16S-062, Jackson ImmunoResearch, West Grove, PA) for 1 hour at room
temperature.
The cells were washed three times with PBS and coverslips were mounted onto
glass
slides using buffered glycerol mounting medium.
S
j. Fibroblast transfection and Western blotting assay
NIH-3T3 cells were plated in 3S mrn dishes at a density of 1.0 X lOS per dish
and incubated overnight at 37 oC in a humid atmosphere containing S% C02.
Cells
were transfected with 1.S -2.2 ug of pSRa-CaMKIIb or pSRa-CaMKIIa or
cotransfected with pSRa-CaMKIIb and pSRa-CaMKIIa using lipofectin plus
(GIBCOlBRL) according to manufacturer's instructions. Cells were harvested 48
hrs
after transfection and extracted with 10 mM Tris-HCI, SO mM KCI, 0.1 mM EGTA,
0.1% Triton, 2 mM PMSF and S ~zg/ml aprotinin, leupeptin and pepstatin at room
temperature for 10 min and centrifuged at 30,000 X g for 30 min at 4 oC.
Various
1 S fractions of the cell extract were resolved by electrophoresis on SDS-
polyacrylamide
gels ( 12%), transferred to nitrocellulose and blotted with monoclonal anti-
CaMKIIa
or anti-CaMKIIb antibody (GIBCO/BRL). The membranes were blotted using a
secondary antibody conjugated to horseradish peroxidase and visualized by ECL
(Amersham).
k. "Pull Out" protein-protein interaction assay
Poly-adenylated mRNA was made as described above. In many experiments,
mRNA species were mixed and used for electroporation at a final concentration
of
typically 1 p,g/~l total. The relative translation efficiency was determined
by a
2S separate in vitro translation reaction using the same mRNA as a template.
Images of
transfected RBL or NIH 3T3 cells were taken on a Zeiss confocal microscope 8-
12
hours after electroporation. 1 ~.M of PMA was added and images of single cells
were
taken under the same configuration. Images were taken before and after PMA
addition and were analyzed using NIH-image software. A plasma membrane
translocation factor was defined as DpM / Ip~e (Figure 2E).
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2. RESULTS
a. Expressed CaMKIIb but not CaMKIIa is enriched in dendritic branches and
cell cortex
The cellular localization of CaMKIIa versus CaMKIIb isoforms was
investigated by constructing CaMKII fusion proteins with GFP (Figure lA). An
earlier study has shown that a GFP-CaMKIIa construct can phosphorylate
substrate
peptides as well as autophosphorylate itself at threonine 28b (Shen and Meyer,
1998).
An additional criterion for functionally intact GFP-CaMKII is the preservation
of
secondary calcium-independent aut(Figures 1B and 1C).
When GFP-tagged CaMKIIa or b isoforms were expressed in cultured
hippocampal neurons, CaMKIIa was largely homogeneous in the soma and main
processes (Figure 1D, left) but was only minimally present in the finer branch
structures. In contrast, CaMKIIb showed a striking enrichment in dendritic
branches
as well as at the cell cortex (Figure 1D, right). When expressed in RBL-cells,
CaMKIIa was nearly homogeneously distributed in the cytosol and CaMKIIb had a
distinct cortical localization (Fig, lE, right).
The differential distribution of the two isoforms suggests that CaMKIIb has
specific binding interactions in cells that do not occur for CaMKIIa.
It was tested more directly whether CaMKIIb has more binding interactions
than CaMKIIa by comparing the local fluorescence recovery after photobleaching
of
GFP-CaMKIIb to that of GFP-CaMKIIa. A 2 ~m diameter laser photobleach spot
was generated in the cell by a short laser pulse and the fluorescence recovery
was
monitored by rapid confocal imaging. Consistent with the hypothesis that
CaMKIIb
but not CaMKIIa undergoes binding interactions, the recovery after
photobleaching
was significantly more rapid for CaKIIa compared to that for CaMKIIb. This
could
be quantitatively shown by a calculated average diffusion coefficient of
CaMKIIb that
was 5 times lower than that of CaMKIIa (Figure 1F, see Procedures above for a
description of the analysis). Nevertheless, the binding interactions of
CaMKIIb were
reversible, since most of the GFP-CaMKIIb fluorescence recovered on the time
scale
of 15 seconds after the laser bleach pulse. Together, these measurements
suggest that
CaMKIIa expressed alone is a highly mobile protein that has only limited
cytosolic
binding interactions, while CaMKIIb is bound in a reversible manner to
dendritic and
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cortical structures.
b. CaMKIIb is an F-actin docking module enriched in dendritic spines
What are the structures in the dendritic branches and cell cortex targeted by
CaMKIIb? The markedly punctuate staining suggested that CaMKIIb is enriched in
dendritic spines. Indeed, an antibody against the postsynaptic protein PSD-95
showed
a clear co-localization between GFP-CaMKIIb and PSD-95 (data not shown). The
arrows in the right panel point to presumed dendritic spines which were highly
enriched in PSD-95 and GFP-CaMKIIb. Some of the neurons also showed an
enriched staining of GFP-CaMKIIb in filopodia like branches (not shown) that
were
reminiscent of developing dendritic spines (Ziv and Smith, 1996). A magnified
image of an arbor of such filopodias is shown in the right panel.
It was verified that the dendritic spines marked by anti-PSD-95 antibodies and
GFP-CaMKIIb corresponded to mature postsynaptic terminals by comparing the
distribution of GFP-CaMKIIb to that of functional pre-synaptic terminals. The
terminals were marked with the fluorescent synaptic vesicle marker FM 4-64
using a
double depolarization protocol {Ziv and Smith, 1996; see Methods). Since this
type
of co-localization study can be performed in livine of an arbor of such
filopodian
artifacts that may arise during the fixation of neurons can be excluded. When
comparing the distribution of GFP-CaMKIIb to the location of active
presynaptic
terminals, the overlayed image showed a marked juxtaposed localization of GFP-
CaMKIIb and loaded FM 4-64 (not shown). This suggests that CaMKIIb is indeed
enriched in mature dendritic spines. In contrast, the uniformly distributed
GFP-
CaMKIIa was not enriched near active synapses {not shown).
Since actin is highly enriched in dendritic spines and cell cortex (Fisher et
al.,
1998; Landis and Reese, 1983, Caceres el al, 1983), it is conceivable that the
localization of CaMKIIb to dendritic spines is mediated by a direct or
indirect binding
interaction of CaMKIIb with F-actin. FM 4-64 was tested. This suggests that
CaMKIIb is indeed enriched in mature dendritic spines. In contrast, the
uniformly
distributed GFP-CaMKIIa was not enriched near active synapses (data not
shown).
Since actin is highly enriched in dendritic spines and cell cortex (Fisher et
al.,
1998; Landis and Reese, 1983, Caceres el al, 1983), it is conceivable that the
localization of CaMKIIb to dendritic spines is mediated by a direct or
indirect binding
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interaction of CaMKIIb with F-actin. The possible co-localization of CaMKIIb
and F-
actin in two model cell lines was tested by using rhodamine-phalloidin as a
marker for
polymerized actin (F-actin). In RBL-cells, which has predominant cortical F-
actin
structures, the cortical rhodamine-phalloidin closely co-localized with GFP-
CaMKIIb.
The same near complete overlap was also observed in NIH-3T3-cells which are
rich
in actin stress fibers (not shown). This suggests that the actin cytoskeleton
co
localization of CaMKIIb is not cell type specific.
Further support for the co-localization of CaMKIIb with F-actin in living
cells
was obtained by comparing cells before and after treatment with the actin
depolarizing drug Iatrunculin (Spector et al. 1989) (not shown). In RBL-cells
(top)
and fibroblasts (bottom), addition of latrunculin led to a near complete loss
in the
cortical as well as stress fiber staining of GFP-CaMKIIb.
We then determined biochemically whether expressed CaMKIIb can bind
directly to purified F-actin. Indeed, Met-35S-labeled CaMKIIb could be
effectively
sedimented by polymerized actin. In contrast, CaMKIIa is much less
sedimentable by
polymerized actin using the same assay. This suggests that the co-localization
of
CaMKIIb with the actin cytoskeleton observed in living cells is the result of
a direct
and reversible binding interaction between CaMKIIb and F-actin.
c. CaMHIIa is targeted to dendritic spines when co-expressed with CaMHIIb
It was then tested whether the co-expression of CaMKIIa and CaMKIIb in the
same cell affects their respective localization. An effective co-expression of
both
isoforms was made possible by using an RNA transfection method. In this
approach,
a large number of translation competent RNA molecules are directly introduced
into
the cytosol of adherent cells by microporation (Teruel and Meyer, 1997; Yokoe
and
Meyer, 1996). Thus, RNA encoding different proteins can be mixed and expressed
at
a defined ratio within each transfected cell. Strikingly, when GFP-CaMKIIa was
expressed together with CaMKIIb (without a GFP-tag) in hippocampal neurons,
GFP-
CaMKIIa became associated with the same dendritic spine and cortical
structures
(data not shown). A largely cortical localization of GFP-CaMKIIa was also
observed
in RBL-cells in the presence of CaMKIIb. In contrast, expression of CaMKIIa
(without a GFP-tag) together with a similar amount of GFP-CaMKIIb did not
affect
the cortical localization of GFP-CaMKIIb (data not shown). Using the same co-
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localization protocols as described above, we also found a marked co-
localization
between GFP-CaMKIIa, co-expressed with CaMKIIb, and anti PSD-95 antibodies
(not shown). In living neurons, GFP-CaMKIIa, coexpressed with CaMKIIb, showed
a marked localization juxtaposed to the presynaptic marker FM 4-64 (not
shown).
Can this effect of CaMKIIb on CaMKIIa localization be confirmed
biochemically? A detergent extraction procedure of NIH-3T3-cells was used to
test
whether CaMKIIb and/or CaMKIIa could be found in an actin enriched pellet
fraction. This extraction protocol has been shown to significantly enrich for
actin and
actin binding proteins (Egelhoff et al., 1991). Expressed CaMKIIb and CaMKIIa
10 isoforms without a GFP-tag were used for these measurements. In agreement
with the
in vivo data, CaMKIIa, when expressed alone, was largely absent from the actin
pellet
while CaMKIIb was highly enriched in the actin pellet (data not shown). When
both
isoforms were expressed together, a significant fraction of CaMKIIa was found
in the
actin cytoskeletal fraction. Taken together, these studies are consistent with
a
targeting mechanism by which CaMKTIa is localized to F-actin if expressed
together
with a sufficient amount of CaMKIIb.
How does CaMKIIb target CaMKIIa to the actin cytoskeleton? A likely
hypothesis is that CaMKIIa does not undergo cytoskeletal binding interactions
of its
own but binds to CaMKIIb which then anchors the complex to F-actin. To
20 understand this heterologous CaMKII targeting mechanism, several important
questions about the binding interaction and oligomerization of CaMKIIa and
CaMKIIb have to be answered: 1. Can CaMKIIb form oligomers on its own and if
it
does, how large are these oligomers compared to those formed by CaMKIIa?, 2.
do
co-expressed CaMKIIa and CaMKIIb isoforms assemble into hetero- or homo-
25 oligomers in living cells?, 3. if they form hetero-oligomers, does the
incorporation of
CaMKIIb into CaMKIIa oligomers occur as a stochastic process?, and 4. what
minimal ratio of CaMKIIb to CaMKIIa is required for targeting CaMKIIa to the
actin
cytoskeleton?
30 d. Oligomer formation can be explored in living cells using a GFP-based
"Pull-
Out" binding assay
To address the question of homo- versus hetero-oligomer formation, an assay
was developed to quantitatively study protein-protein binding interactions in
living
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cells {"Pull-Out" binding assay, Figure 2A). The strategy was to use a protein-
domain
(PM-domain) that translocates to the plasma membrane in response to the
addition of
a drug. The potential binding interaction between the investigated Protein X
and
Protein Y could then be investigated by fusing this PM-domain to Protein X and
a
5 GFP-tag to Protein Y (or vice versa). Both fusion proteins could then be
expressed in
the same cell and their binding interaction investigated. 1. If the initially
cytosolic
GFP-Protein Y remains cytosolic after drug addition, no significant binding
interaction occurs between Proteins X and Y. 2. If the drug addition leads to
the GFP-
Protein Y translocation to the plasma membrane (along with the non-fluorescent
PM-
10 Protein X), Proteins X and Y bind to each other.
We used the first phorbol-ester binding domain of protein kinase C as such a
PM-domain. This small 6 kDa domain is an initially cytosolic protein that
binds
nearly irreversibly to the plasma membrane after phorbol ester addition
{Oancea et al.,
1998). The distinct property of this domain is shown in Figure 2B. Before
phorbol
15 ester addition, a fusion protein of the phorbol ester binding domain with
GFP is a
cytosolic protein (left panel) that is "pulled" from the cytosol to the plasma
membrane
after addition of phorbol ester (right panel). This translocation process
occurs in less
than a minute and is mediated by a diffusion-dependent high affinity binding
interaction of the fusion protein with plasma membrane localized phorbol ester
20 (Oancea et al., 1998). It should be noted that the also visible nuclear
localized GFP
fusion protein translocates to the plasma membrane much slower due to its slow
diffusion through nuclear pores.
This PM-domain was first used to determine whether most of the expressed
CaMKIIa isoforms is present in an oligomeric state. To test for
oligornerization in
25 vivo, PM-tagged CaMK.IIa and GFP-CaMKIIa were expressed in the same cell
(Figure 2C, left). As expected, addition of phorbol ester led to a marked
translocation of the initially cytosolic GFP-CaMKIIa to the plasma membrane
(Figure 2C, right). This change in the distribution before and after addition
of
phorbol ester can be more quantitatively measured in a line profile analysis
comparing
30 the fluorescence intensity across the cell before and after phorbol ester
addition
(Figure SC, bottom). In control experiments, GFP was expressed alone and no
plasma
membrane translocation of GFP was observed after phorbol ester addition
(Figure 2
D). The finding that most GFP-CaMKIIa is pulled to the plasma membrane by PM-
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CaMKIIa strongly suggests that most of the expressed CaMKIIa is present in the
cell
in an oligomeric state.
e. CaMKIIb homo-oligomers are significantly smaller than CaMKIIa homo-
oligomers
While CaMKIIa has been proposed to be an homo-oligomer with 8 to 12
subunits, it is controversial if CaMKIIb forms oligomers and how large these
potential
oligomers are (Yamauchi et al., 1989). To measure the apparent oligomer sizes
of
CaMKIIa and CaMKIIb in living cells, we expressed GFP-CaMKII together with a
decreasing amount of PM-CaMKII and measured the phorbol ester induced
translocation to the plasma membrane (a schematic view of the expected
oligomer
translocation process is shown in Figure 3A). Since the phorbol ester-induced
plasma membrane affinity of the PM-domain is nearly irreversible, it is likely
that a
single subunit of PM-CaMKIIa is sufficient to induce the plasma membrane
translocation of a CaMKIIa oligomer. As discussed above, the RNA transfection
method allows one to quantitatively titrate the amount of the two CaMKII
fusion
proteins in the same cell. To determine the respective expression leveis for
the two
microporated RNA species, the concentration of the translated proteins was
measured
in parallel by in vitro translation of the same RNAs in the presence of 35S-
Met
(Figure 3B).
Figure 3C shows the GFP-CaMKIIa plasma membrane translocation at
increasing dilutions of co-expressed PM-CaMKIIa. The left images show the
distribution before and the right images after phorbol ester addition.
Interestingly, the
phorbol ester induced targeting to the plasma membrane was still measurable
when
PM-CaMKIIa was diluted to less than 3% of GFP-CaMKIIa. The sequential
reduction in plasma membrane translocation can be seen more clearly in
fluorescence
line intensity traces (Figure 3D). The loss in phorbol ester-mediated plasma
membrane targeting at increasing PM-CaMKIIa dilutions was analyzed by dividing
the relative plasma membrane fluorescence intensity of GFP-CaMKIIa (DPM) by
the
average cytosolic fluorescence intensity before phorbol ester addition (Ipre)
(Figure
6E). A DPM / Ipre ratio of 0 indicates that no plasma membrane translocation
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occurred and a DPM / Ip~ ratio of ~ 3 corresponds to the translocation
observed for
the PM-GFP construct itself.
Based on this analysis, a titration curve can be obtained, showing the
relative
plasma membrane translocation at decreasing dilution ratios of PM-CaMKIIa to
GFP-
CaMKIIa (Figure 3F). A 50% reduction in plasma membrane targeting was observed
at an approximate dilution ratio of one PM-CaMKIIa per fourteen GFP-CaMKIIa. A
Poisson distribution model would predict that the probability (p) to introduce
at least
one subunit into an oligomer with N subunits is p = 1 - (R/(R+1))N , with R as
the
dilution ratio. Using this model, a best fit to the data was obtained for N =
13.5, in
close agreement with a previous estimate of 12 subunits for purified CaMKIIa
(Kanaseki et al., 1991; Yamauchi et al., 1989).
We then used the same approach to determine if CaMKIib forms oligomers.
Although GFP-CaMKIIb has a partial cortical and internal F-actin localization,
this
binding interaction was reversible and a much more pronounced plasma membrane
localization can be induced by addition of phorbol ester to a PM-tagged
CaMKIIb.
Using this phorbol ester triggered increase in plasma membrane translocation
of GFP-
CaMKIIb, a titration curve was obtained for the relative plasma membrane
translocation at decreasing dilution ratios of PM-CaMKIIb to GFP-CaMKIIb.
Interestingly, the apparent average size of CaMKIIb oligomers was 4.2,
significantly
smaller than that of the a-isoform (Figure 3F). Thus, these in vivo
measurements
clearly show that CaMKIIb can form oligomers, albeit with a significantly
smaller
apparent size than those formed by CaMKIIa. This apparent size of CaMKIIb
oligomers was consistent with our finding that CaMKIIb purfied from
Baculovirus
transfected Sf9 cells had a size much smaller than that of CaMKIIa expressed
by the
same method. (unpublished results by Kang Shen).
f. CaMKIIa and b form stochastic hetero-oligomers
Since the relative expression of CaMKIIa to b is highly variable between
different types of neurons, next determined was how efficient is hetero-
oligomer
formation compared to homo-oligomer formation. The same dilution approach as
described in Figure 3 was pursued but now for the hetero-oligomers. The
calculated
line plots in Figure 4A show the curves expected for the insertion of PM-
CaMKIIb
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into GFP-CaMKIIa oligomers fox a stochastic insertion mechanism {dashed
curve).
The measured relative plasma membrane translocation for decreasing ratios of
PM-
CaMKIIb to GFP-CaMKIIa closely matched a predicted stochastic insertion
mechanism. The same measurements were then made for PM-CaMKIIa insertion into
GFP-CaMKIIb oligomers by dilution of the PM-CaMKIIa fusion protein at a
constant
concentration of CaMKIIb (Figure 4B). The calculated curve for a stochastic
insertion mechanism is overlapping with the fitted one.
Together, this titration approach suggests that if CaMKIIa and CaMKIIb
isoforms are expressed at the same time and place, they form mixed oligomers
with a
stochastic probability for the insertion of either one of the isoforms. This
also
suggests that most CaMKII oligomers in neurons contain a variable fraction of
CaMKIIb that is defined by the relative expression level of locally translated
CaMKIIb versus CaMKIIa. For the physiological situation, it is then important
to
know how many CaMKIIb isoforms have to be inserted into mostly CaMKIIa hetero-
oligomers to still effectively target CaMKII to its cytoskeletal docking site.
g. A small number of CaMKIIb isoforms are sufficient to target CaMKII hetero-
oligomers to the actin-cytoskeleton
While the previous studies were useful to dissect the oligomer formation of
CaMKIIa and CaMKIIb, they did not resolve whether individual or multiple
CaMKIIb subunits are zequired for the targeting of CaMKIIa/b hetero-oligomers
to
the actin cytoskeleton. The same RNA dilution strategy was used to determine
at
which ratio of GFP-CaMKIIa to CaMKIIb the cytoskeletal localization still
occurs.
The distinct cortical actin cytoskeleton localization of CaMKIIb in RBL-cells
was
used in this assay (Figure 4C). While CaMKIIb was less potent in targeting GFP-
CaMKIIa to the plasma membrane than the PM-CaMKII constructs, SO%
translocation to the cortical actin cytoskeleton required a ratio of
approximately 6.5
1 of GFP-CaMKIIa to CaMKIIb. Since CaMKIIa isoforms contain approximately 13
subunits, this suggests that a small number of CaMKIIb subunits are sufficient
to
target CaMKIIa/b hetero-oligomers to the actin cytoskeleton.
In a second independent approach to understand the cytoskeletal targeting of
CaMKIIa by CaMKIIb, we measured the binding interactions of the hetero-
oligomers
by measuring their diffusion coefficients. As shown in Figure 1F, GFP-CaMKIIa
has
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a S-fold faster apparent diffusion than GFP-CaMKIIb. Diffusion coefficients
were
then measured at increasing dilutions of GFP-CaMKIIa to CaMKIIb. Similar to
the
results with the localization to the cortical cytoskeleton, the diffusion
coefficient of
CaMKIIa was reduced by SO% when the concentration of CaMKIIb exceeded 1 S% of
S that of GFP-CaMKIIa (Figure 4D). Together, these measurements show that
CaMKIIb is a potent targeting domain that can localize a much larger number of
CaMKIIa isoforms to the actin cytoskeleton.
3.REFERENCES
Barria, A., et al., (1997). Science 276, 2042-S.
Bayer, K. U., et al., (1996). Mol. Cell. Biol. 16, 29-36.
Benfenati, F., et al., (1992). Nature 359, 417-20.
Bennett, M., et al., (1983). J Biol Chem. 258:12735-44
Braun, A. P., and Schulman, H. (1995). Annu.~Rev. Physiol. 57, 417-4S.
1 S Brocke, L., et al., ( 1995). J. Neurosci. 1 S, 6797-808.
Burgin K. E., et al., (1990) J. Neurosci. 10, 1788-98.
Caceres, A., et al., (1983). Proc Natl Acad Sci U S A. 80:1738-42
Chapman, P. F., et al., (1995). Neuron 14, 591-7.
Colbran, R. J., and Soderling, T. R. (1990). J. Biol. Chem. 265:11213-9.
De Koninck, P., and Schulman, H. (1998). Science 279, 191-2
Egelhoff, T. T., et al., (1991). J. Cell Biol.112, 677-88.
Fisher, M., et al., ( 1998). Neuron 20:847-S4
Giese, K. P., et al., (i998). Science 279, 873-6.
Glazewski, S., et al., (1996). Science 272, 421-3.
2S Gordon, J. A., et al., (1996). Neuron 17, 491-9.
GuptaRoy, B., and Griffith, L. C. (1996). J. Neurochem. 66, 1282-8.
Hanson, PL, and Schulman, H. (1992). J. Biol. Chem. 267, 17216-24.
Hanson, P. L, et al., (1994). Neuron 12, 943-S6.
Chen, H.J., et al., (1998). Neuron 20, 895-904.
Kanaseki, T., et al., (1991). J. Cell Biol. 115, 1049-60.
Kelly, P. T., et al., (I987). J. Neurochem. 49, 1927-40.
Kelly, P. T., and Vernon, P. (1985). Brain Res. 350, 211-24.
CA 02345392 2001-03-23
WO 00/17221 PCT/US99/19118
- 30 -
Lai, Y., et al., (1986). Proc. Natl. Acad. Sci. U S A. 83, 4253-7.
Landis, D., and Reese, T. (1983). J Cell Biol. 97:1169-78
Llinas, R., et al., (1991). J. Physiol. 43G, 257-82.
Lou, L. L., et al., (1986). Proc. Natl. Acad. Sci. U S A. 83, 9497-501.
Mayford, M., et al., (1996). Science 274, 1678-83.
Mayford, M., et aL, (1995}. Cell 81, 891-904.
McGlade-McCulloh, E., et al., {1993). Nature. 362, 640-2.
McNeill, R. B. and Colbran, R. J. (1995). J. Biol. Chem. 270, 10043-9.
Meyer, T., et al., (1992). Science 256, 1199-202.
Miller, S. G., and Kennedy, M. B. (1985). J. Biol. Chem. 260, 9039-46.
Miller, S. G., and Kennedy, M. B. (1986). Cell 44, 861-70.
Mukherji, S., and Soderling, TR. (1994). J. Biol. Chem. 269, 13744-7.
Nicoll, R. A., and Malenka, R. C. (1995). Nature. 377, 115-8
Nomura, T., et al., (1997). Brain Res. 766,129-41
Oancea, E., et al., (1998). J. Cell Biol. 140, 485-98.
Ohta, Y., et al., (1986). Febs. Lett. 208, 423-6.
Ramakrishnapillai, V., et al., (1996). J. Biol. Chem. 271: 31670-8
Ryan, T.A. and Smith, S.J. (1995) Neuron 14:983-989.
Scholz, W. K., et al., (1988). J. Neurosci. 8, 1039-51.
Schworer, C. M., et al., (1986). J. Biol. Chem. 261, 8581-4.
Shen, K., and Meyer, T. (1998) J. Neurochem. 70, 96-104.
Silva, A. J., et al., (1992). Science 257, 201-6.
Soderling, T. R. (1993). Mol. Cell. Biochem. 128, 93-101.
Spector, L, et al., (1989). Cell Motil. Cytoskeleton 13, 127-44,
Srinivasan, M., et al., (1994). J. Cell Biol. 126, 839-52.
Strack, S., et al., (1997). J. Biol. Chem. 272, 13467-70.
Subramanian, K., and Meyer, T. (1997). Cell 89, 963-971.
Sugai, R., et al., (1996). J. Biochem. 120, 773-9.
Teruel, M. N., and Meyer, T. (1997). Biophys. J. 73, 1785-1796.
Vallano, M. L., et al., (1986). Ann. N. Y. Acad. Sci. =166, 357-74.
Wu, G. Y., and Cline, H. T. (1998). Science 279, 222-6.
Wyszynski, M., et al., (1997). Nature 38.5, 439-42.
Yamauchi, T., et al., (1989). J Biol. Chem. 264, 19108-16.
CA 02345392 2001-03-23
WO 00/17221 PCT/US99/19118
-31 -
Yokoe, H., a.nd Meyer, T. {1996). l;t, 1252-1256.
The foregoing is illustrative of the present invention, and is not to be
construed
as limiting thereof. The invention is defined by the following claims, with
S equivalents of the claims to be included therein.
