4E-BP3, A NEW MEMBER OF THE EUKARYOTIC INITIATION FACTOR 4E-BINDING PROTEIN FAMILY AND USE THEREOF

4E-BP3, UN NOUVEAU MEMBRE DE LA FAMILLE DES PROTEINES EUCARYOTES LIEES AU FACTEUR D'INITIATION 4E ET UTILISATIONS CONNEXES

English Abstract

The present invention relates to the process of protein
synthesis in eukaryotic cells. More particularly, the present invention
relates to translation and more specifically to the initiation of translation
in such cells. More specifically, the present invention relates to proteins
involved in the initiation of translation in animal cells, to the modulation
of
translation thereby and to the interaction and sequestering of a limiting
factor of initiation of translation, elF-4E. The present invention also
relates to isolated nucleic acid molecules encoding a human protein, a
4E-binding protein (4E-BP3), as well as vectors and host cells harboring
same. In addition, the present invention relates to screening assays for
identifying modulators of 4E-BP3 activity and to the identification of
mutants thereof which abrogate its interaction with elF-4E.

Claims

50


The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:

1. An isolated 4E-BP3 protein exhibiting homology to
4E-BP1 and 4E-BP2 and interacting with elF4E.

2. The isolated 4E-BP3 of claim 1 having an amino
acid sequence at least 95% identical to a sequence selected from the
group consisting of:
(a) amino acids from about 1 to about 100 of Figure 1A;
(b) amino acids from about 2 to about 100 of Figure 1A;
(c) amino acids from about 37 to about 47 of Figure 1A;
(d) amino acids from about 1 to about 101 of M4E-BP3
of Figure 1C;
(e) amino acids from about 2 to about 101 of M4E-BP3
of Figure 1C;
(f) amino acids from about 37 to about 47 of M4E-BP3
of Figure 1C; and
(g) the amino acid sequence of an epitope-bearing
portion of any one of the polypeptides of (a), (b), (c), (d), (e) or (f).

3. An isolated nucleic molecule comprising a
polynucleotide sequence at least 95% identical to a sequence selected
from the group consisting of:



51


(a) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 1 to about 100 of
Figure 1A;
(b) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 2 to about 100 in
Figure 1A;
(c) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 37 to about 47 in
Figure 1A;
(d) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 1 to about 101 of
M4E-BP3 of Figure 1C;
(e) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 2 to about 101 of
M4E-BP3 of Figure 1C;
(f) a nucleotide sequence encoding a 4E-BP3
polypeptide comprising amino acids from about 37 to about 47 of
M4E-BP3 of Figure 1C; and
(g) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), (c), (d), (e) or (f).

4. A recombinant vector comprising said isolated
nucleic acid molecule of claim 3.

5. A method of making a recombinant host cell
comprising introducing the recombinant vector of claim 4 into a host cell.



52


6. A recombinant host cell produced by the method of
claim 5.

7. A recombinant method for producing 4E-BP3
polypeptide, comprising culturing said host cell of claim 6 under
conditions such that said polypeptide is expressed and recovering said
4E-BP3 polypeptide.

8. A method for treating an animal in need of
modulation of 4E-BP3 level and/or activity and/or in need of modulation
of elF-4E level and/or activity, comprising administering thereinto a
therapeutically effective amount of a 4E-BP3 polypeptide, mutant,
fragment or derivative thereof, and/or a nucleic acid molecule encoding
same and/or 4E-BP3-activity modulator together with a pharmaceutically
acceptable carrier.

9. An antibody directed against 4E-BP3.

10. The antibody of claim 9, selected from the group
consisting of antibody 1791 and 1862.

11. A method of promoting cap-independent translation
in a cell or extract comprising an addition thereto of an effective amount
of 4E-BP.




53


12. The method of claim 11, wherein said 4E-BP is
4E-BP3.

Description

CA 02238217 1998-OS-22
1
TITLE OF THE INVENTION
4E-BP3, A NEW MEMBER OF THE EUKARYOTIC
INITIATION FACTOR 4E-BINDING PROTEIN FAMILY AND USE
THEREOF.
FIELD OF THE INVENTION
The present invention relates to the process of protein
synthesis in eukaryotic cells. More particularly, the present invention
relates to translation and more specifically to the initiation of translation
in such cells. More specifically, the present invention relates to proteins
involved in the initiation of translation in animal cells, to the modulation
of
translation thereby and to the interaction and sequestering of a limiting
factor of initiation of translation, eIF-4E. The present invention also
relates to isolated nucleic acid molecules encoding a human protein, a
4E- binding protein (4E-BP3), as well as vectors and host cells harboring
same. In addition, the present invention relates to screening assays for
identifying modulators of 4E-BP3 activity and to the identification of
mutants thereof which abrogate its interaction with eIF-4E.
BACKGROUND OF THE INVENTION
Translation initiation in eukaryotes is mediated by the
cap structure (m'GpppN, where N is any nucleotide) present at the 5' end
of all cellular mRNAs, except organellar. The cap is recognized by
eukaryotic initiation factor 4F (eIF4F), which consists of three
polypeptides, including eIF4E, the cap binding protein subunit. The
interaction of the cap with eIF4E facilitates the binding of the ribosome to
the mRNA. eIF4E activity is regulated in part by two translational


CA 02238217 1998-OS-22
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repressors, 4E-BP1 and 4E-BP2, which bind to it and prevent its
assembly into eIF4F.
Modulation of gene expression in cells is partly achieved
by regulating translation rates. Translation initiation is the major target of
such regulation in eukaryotic cells (Mathews et al., 1996 in Translational
Control Hershey et al. eds, pp. 1-30, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY). The rate limiting step in translation
initiation is the binding of the 43S pre-initiation complex to the mRNA.
This process is facilitated by the presence of the mRNA 5' cap structure
(Shatkin, 1985, Cell 402:223-224). Ribosome binding is mediated by
eIF4F~ (Merrick et al., 1996, in Translational Control Hershey et al. eds,
pp. 31-70, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;
Sonenberg, 1996, in Translational Control Hershey et al. eds, pp. 245-
270, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY),
which consists of three subunits: eIF4E, eIF4A, and eIF4G. eIF4F, in
association with eIF4B, is thought to unwind the mRNA secondary
structure in the vicinity of the cap to allow the binding of the 43S pre-
initiation complex (reviewed in refs. Sonenberg, 1996, in Translational
Control Hershey et al. eds, pp. 245-270, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY; Pain , 1996, Eur. J. Biochem. 236 3 :747-
771 ).
eIF4E is present in rate-limiting amounts in most cells
(Duncan et al., 1987, J. Biol. Chem. 262(11:380-388; Hiremath et al.,
1985, J. Biol. Chem. 260 13 :7843-7849, but see Rau et al., 1996, J Biol
Chem 271 15 :8983-90, for an exception in rabbit reticulocytes), and plays
a central role in translational control (Sonenberg, 1996, in Translational
Control Hershey et al. eds, pp. 245-270, Cold Spring Harbor Laboratory


CA 02238217 1998-OS-22
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Press, Cold Spring Harbor, NY; Pain , 1996, supra). The key role of
eIF4E regulation in protein synthesis is underscored by the presence of
two related eIF4E binding proteins, 4E-BP1 and 4E-BP2 (Pause et al.,
1994, Nature 3716500:762-767; Lin et al., 1994, Science
266 5185 :653-656). These proteins specifically inhibit eIF4E-dependent
translation initiation (Pause et al., 1994, supra) by competing with eIF4G
for binding to eIF4E (Haghighat et al., 1995, EMBO J. 14i(22):5701-5709).
Consequently, 4E-BPs prevent eIF4E association with eIF4G to form the
eIF4F complex, resulting in the subsequent inhibition of the 43S pre-
initiation complex binding to the mRNA. The competition between 4E-BPs
and eIF4G is explained by the presence of a common eIF4E binding site
in 4E-BPs and eIF4G (Mader et al., 1995, Mol. Cell. Biol. 15(9):4990-
4997).
The binding of 4E-BP1 and 4E-BP2 to eIF4E is
controlled by their phosphorylation state. The underphosphorylated forms
of 4E-BPs interact with eIF4E, whereas the hyperphosphorylated forms
do not (Pause et al., 1994, supra; Lin et al., 1994, supra; Gingras et al.,
1997, Virology 237:182-186). Upon cell stimulation with serum, growth
factors or hormones, 4E-BP1 becomes hyperphosphorylated and
dissociates from eIF4E to relieve the translational inhibition (Pause et al.,
1994, supra; Lin et al., 1994, supra; Fleurent et al., 1997, J. Biol. Chem.
272(7):4006-4012). The regulation of eIF4E activity by 4E-BPs is of
particular significance considering the transforming potential of eIF4E
(Lazaris-Karatzas et al., 1990, Nature 345(6275:544-547). Indeed, the
overexpression of 4E-BP1 and 4E-BP2 in cells transformed by eIF4E or
v-src resulted in a significant reversion of the transformed phenotype
(Rousseau et al., 1996, Oncogene 111):2415-2420).


CA 02238217 1998-OS-22
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While 4E-BP1 and 4E-BP2 have been shown to affect
the cap-dependent translation of messages, an effect of these proteins
on cap-independent translation has not been reported.
There thus remains a need to identify factors which
could affect the translation of capped messages and messages translated
through internal initiation (Pause et al. 1994, supra; Ohlmann et al., 1996,
EMBO J. 15(6):1371-1382; Pelletier et al., 1985, supra).
There also remains a need to identify residues in 4E-
BPs which modulate its interaction with eIF-4E and/or modulate its effect
on translation initiation.
The present invention seeks to meet these and other
needs.
The present description refers to a number of
documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
The invention concerns a novel eukaryotic translation
factor termed 4E-BP3, a new member of the 4E-BP family. 4E-BP3 is
homologous to 4E-BP1 and 4E-BP2, exhibiting 57% and 59% identity,
respectively. The homology is most striking in the middle region of the
protein, which contains the eIF4E binding motif and residues which are
phosphorylated in 4E-BP1. 4E-BP3 is a heat stable protein that binds to
eIF4E in vitro as well as in vivo. Further, 4E-BP3 overexpression
specifically reduces eIF4E-dependent translation. Strikingly, 4E-BP3 also
reduces translation mediated by internal initiation. The overlapping
function and expression of the different 4E-BP family members imply that


CA 02238217 1998-OS-22
there is redundancy in this translational control mechanism, underscoring
its importance.
More particularly, the present invention provides isolated
polypeptides having the amino acid sequences shown in Figure 1.
5 The present invention further relates to isolated nucleic
acid molecules comprising polynucleotides which encode a 4E-BP3
polypeptide, and more particularly to isolated nucleic acid molecules
encoding the 4E-BP3 polypeptide having the amino acid sequence shown
in Figure 1.
The invention in addition relates to recombinant vectors
harboring the isolated nucleic acid molecules of the present invention.
More particularly, the invention relates to expression vectors which
express the 4E-BP3 polypeptides of the present invention. The present
invention further relates to host cells containing such recombinant vectors
or expression vectors, to methods of making such host cells, and to
methods of making such vectors.
Further, the present invention provides screening assays
and methods for identifying modulators of 4E-BP activity and especially
of 4E-BP3. More particularly, the present invention relates to assays and
methods for screening and identifying compounds which can enhance or
inhibit the biological activity of 4E-BPs and especially 4E-BP3. In one
particular embodiment of the present invention, the screening assay for
identifying modulators of 4E-BP activity comprises contacting cells or
extracts containing a 4E-BP and a candidate compound, assaying a
cellular response or biological function of 4E-BP, wherein the potential
modulating compound is selected when the cellular response or 4E-BP
biological activity in the presence of the candidate compound is


CA 02238217 1998-OS-22
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measurably different than in the absence thereof, and whereby an
increase in cellular response or 4E-BP biological activity over the control
without compound, indicates that the compound is an agonist while a
decrease in cellular response or 4E-BP biological activity indicates that
the compound is an antagonist.
In addition, the present invention relates to methods for
treating an animal (such as a human) in need of a modulation of 4E-BP
level and/or activity, which comprises administration thereto of a
composition comprising a therapeutically effective amount of 4E-BP (such
as 4E-BP3) polypeptide, and /or 4E-BP nucleic acid molecule encoding
same, and/or modulators of 4E-BP activity. In one embodiment, the
present invention relates to an administration of a 4E-BP mutant, thereby
relieving the negative translational control of a native 4E-BP.
The invention further relates to the use of polypeptides
of the present invention, and/or modulators of 4E-BP activity in in vitro
translation systems, and to methods of modulating translation in cells or
extracts thereof comprising an addition of the polypeptides, and/or nucleic
acid molecules, and/or modulators of 4E-BP activity of the present
invention, to affect cap-dependent and/or cap-independent translation.
In accordance with the present invention, there is
therefore provided, an isolated 4E-BP3 protein exhibiting homology to 4E-
BP1 and 4E-BP2 and interacting with eIF4E.
In accordance with the present invention, there is also
provided, an isolated nucleic acid molecule comprising a polynucleotide
sequence encoding 4E-BP3.
In accordance with another aspect of the present
invention, there is provided, an isolated nucleic acid molecule comprising


CA 02238217 1998-OS-22
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a polynucleotide sequence which hybridizes under stringent conditions to
a polynucleotide sequence encoding 4E-BP3 or to a sequence which is
complementary thereto.
In accordance with yet another aspect of the present
invention, there is provided a method of constructing a recombinant
vector which comprises inserting an isolated nucleic acid molecule
encoding 4E-BP3 (or a mutant thereof) into a vector. In addition, there is
also provided a recombinant vector harboring an isolated nucleic acid
molecule encoding a 4E-BP1 or 4E-BP2 mutant.
In accordance with a further aspect of the present
invention, there is provided a method for making a recombinant cell
comprising introducing thereinto a recombinant vector harboring a nucleic
acid sequence encoding a 4E-BP of the present invention.
In accordance with an additional aspect of the present
invention, there is provided an antibody which recognizes specifically a
4E-BP3 polypeptide or derivative thereof.
In accordance with yet an additional aspect of the
present invention, there is provided a method for treating an animal in
need of modulation of 4E-BP3 level and/or activity, comprising
administering thereinto a therapeutically effective amount of a 4E-BP3
polypeptide, mutant, fragment or derivative thereof and/or 4E-BP3
encoding nucleic acid molecule and/or a 4E-BP3-activity modulator
together with a pharmaceutically acceptable carrier.
Also, there is provided a method for treating an animal
in need of modulation of eIF-4E level and/or activity, comprising
administering thereinto a therapeutically effective amount of a 4E-BP3
polypeptide, mutant, fragment or derivative thereof, and/or a nucleic acid


CA 02238217 1998-OS-22
molecule encoding same, and/or a 4E-BP1 and/or 4E-BP2 polypeptide,
mutant, fragment or derivative thereof, and/or a nucleic acid molecule
encoding same and/or a 4E-BP3-activity modulator together with a
pharmaceutically acceptable carrier.
In accordance with a further additional aspect of the
present invention, there is provided a method of increasing the
translational efficiency in a cell by introducing thereinto an effective
amount of a 4E-BP polypeptide, mutant, fragment or derivative thereof
or a nucleic acid molecule encoding same.
In accordance with yet a further additional aspect of the
present invention, there is provided a method to increase cap-
independent translation in vivo and/or in vitro by adding an effective
amount of a 4E-BP polypeptide or 4E-BP-encoding nucleic acid molecule.
The 4E-BP3 polypeptides and nucleic acid molecules of
the instant invention have been isolated from human. Nevertheless, it will
be clear to the person of ordinary skill that the present invention should
not be so limited. Indeed, using the teachings of the present invention
and those of the art, homologues of 4E-BP and 4E-BP3 can be identified
and isolated from other animal species. Non-limiting examples thereof
include monkey, mouse, rat, rabbit, and frog. The significant identity
between the human and mouse 4E-BP3 protein validates this contention.
It would also be clear to the person of ordinary skill that
the combination of more than one 4E-BP, mutant, fragment or derivative
thereof to practice the present invention is also encompassed as being
within the scope of the instant invention.


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BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
Fig. 1. shows the Sequence of 4E-BP3. A, nucleotide
and deduced amino acid sequence of human 4E-BP3. The
polyadenylation signal is underlined. The sequence corresponding to the
peptide used to generate antiserum 1791 is shown in bold type. 8,
sequence alignment of human 4E-BPs. Identical (black box) and
conserved (shaded box) amino acids are highlighted. Amino acids
involved in eIF4E binding are boxed. Phosphorylation sites of 4E-BP1 are
denoted by asterisks. C, sequence alignment of human 4E-BP3 and
mouse 4E-BP3 (EST W18851, GeneBank).
Fig. 2. shows the Northern analysis. Tissue distribution
of 4E-BP3 mRNA was analyzed using Clontech MTN1 (A) and MTN2 (8)
Northern blots. The analysis was performed as described in Example 1.
Fig. 3. shows the Identification of 4E-BP3 in cells. A, an
extract (100 pg) from LNCaP cells was resolved by SDS-PAGE and
Western blotting was performed with the following antisera: antiserum
1862 pre-adsorbed with GST-4E-BP3 (lane 1 ), antiserum 1862 pre-
adsorbed with GST-HMK (lane 2), antiserum 1791 (lane 3) and antiserum
1791 pre-adsorbed with its cognate peptide (lane 4). 8, an extract (20 pg)
from HeLa cells transfected with pcDNA3 (lane 1 ) or with pcDNA3-4E-
BP3 (lane 2) and total (lane 3) or heat treated (lane 4) extract (250 pg)
from LNCaP cells was resolved by SDS-PAGE. Western blotting was
performed with antiserum 1791 (1:750).


CA 02238217 1998-OS-22
Fig. 4. shows the Interaction of 4E-BP3 with eIF4E. A,
In vitro synthesized capped mRNAs were translated in a wheat germ
extract and proteins were analyzed by SDS-PAGE as follows: lane 1, 4E-
BP3; lane 2, 4E-BP3 + FLAG eIF4E; lane 3, 4E-BP3-Y40A + FLAG
5 eIF4E; lane 4, 4E-BP3-L45A + FLAG-eIF4E; Lane 5, FLAG-eIF4E. 8, In
vitro translated mixture as described in A were incubated with m'GDP-
agarose resin. After washing, bound proteins were resolved by SDS-
PAGE. C, HA-tagged protein expression plasmids, pcDNA3-HA (lane 1 ),
pcDNA3-HA-La (lane 2), pcDNA3-HA-4E-BP3 (lane 3), pcDNA3-HA-4E-
10 BP3-Y40A (lane 4) or pcDNA3-HA-4E-BP3-L45A (lane 5) were
transfected into HeLa cells together with pcDNA3-FLAG-eIF4E. Total cell
extracts (24 Ng) were resolved by SDS-PAGE and analyzed by Western
blotting for the presence of HA-tagged protein (top panel) or FLAG-eIF4E
(bottom panel). D, HeLa cell extracts (300 pg) as described in C were
immunoprecipitated with anti-HA antibody and immunoprecipitates were
resolved by SDS-PAGE. Western blotting analysis was performed using
anti-HA antibody (top panel) and anti-eIF4E antibody (bottom panel). E,
LNCaP cell extract was precipitated with m'GDP-agarose and the bound
proteins were resolved by SDS-PAGE. Western blotting was performed
using anti-4E-BP3 antibody 1791 (1:1000) and anti-eIF4E antibody 5853
(1:2500). Lane 1, LNCaP extract (150 pg). Lane 2, m'GDP-precipitated
material.
Fig. 5. shows that the 4E-BP3 inhibits cap-dependent
translation in vivo. HeLa cells were infected with recombinant vaccinia
virus vTF7-3, which expresses the T7 RNA polymerase, and then
transfected with pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-4E-BP3.
A, pcDNA3-RLUC-POLIRES-FLOC reporter vector. RLUC, Renilla


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reniformis luciferase ORF; FLUC, Firefly luciferase ORF; POLIRES,
poliovirus 5'UTR, see Example 1 for details. 8, Western blot analysis of
transfected cell extracts (10 pg) for the presence of 4E-BP3 with anti-4E-
BP3 1791 (1:750). Lane 1, pcDNA3; lane 2, pcDNA3-4E-BP3; lane 3,
pcDNA3-4E-BP3-Y40A; lane 4, pcDNA3-4E-BP3-L45A. C and D, RLUC
and FLUC activity was measured after 17 h as described in Experimental
Procedures. The activity of the RLUC cistron is reported in C and the
activity of the FLUC cistron is reported in D. The RLUC and FLUC activity
of pcDNA3 transfected cells is set at 100%. Each experiment was carried
out twice in triplicate. The error bars represent the standard deviation of
the mean.
Fig. 6. shows the Metabolic labelling of 4E-BP3. HeLa
cells transfected with pcDNA3 (lane 1 ) or with pcDNA3-4E-BP3 (lane 2)
or LNCaP cells (lane 3) were labelled with [3ZP] orthophosphate and 4E-
BP3 was immunoprecipitated using 1862 antiserum as described in
Example 1
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference to the
accompanying drawing which is exemplary and should not be interpreted
as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
A polypeptide which interacts with eIF-4E exhibits
homology to 4E-BP1, and 4E-BP2 has thus been identified in animal cells
and termed 4E-BP3. The nucleic acid and amino acid sequences of a


CA 02238217 1998-OS-22
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human 4E-BP3 is described hereinbelow and the functional role of this
factor assessed.
Nucleotide sequences are presented herein by single
strand, in the 5' to 3' direction, from left to right, using the one letter
nucleotide symbols as commonly used in the art and in accordance with
the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission.
Unless defined otherwise, the scientific and
technological terms and nomenclature used herein have the same
meaning as commonly understood by a person of ordinary skill to which
this invention pertains. Generally, the procedures for cell cultures,
infection, molecular biology methods and the like are common methods
used in the art. Such standard techniques can be found in reference
manuals such as for example Sambrook et al. (1989, Molecular Cloning
- A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et
al. (1994, Current Protocols in Molecular Biology, Wiley, New York).
The present description refers to a number of routinely
used recombinant DNA (rDNA) technology terms. Nevertheless,
definitions of selected examples of such rDNA terms are provided for
clarity and consistency.
As used herein, "nucleic acid molecule", refers to a
polymer of nucleotides. Non-limiting examples thereof include DNA (i.e.
genomic DNA, cDNA) and RNA molecules (i.e. mRNA). The nucleic acid
molecule can be obtained by cloning techniques or synthesized. DNA can
be double-stranded or single stranded (coding strand or non-coding
strand [antisense]).


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The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule purified from its natural environment. Non-limiting
examples of an isolated nucleic acid molecule is a DNA sequence
inserted into a vector, and a partially purified polynucleotide sequence in
solution.
The term "recombinant DNA" as known in the art refers
to a DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering.
The term "DNA segment", is used herein, to refer to a
DNA molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can
encode a linear stretch or sequence of amino acids which can be referred
to as a polypeptide, protein, protein fragment and the like.
The terminology "amplification pair" refers herein to a
pair of oligonucleotides (oligos) of the present invention, which are
selected to be used together in amplifying a selected nucleic acid
sequence by one of a number of types of amplification processes,
preferably a polymerase chain reaction. Other types of amplification
processes include ligase chain reaction, strand displacement
amplification, or nucleic acid sequence-based amplification, as explained
in greater detail below. As commonly known in the art, the oligos are
designed to bind to a complementary sequence under selected
conditions. For example, homologs of human or mouse 4E-BP3 could be
isolated using an amplification method such as PCR with an amplification
pair designed by comparing the homology of the human and mouse
sequences.


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The nucleic acid (i.e. DNA or RNA) for practicing the
present invention may be obtained according to well known methods.
As used herein, the term "physiologically relevant" is
meant to describe interactions which can modulate transcription of a gene
in its natural setting.
Oligonucleotide probes or primers of the present
invention may be of any suitable length, depending on the particular
assay format and the particular needs and targeted genomes employed.
In general, the oligonucleotide probes or primers are at least 12
nucleotides in length, preferably between 15 and 24 molecules, and they
may be adapted to be especially suited to a chosen nucleic acid
amplification system. As commonly known in the art, the oligonucleotide
probes and primers can be designed by taking into consideration the
melting point of hydrizidation thereof with its targeted sequence (see
below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory
Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current
Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
The terms "DNA oligonucleotide", or "DNA molecule" or
"DNA sequence" refer to a molecule comprised of the
deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or
cytosine (C). Oligonucleotide or DNA can be found in linear DNA
molecules or fragments, viruses, plasmids, vectors, chromosomes or
synthetically derived DNA.
"Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions will
form a thermodynamically favored double-stranded structure. Examples


CA 02238217 1998-OS-22
of hybridization conditions can be found in the two laboratory manuals
referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989,
supra) and are commonly known in the art. In the case of a hybridization
to a nitrocellulose filter, as for example in the well known Southern
5 blotting procedure, a nitrocellulose filter can be incubated overnight at
65°C with a labeled probe in a solution containing high salt ( 5 x SSC
or
5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 pg/ml denatured
carried DNA ( i.e. salmon sperm DNA). The non-specifically binding
probe can then be washed off the filter by several washes in 0.2 x
10 SSC/0.1 % SDS at a temperature which is selected in view of the desired
stringency: room temperature (low stringency), 42°C (moderate
stringency) or 65°C (high stringency). The selected temperature is
based
on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA
hybrids can also be formed and detected. In such cases, the conditions
15 of hybridization and washing can be adapted according to well known
methods by the person of ordinary skill. Stringent conditions will be
preferably used (Sambrook et al.,1989, supra). As well known in the art
other stringent hybridization conditions can be used (i.e. 42°C in the
presence of 50% of formamide).
Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
a-nucleotides and the like. Modified sugar-phosphate backbones are
generally taught by Miller, 1988 (Ann. Reports Med. Chem. 23:295) and
Moran et al., 1987 (Nucl. Acids Res., 14:5019). Probes of the invention
can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA), and preferably of DNA.


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The types of detection methods in which probes can be
used include Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection). labeled proteins could also be
used to detect a particular nucleic acid sequence to which it binds. Other
detection methods include kits containing probes on a dipstick setup and
the like.
Although the present invention is not specifically
dependent on the use of a label for the detection of a particular nucleic
acid sequence, such a label might be beneficial, by increasing the
sensitivity of the detection. Furthermore, it enables automation. Probes
can be labeled according to numerous well known methods (Sambrook
et al., 1989, supra). Non-limiting examples of labels include 3H, '4C, 32P,
and 35S. Non-limiting examples of detectable markers include ligands,
fluorophores, chemiluminescent agents, enzymes, and antibodies. Other
detectable markers for use with probes, which can enable an increase in
sensitivity of the method of the invention, include biotin and
radionucleotides. It will become evident to the person of ordinary skill that
the choice of a particular label dictates the manner in which it is bound to
the probe.
As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods. Non-limiting
examples thereof include kinasing the 5' ends of the probes using gamma
s2P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of
E. coli in the presence of radioactive dNTP (i.e. uniformly labeled DNA
probe using random oligonucleotide primers in low-melt gels), using the
SP6/T7 system to transcribe a DNA segment in the presence of one or
more radioactive NTP, and the like.


CA 02238217 1998-OS-22
17
As used herein, "oligonucleotides" or "oligos" define a
molecule having two or more nucleotides (ribo or deoxyribonucleotides).
The size of the oligo will be dictated by the particular situation and
ultimately on the particular use thereof and adapted accordingly by the
person of ordinary skill. An oligonucleotide can be synthetised chemically
or derived by cloning according to well known methods.
As used herein, a "primer" defines an oligonucleotide
which is capable of annealing to a target sequence, thereby creating a
double stranded region which can serve as an initiation point for DNA
synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid
sequence may be carried out by a number of suitable methods. See
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous
amplification techniques have been described and can be readily adapted
to suit particular needs of a person of ordinary skill. Non-limiting examples
of amplification techniques include polymerise chain reaction (PCR),
ligase chain reaction (LCR), strand displacement amplification (SDA),
transcription-based amplification, the Q~i replicase system and NASBA
(Kwoh et al., 1989, Proc. Natl. Acid. Sci. USA 86, 1173-1177; Lizardi et
al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.
Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,
amplification will be carried out using PCR.
Polymerise chain reaction (PCR) is carried out in
accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S.
Patent are incorporated herein by reference). In general, PCR involves,
a treatment of a nucleic acid sample (e.g., in the presence of a heat


CA 02238217 1998-OS-22
18
stable DNA polymerise) under hybridizing conditions, with one
oligonucleotide primer for each strand of the specific sequence to be
detected. An extension product of each primer which is synthesized is
complementary to each of the two nucleic acid strands, with the primers
sufficiently complementary to each strand of the specific sequence to
hybridize therewith. The extension product synthesized from each primer
can also serve as a template for further synthesis of extension products
using the same primers. Following a sufficient number of rounds of
synthesis of extension products, the sample is analysed to assess
whether the sequence or sequences to be detected are present.
Detection of the amplified sequence may be carried out by visualization
following EtBr staining of the DNA following gel electrophores, or using
a detectable label in accordance with known techniques, and the like. For
a review on PCR techniques (see PCR Protocols, A Guide to Methods
and Amplifications, Michael et al. Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) is carried out in accordance
with known techniques (Weiss, 1991, Science 254:1292). Adaptation of
the protocol to meet the desired needs can be carried out by a person of
ordinary skill. Strand displacement amplification (SDA) is also carried out
in accordance with known techniques or adaptations thereof to meet the
particular needs (Walker et al., 1992, Proc. Natl. Acid. Sci. USA
89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696.
As used herein, the term "gene" is well known in the art
and relates to a nucleic acid sequence defining a single protein or
polypeptide. A "structural gene" defines a DNA sequence which is
transcribed into RNA and translated into a protein having a specific amino
acid sequence thereby giving rise the a specific polypeptide or protein. It


CA 02238217 1998-OS-22
19
will readily recognized by the person of ordinary skill, that the nucleic acid
sequence of the present invention can be incorporated into anyone of
numerous established kit formats which are well known in the art.
A "heterologous" (i.e. a heterologous gene) region of a
DNA molecule is a subsegment segment of DNA within a larger segment
that is not found in association therewith in nature. The term
"heterologous" can be similarly used to define two polypeptidic segments
not joined together in nature. Non-limiting examples of heterologous
genes include reporter genes such as luciferase, chloramphenicol acetyl
transferase, ~i-galactosidase, and the like which can be juxtaposed or
joined to heterologous control regions or to heterologous polypeptides.
The term "vector" is commonly known in the art and
defines a plasmid DNA, phage DNA, viral DNA and the like, which can
serve as a DNA vehicle into which DNA of the present invention can be
cloned. Numerous types of vectors exist and are well known in the art.
The term "expression" defines the process by which a
structural gene is transcribed into mRNA (transcription), the mRNA is then
being translated (translation) into one polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or
vehicle as described above but designed to enable the expression of an
inserted sequence following transformation into a host. The cloned gene
(inserted sequence) is usually placed under the control of control element
sequences such as promoter sequences. The placing of a cloned gene
under such control sequences is often refered to as being operably linked
to control elements or sequences.
Operably linked sequences may also include two
segments that are transcribed onto the same RNA transcript. Thus, two


CA 02238217 1998-OS-22
sequences, such as a promoter and a "reporter sequence" are operably
linked if transcription commencing in the promoter will produce an RNA
transcript of the reporter sequence. In order to be "operably linked" it is
not necessary that two sequences be immediately adjacent to one
5 another.
Expression control sequences will vary depending on
whether the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can
additionally contain transcriptional elements such as enhancer elements,
10 termination sequences, tissue-specificity elements, and/or translational
initiation and termination sites. Typically, expression vectors are
prokaryote specific or eukaryote specific although shuttle vectors are also
widely available.
Prokaryotic expression are useful for the preparation of
15 large quantities of the protein encoded by the DNA sequence of interest.
This protein can be purified according to standard protocols that take
advantage of the intrinsic properties thereof, such as size and charge (i.e.
SDS gel electrophoresis, gel filtration, centrifugation, ion exchange
chromatography...). In addition, the protein of interest can be purified via
20 affinity chromatography using polyclonal or monoclonal antibodies. The
purified protein can be used for therapeutic applications.
The DNA construct can be a vector comprising a
promoter that is operably linked to an oligonucleotide sequence of the
present invention, which is in turn, operably linked to a heterologous
gene, such as the gene for the luciferase reporter molecule. "Promoter"
refers to a DNA regulatory region capable of binding directly or indirectly
to RNA polymerase in a cell and initiating transcription of a downstream


CA 02238217 1998-OS-22
21
(3' direction) coding sequence. For purposes of the present invention, the
promoter is bound at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of bases
or elements necessary to initiate transcription at levels detectable above
background. Within the promoter will be found a transcription initiation site
(conveniently defined by mapping with S1 nuclease), as well as protein
binding domains (consensus sequences) responsible for the binding of
RNA polymerase. Eukaryotic promoters will often, but not always, contain
"TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain
Shine-Dalgarno sequences in addition to the -10 and -35 consensus
sequences.
As used herein, the designation "functional derivative"
denotes, in the context of a functional derivative of a sequence whether
an nucleic acid or amino acid sequence, a molecule that retains a
biological activity (either function or structural) that is substantially
similar
to that of the original sequence. This functional derivative or equivalent
may be a natural derivatives or may be prepared synthetically. Such
derivatives include amino acid sequences having substitutions, deletions,
or additions of one or more amino acids, provided that the biological
activity of the protein is conserved. The same applies to derivatives of
nucleic acid sequences which can have substitutions, deletions, or
additions of one or more nucleotides, provided that the biological activity
of the sequence is generally maintained. When relating to a protein
sequence, the substituting amino acid as chemico-physical properties
which are similar to that of the substituted amino acid. The similar
chemico-physical properties include, similarities in charge, bulkiness,
hydrophobicity, hydrophylicity and the like. The term "functional


CA 02238217 1998-OS-22
22
derivatives" is intended to include "functional fragments", "functional
segments", "functional variants", "functional analogs" or "functional
chemical derivatives" of the subject matter of the present invention.
"Fragments" of the nucleic acid molecules according to the present
invention refer to such molecules having at least 12 nt, more particularly
at least 18 nt, and even more preferably at least 24 nt which have utility
as diagnostic probes and/or primers. It will become apparent to the
person of ordinary skill that larger fragments of 100 nt, 1000 nt, 2000 nt
and more also find utility in accordance with the present invention.
The term "at least 24 nt" is meant to refer to 24
contiguous nt of a chosen sequence such as shown for example in
Figure 1.
The term "functional variant" refers herein to a protein
or nucleic acid molecule which is substantially similar in structure and
biological activity to the protein or nucleic acid of the present invention.
The functional derivatives of the present invention can
be synthesized chemically or produced through recombinant DNA
technology, all these methods are well known in the art.
The term "molecule" is used herein in a broad sense and
is intended to include natural molecules, synthetic molecules, and mixture
of natural and synthetic molecules. The term "molecule" is also meant to
cover a mixture of more than one molecule such as for example pools or
libraries of molecules. Non-limiting examples of molecules include
chemicals, biological macromolecules, cell extracts and the like. The term
"compound" is used herein interchangeably with molecule and is similarly
defined.


CA 02238217 1998-OS-22
23
Nucleic acid fragments in accordance with the present
invention include epitope-encoding portions of the polypeptides of the
invention. Such portions can be identified by the person of ordinary skill
using the nucleic acid sequences of the present invention in accordance
with well known methods. Such epitopes are useful in raising antibodies
that are specific to the polypeptides of the present invention. The
invention also provides nucleic acid molecules which comprise
polynucleotide sequences capable of hybridizing under stringent
conditions to the polynucleotide sequences of the present invention or to
portions thereof.
The term hybridizing to a "portion of a polynucleotide
sequence" refers to a polynucleotide which hybridizes to at least 12 nt,
more preferably at least 18 nt, even more preferably at least 24 nt and
especially to about 50 nt of a polynucleotide sequence of the present
invention.
The present invention further provides isolated nucleic
acid molecules comprising a polynucleotide sequences which is at least
95% identical, and preferably from 96% to 99% identical to the
polynucleic acid sequence encoding the full length 4E-BP3 polypeptide
or fragments and/or derivatives thereof. Methods to compare sequences
and determine their homology/identity are well known in the art.
As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject matter of the
invention. Such moieties could affect the physico-chemical characteristic
of the derivative (i.e. solubility, absorption, half life and the like,
decrease
of toxicity). Such moieties are exemplified in Remington's Pharmaceutical


CA 02238217 1998-OS-22
24
Sciences (1980). Methods of coupling these chemical-physical moieties
to a polypeptide are well known in the art.
The term "allele" defines an alternative form of a gene
which occupies a given locus on a chromosome.
As commonly known, a "mutation" is a detectable
change in the genetic material which can be transmitted to a daughter
cell. As well known, a mutation can be, for example, a detectable change
in one or more deoxyribonucleotide. For example, nucleotides can be
added, deleted, substituted for, inverted, or transposed to a new position.
Spontaneous mutations and experimentally induced mutations exist. The
result of a mutations of nucleic acid molecule is a mutant nucleic acid
molecule. A mutant polypeptide can be encoded from this mutant nucleic
acid molecule.
As used herein, the term "purified" refers to a molecule
having been separated from a cellular component. Thus, for example, a
"purified protein" has been purified to a level not found in nature. A
"substantially pure" molecule is a molecule that is lacking in all other
cellular components.
The term "isolated polypeptide" refers to a polypeptide
removed from its natural environment. Non-limiting examples of isolated
polypeptides include a polypeptide produced recombinantly in a host cell
and partially or substantially purified polypeptides from such host cells.
The polypeptides of the present invention comprise polypeptides encoded
by the nucleic acid molecules of the present invention, as shown for
example in Figure 1. The present invention also provides polypeptides
comprising amino acids sequences which are at least 95% homologous,
preferably from 96-99% homologous, even more preferably at least 95%


CA 02238217 1998-OS-22
identical and especially preferably from 96% to 99% identical to the full
length 4E-BP3 polypeptide sequence or fragments or derivatives thereof.
As used herein, the terms "molecule", "compound" or
"ligand" are used interchangeably and broadly to refer to natural,
5 synthetic or semi-synthetic molecules or compounds. The term "molecule"
therefore denotes for examples chemicals, macromolecules, cell or tissue
extracts (from plants or animals) and the like. Non-limiting examples of
molecules include nucleic acid molecules, peptides, antibodies,
carbohydrates and pharmaceutical agents. The agents can be selected
10 and screened by a variety of means including random screening, rational
selection and by rational design using for example protein or ligand
modelling methods such as computer modelling. The terms "rationally
selected" or "rationally designed" are meant to define compounds which
have been chosen based on the configuration of the interaction domains
15 of the present invention. As will be understood by the person of ordinary
skill, macromolecules having non-naturally occurring modifications are
also within the scope of the term "molecule". For example,
peptidomimetics, well known in the pharmaceutical industry and generally
referred to as peptide analogs can be generated by modelling as
2o mentioned above. Similarly, in a preferred embodiment, the polypeptides
of the present invention are modified to enhance their stability. It should
be understood that in most cases this modification should not alter the
biological activity of the interaction domain. The molecules identified in
accordance with the teachings of the present invention have a therapeutic
25 value is diseases or conditions in which the physiology or homeastasis of
the cell and/or tissue is compromised by a defect in in modulating gene
expression and/or translation. Alternatively, the molecules identified in


CA 02238217 1998-OS-22
26
accordance with the teachings of the present invention find utility in the
development of more efficient cell lines or cell extracts for translating
mRNAs. Non-limiting examples of diseases and/or conditions in which
the protein and/or nucleic acid molecules of the present invention find
utility include cancer, apoptosis and aberrant proliferation of cells.
As used herein, agonists and antagonists of translation
activity also include potentiators of known compounds with such agonist
or antagonist properties. In one embodiment, agonists can be detected
by contacting the indicator cell with a compound or mixture or library of
molecules, for a fixed period of time, and then determining the effect of
the compound on the cell.
The level of gene expression of the reporter gene (e.g.
the level of luciferase, or (3-gal, produced) within the treated cells can be
compared to that of the reporter gene in the absence of the molecule(s).
The difference between the levels of gene expression indicates whether
the molecules) of interest agonizes the aforementioned interaction. The
magnitude of the level of reporter gene product expressed (treated vs.
untreated cells) provides a relative indication of the strength of that
molecules) as an agonist. The same type of approach can also be used
2o in the presence of an antagonist(s).
Alternatively, an indicator cell in accordance with the
present invention can be used to identify antagonists. For example, the
test molecule or molecules are incubated with the host cell in conjunction
with one or more agonists held at a fixed concentration. An indication and
relative strength of the antagonistic properties of the molecules) can be
provided by comparing the level of gene expression in the indicator cell
in the presence of the agonist, in the absence of test molecules vs in the


CA 02238217 1998-OS-22
27
presence thereof. Of course, the antagonistic effect of a molecule can
also be determined in the absence of agonist, simply by comparing the
level of expression of the reporter gene product in the presence and
absence of the test molecule(s).
It shall be understood that the "in vivo" experimental
model can also be used to carry out an "in vitro" assay. For example,
cellular extracts from the indicator cells can be prepared and used in one
of the aforementioned "in vitro" tests (such as binding assays or in vitro
translations).
As used herein the recitation "indicator cells" refers to
cells that express 4E-BPs and eIF-4E, and wherein an interaction
between the domains responsible for their interaction is coupled to an
identifiable or selectable phenotype or characteristic such that it provides
an assessment of the interaction between these domains. Such indicator
cells can be used in the screening assays of the present invention. In
certain embodiments, the indicator cells have been engineered so as to
express a chosen derivative, fragment, homolog, or mutant of 4E-BP (i.e.
eIF4E interacting domains). The cells can be yeast cells or higher
eukaryotic cells such as mammalian cells (WO 96/41169). In one
particular embodiment, the indicator cell is a yeast cell harboring vectors
enabling the use of the two hybrid system technology, as well known in
the art (Ausubel et al., 1994, supra) and can be used to test a compound
or a library thereof. In one embodiment, a reporter gene encoding a
selectable marker or an assayable protein can be operably linked to a
control element such that expression of the selectable marker or
assayable protein is dependent on the interaction of the 4E-BP3 domain
with its binding partner (i.e. eIF-4E). Such an indicator cell could be used


CA 02238217 1998-OS-22
28
to rapidly screen at high-throughput a vast array of test molecules. In a
particular embodiment, the reporter gene is luciferase or (3-Gal.
As exemplified herein below in one embodiment, at least
one of 4E-BP3 domain may be provided as a fusion protein. The design
of constructs therefor and the expression and production of fusion
proteins are exemplified herein (i.e. Example 1 ) and are well known in the
art (Sambrook et al., 1989, supra; and Ausubel et al., 1994, supra). In a
particular embodiment, both the eIF-4E interaction domain of 4E-BP3 and
eIF-4E are part of fusion proteins. For example, in a particular
embodiment, the fusions are a LexA-4E-BP3 fusion (DNA-binding domain
-4E-BP3; bait) and a B42-eIF-4E fusion (transactivator domain - 4E-BP3;
prey). In still a particularly preferred embodiment, the LexA-4E-BP3 and
B42-eIF-4E fusion proteins are expressed in a yeast cell also harboring
a reporter gene operably linked to a LexA operator and/or LexA
responsive element. As exemplified hereinbelow, the fusions are GST-
4E-BP3 and FLAG-eIF-4E.
Non-limiting examples of such fusion proteins include
Gluthione-S-transferase (GST) fusions, HIS fusions, FLAG fusions,
hemaglutinin A (HA) fusions and Maltose binding protein (MBP) fusions.
In certain embodiments, it might be beneficial to introduce a protease
cleavage site between the two polypeptide sequences which have been
fused. Such protease cleavage sites between two heterologously fused
polypeptides are well known in the art.
In certain embodiments, it might also be beneficial to
fuse the interaction domains of the present invention to signal peptide
sequences enabling a secretion of the fusion protein from the host cell.
Signal peptides from diverse organisms are well known in the art.


CA 02238217 1998-OS-22
29
Bacterial OmpA and yeast Suc2 are two non-limiting examples of proteins
containing signal sequences. In certain embodiments, it might also be
beneficial to introduce a linker (commonly known) between the interaction
domain and the heterologous polypeptide portion. Such fusion protein find
utility in the assays of the present invention as well as for purification
purposes, detection purposes and the like.
For certainty, the sequences and polypeptides useful to
practice the invention include without being limited thereto mutants,
homologs, subtypes, alleles and the like. It shall be understood that
generally, the sequences of the present invention should encode a
functional (albeit defective) interaction domain. It will be clear to the
person of ordinary skill that whether an interaction domain of the present
invention, variant, derivative, or fragment thereof retains its function in
binding to its partner can be readily determined by using the teachings
and assays of the present invention and the general teachings of the art.
As exemplified herein below, the interaction domains of
the present invention can be modified, for example by in vitro
mutagenesis, to dissect the structure-function relationship thereof and
permit a better design and identification of modulating compounds.
However, some derivative or analogs having lost their biological function
of interacting with their respective interaction partner may still find
utility,
for example for raising antibodies. Such analogs or derivatives could be
used for example to raise antibodies to the interaction domains of the
present invention. These antibodies could be used for detection or
purification purposes. In addition, these antibodies could also act as
competitive or non-competitive inhibitor and be found to be modulators of
4E-BP3 activity and/or cap-dependent and/or cap-independent


CA 02238217 1998-OS-22
translation. A non-limiting example of an antibody reacting with 4E-BP3
is exemplified hereinbelow (antiserum 1791 and 1862).
A host cell or indicator cell has been "transfected" by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA
5 has been introduced inside the cell. The transfecting DNA may or may not
be integrated (covalently linked) into chromosomal DNA making up the
genome of the cell. In prokaryotes, yeast, and mammalian cells for
example, the transfecting DNA may be maintained on an episomal
element such as a plasmid. With respect to eukaryotic cells, a stably
10 transfected cell is one in which the transfecting DNA has become
integrated into a chromosome so that it is inherited by daughter cells
through chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones comprised of
a population of daughter cells containing the transfecting DNA.
15 Transfection methods are well known in the art (Sambrook et al., 1989)
supra; Ausubel et al., 1994, supra). It will be understood that extracts
from animal cells or mammalian cells for example could be used in certain
embodiments, to compensate for the lack of certain factors in lower
eukaryotic indicator cells.
20 In general, techniques for preparing antibodies
(including monoclonal antibodies and hybridomas) and for detecting
antigens using antibodies are well known in the art (Campbell, 1984, In
"Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology", Elsevier Science Publisher,
25 Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody -
A Laboratory Manual, CSH Laboratories). The present invention also
provides polyclonal, monoclonal antibodies, or humanized versions


CA 02238217 1998-OS-22
31
thereof, chimeric antibodies and the like which inhibit or neutralize their
respective interaction domains and/or are specific thereto. A non-limiting
example of an antibody reacting with 4E-BP3 is exemplified hereinbelow
(antiserum 1791 and 1862).
The present invention also provides antisense nucleic
acid molecules which can be used for example to decrease or abrogate
the expression of 4E-BP3. An antisense nucleic acid molecule according
to the present invention refers to a molecule capable of forming a stable
duplex or triplex with a portion of its targeted nucleic acid sequence (DNA
or RNA). The use of antisense nucleic acid molecules and the design and
modification of such molecules is well known in the art as described for
example in WO 96/32966, WO 96/11266, WO 94/15646, WO 93/08845,
and USP 5,593,974. Antisense nucleic acid molecules according to the
present invention can be derived from the nucleic acid sequences and
modified in accordance to well known methods. For example, some
antisense molecules can be designed to be more resistant to degradation
to increase their affinity to their targeted sequence, to affect their
transport to chosen cell types or cell compartments, and/or to enhance
their lipid solubility by using nucleotide analogs and/or substituting chosen
2o chemical fragments thereof, as commonly known in the art.
From the specification and appended claims, the term
therapeutic agent should be taken in a broad sense so as to also include
a combination of at least two such therapeutic agents. Further, the DNA
segments or proteins according to the present invention can be
introduced into individuals in a number of ways. For example,
erythropoietic cells can be isolated from the afflicted individual,
transformed with a DNA construct according to the invention and


CA 02238217 1998-OS-22
32
reintroduced to the afflicted individual in a number of ways, including
intravenous injection. Alternatively, the DNA construct can be
administered directly to the afFlicted individual, for example, by injection
in the bone marrow. The DNA construct can also be delivered through a
vehicle such as a liposome, or nanoerythrosome which can be designed
to be targeted to a specific cell type, and engineered to be administered
through different routes.
For administration to humans, the prescribing medical
professional will ultimately determine the appropriate form and dosage for
a given patient, and this can be expected to vary according to the chosen
therapeutic regimen (i.e DNA construct, protein, cells), the response and
condition of the patient as well as the severity of the disease.
Composition within the scope of the present invention
should contain the active agent (i.e. fusion protein, nucleic acid, and
molecule) in an amount effective to achieve an inhibitory effect on HIV
and related viruses while avoiding adverse side effects. Typically, the
nucleic acids in accordance with the present invention can be
administered to mammals (i.e. humans) in doses ranging from 0.005 to
1 mg per kg of body weight per day of the mammal which is treated.
Pharmaceutically acceptable preparations and salts of the active agent
are within the scope of the present invention and are well known in the art
(Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For the
administration of polypeptides, antagonists, agonists and the like, the
amount administered should be chosen so as to avoid adverse side
effects. The dosage will be adapted by the clinician in accordance with
conventional factors such as the extent of the disease and different


CA 02238217 1998-OS-22
33
parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be
administered to the mammal.
As used herein, "cap-independent translation" is meant
to include translation which is not dependent on eIF-4E such as
translation through an internal ribosomal entry site (IRES; or internal
translation) and non-IRES dependent translation such as, for example,
translation of AMV-4 messages.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Experimental procedures
Cloning of 4E-BP3 cDNA
The cDNA clone #87895 (Human Genome Sciences
Inc.) was obtained from a prostate carcinoma cell line (LNCaP) EST
cDNA library as described (Adams et al., 1995, Nature 377(6547)
Suppl:3-174). Sequence alignment of 4E-BPs was performed using
CLUSTAL W Multiple Sequence Alignment Program (version 1.7)
(Thompson et al., 1994, Nucleic Acids Res. 22(22):4673-4680).
Northern blotting analysis
Human 4E-BP3 mRNA distribution was analyzed by
Northern hybridization using commercial blots (Multiple Tissue Northern
Blot 1 and 2, Clontech), with nucleotides 1-597 of the cDNA clone used
as a probe, according to the Manufacturer's instructions.
Antibodies
Anti-4E-BP3 antibody #1791 was raised in a rabbit
against a synthetic peptide (CVTTPPTAPLSKLEE) comprising amino


CA 02238217 1998-OS-22
34
acids 67 to 80 of 4E-BP3. The peptide (2 mg) was cross-linked to keyhole
limpet hemocyanin (KLH) using a commercial kit (Pierce Chemical
Company) according to the Manufacturer's instructions. Anti-4E-BP3
antibody #1862 was raised in a rabbit against a GST-4E-BP3 fusion. Anti-
eIF4E antibody #5853 was described previously (Thompson et al., 1994,
supra).
Plasmids
pcDNA3-4E-BP3 was generated by cloning the coding
sequence of 4E-BP3 into pcDNA3 (Invitrogen) using PCR. Mutants of 4E-
BP3 (Y40A or L45A) were generated by PCR mutagenesis and
subcloned into pcDNA3. For N-terminally HA-tagged constructs, the
coding sequence of 4E-BP3, 4E-BP3-Y40A or 4E-BP3-L45A was
subcloned into pcDNA3-HA (Imataka et al., 1997, EMBO J. 1~:817-
825). pcDNA3-FLAG-eIF4E was constructed by cloning the entire coding
sequence of mouse eIF4E into pcDNA3-FLAG using PCR. The bicistronic
reporter plasmid pcDNA3-RLUC-POLIRES-FLUC was generated by
subcloning the coding sequence for Firefly Luciferase (FLOC) from
pGEM-LUC (Promega), the coding sequence for Renilla reniformis
Luciferase (RLUC) from pRL-CMV (Promega) and the complete
Poliovirus type II Lansing 5'UTR (POLIRES) (nucleotides 1-737) (La
Monica et al., 1986, J. Virol. 5712):515-525) into pcDNA3.
Western blotting analysis
Polypeptides were resolved on SDS-15%
polyacrylamide gels and transferred onto a 0.22 pm nitrocellulose
membrane. The membrane was blocked for 16 h at 4°C with 5% milk in
Tris-buffered saline containing 0.5% Tween 20 (TBST). The membrane
was incubated for 2 h with primary antibody 1862 (1:1000 in TBST with


CA 02238217 1998-OS-22
1 % BSA) or 1791 (1:750 in TBST with 1 % BSA). Incubation with
secondary antibody was performed with peroxidase-coupled donkey anti-
rabbit Ig (Amersham)(1:5000 in TBST). Detection was performed with
ECL (Amersham). For adsorption experiments, antiserum (10 pl) was pre-
y incubated on ice for 20 min with GST-HMK (20 pg) or GST-BP3 (20 pg)
for antiserum 1862 or with the cognate peptide (20 pg) for antiserum
1791.
In vitro translation and cap-affinity assay
Capped RNA was synthesized with T7 RNA polymerase
10 in the presence of the cap analog, m'GpppG (Pelletier et al., 1985, Cell
40(3):515-526). Wheat germ extract (Promega) (25 pl) was programmed
with mRNA in the presence of [35S] cysteine or methionine (25 pCi)
according to the Manufacturer's instructions. Following translation,
extracts programmed with FLAG-eIF4E or 4E-BP3 mRNA were mixed
15 and incubated on ice for 1 h to allow for protein interaction. Protein
complexes were recovered at 4°C for 60 min with 20 pl of m'GDP-
agarose resin (Edery et al., 1988, Gene 7_ 4(2):517-525) previously
washed in buffer A (50mM Tris-HCI, pH 7.5, 150 mM KCI, 1 mM DTT, 1
mM EDTA, 1 mM PMSF). The resin was washed 3 times with 1 ml buffer
20 A, resuspended in Laemmli sample buffer (Laemmli, 1970, Nature
22 X259):680-685), boiled and proteins were resolved by SDS-PAGE.
Gels were processed for fluorography with En3Hance (DuPont).
Cell Culture, transfections and extracts preparation
HeLa cells were grown in DMEM containing 10% FBS.
25 Cells were infected for 1 h in serum free medium with recombinant
vaccinia virus vTF7-3 (Fuerst et al., 1986, Proc. Natl. Acad. Sci. USA
83(21 ):8122-8126; Belsham et al., 1990, J. Virol. 64(11 ):5389-5395).


CA 02238217 1998-OS-22
36
Cells were then transiently transfected with pcDNA3-RLUC-POLIRES-
FLUC (0.75 pg) and either pcDNA3, pcDNA3-4E-BP3, pCDNA3-4E-BP3-
Y40A or pcDNA3-4E-BP3-L45A (2 pg) using Lipofectin (Gibco-BRL)(3.5
pl) as previously described (Pause et al., 1994, supra). Cell extracts were
prepared in Passive Lysis Buffer (Promega) 17 h after infection and
assayed for RLUC and FLUC activity in a luminometer (BIOORBIT) using
a Dual-Luciferase Reporter Assay System (Promega).
HeLa cells (6 cm dish) were transiently transfected with
pcDNA3, pcDNA3-4.E-BP3 (6 pg) or with pcDNA3-FLAG-eIF4E (3 pg) and
pcDNA3-HA-La, pcDNA3-HA-4E-BP3, pcDNA3-HA-4E-BP3-Y40A or
pcDNA3-HA-4E-BP3-L45A (3 pg) using Lipofectamine (Gibco-BRL) (10
ill) according to the Manufacturer's instructions. Cell extracts were
prepared after 24 h by scraping cells in cold buffer A and subjecting the
suspension to three freeze-thaw cycles. Cell debris was pelleted by
centrifugation and the protein concentration in the supernatant was
determined by the Bio-Rad assay.
LNCaP cells were grown in RPMI 1640 containing 10%
FBS. Cell extracts were prepared in buffer A as described above. LNCaP
cell extract (5 mg/ml) was boiled for 8 min and then incubated on ice for
10 min. The precipitated material was removed by centrifugation and
Laemmli sample buffer was added to the supernatant before boiling for
5 min.
Co-immunoprecipitation and m'GDP-agarose precipitation
For co-immuno-precipitation experiments, HeLa cells (6
cm dish) were lysed 24 h after transfection in 500 ill of cold NP40 buffer
(50mM Tris-HCI, pH 7.5, 100 mM KCI, 0.5% Nonidet P-40, 1 mM DTT, 0.5
mM EDTA, 1 mM PMSF) and debris was spun down. Extracts were


CA 02238217 1998-OS-22
37
immunoprecipitated with anti-HA antibody HA.11 (BAbCO)(1.25 Ng) for
1 h at 4°C. Protein G-sepharose (10 pl) was added and the mixture was
incubated for 1 h at 4°C. After washing 3 times with 1 ml NP40 buffer,
immunoprecipitates were eluted in Laemmli sample buffer and proteins
were subjected to SDS-PAGE.
For m'GDP-agarose precipitation, 1.5 mg of LNCaP cell
extract, prepared in buffer A as described above, was incubated with 30
pl of m'GDP-agarose resin for 90 min at 4°C. The resin was washed 3
times with 1 ml buffer A, resuspended in Laemmli sample buffer and
proteins were subjected to SDS-PAGE.
Metabolic labelling and immunoprecipitation
LNCaP cells or transfected (pcDNA3 or pcDNA3-4E-
BP3) HeLa cells (10 cm dish) were incubated for 5 h at 37°C in
serum-
free EMEM containing 0.5 mCi/ml [32P] orthophosphate (DuPont-NEN;
3000 mCi/mmole). The medium was removed and the cells rinsed twice
with PBS. Cells were lysed in lysis buffer [10% glycerol, 50 mM Tris (pH
7.5), 60 mM KCI, 2 mM CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-
tetraacetic acid), 1 % Triton X-100, 2mM DTT, 50 mM [3-
glycerolphosphate, 0.1 mM sodium orthovanadate, 1 mM EGTA, 10 mM
sodium pyrophosphate, 50 mM sodium fluoride, 1 mM PMSF] for 30 min
at 4°C. Cells were harvested by scraping and cell debris was removed by
centrifugation. The extract was precleared at 4°C for 1 h by incubation
with pre-immune serum (7 pl) bound to protein A beads (15 pl). The
supernatant was transferred to a fresh tube together with 1862 antiserum
(10 fll) bound to protein A beads (25 NI). Incubation end-over-end was
carried out for 4 h at 4°C. Beads were spun down and washed 3 times
with lysis buffer, 3 times with RIPA buffer and 3 times with LiCI buffer (500


CA 02238217 1998-OS-22
38
mM LiCI, 50 mM Tris (pH 7.5)). Immuno-precipitated material was
subjected to SDS-15% PAGE and transferred to a nitrocellulose
membrane which was dried and autoradiographed.
EXAMPLE 2
Cloning of 4E-BP3 cDNA
A cDNA for 4E-BP3 (Fig. 1A) was isolated from a
LNCaP cDNA library. The cDNA is 698 by long and consists of a 5'
untranslated region (UTR) of 72 bp, an open reading frame (ORF) of 300
bp, and a 3'UTR of 326 bp. Whether the 5'UTR of the cDNA is full length
remains to be confirmed. It is most probably close to full length, as the
size of the mRNA observed in Fig. 3 (800 bp) is close to the size of the
cDNA (698 bp), taking into consideration the presence of a poly A tail on
the mRNA. The 3'UTR of the cDNA contains a polyadenylation signal
(AGUAAA, underlined in Fig. 1A) located 9 nucleotides upstream of the
polyA tail. The sequence, although not identical to the consensus
polyadenylation sequence (AAUAAA), has been demonstrated to function
in some viral cDNAs (Guntaka, 1993, Microbiol. Rev. 5713):511-521 ) and
has not been excluded as a putative polyadenylation signal in eukaryotic
mRNAs (Birnstiel et al., 1985, Cell 41 (2):349-359). The ORF encodes a
predicted 100 amino acid protein with a molecular weight of 10,873. This
putative polypeptide is highly homologous to the previously cloned 4E-
BP1 and 4E-BP2 proteins, sharing 57% and 59% identity, respectively
(Fig. 1 B)(Pause et al., 1994, supra). The homology between the 4E-BPs
is highest in the middle portion, which contains the eIF4E binding region
(Mader et al., 1995, supra) (Fig. 1 B, boxed residues). It is also of interest
that all of the phosphorylation sites reported for rat 4E-BP1 (Fadden et


CA 02238217 1998-OS-22
39
al., 1997, J. Biol. Chem. 272 15 :10240-10247) are conserved in 4E-BP2
and 4E-BP3 (Fig. 1 B, denoted by asterisks), with the exception of a
conservative change, serine 83 (in 4E-BP1 ) to a threonine in 4E-BP3. All
of the phosphorylation sites contain a (Ser/Thr)-Pro motif.
Database searches in GeneBank identified several
mouse ESTs that are predicted to encode for a protein highly homologous
to the human 4E-BP3. The longest EST (GeneBank Accession Number
W18851) is 623 by long and could encode a protein of 101 amino acids
with 86% identity to the human 4E-BP3 (Fig. 1 C). It is therefore most
probably the mouse homologue of human 4E-BP3.
A new cDNA encoding for a protein that shares a high
degree of identity with the translational inhibitors 4E-BP1 and 4E-BP2
(Pause et al., 1994, supra), and was thus termed 4E-BP3 has been
isolated. Database searches (NCBI database and Human Genome
Sciences database) have not revealed any homologies other than to 4E-
BP1, 4E-BP2 and to ESTs encoding for the three 4E-BPs.
4E-BP3 was identified by Western blotting as a
polypeptide having an apparent molecular mass of 14.6 kDa, which
differs from the predicted molecular mass of 10.9 kDa. However,
overexpression of 4E-BP3 in HeLa cells and in vitro translation of 4E-BP3
also yielded a polypeptide having an apparent molecular mass of 14.6
kDa, suggesting that the cDNA indeed encodes for a full-length protein.
This type of aberrant migration is also observed for 4E-BP1 and 4E-BP2
(Pause et al., 1994, supra; Hu et al., 1994, Proc. Natl. Acad. Sci. USA
91 (9):3730-3734; Lin et al., 1996, J. Biol. Chem. 271 (47):30199-30204),
and is thought to be due to the high proline content of the proteins, which
might affect their mobility in SDS-PAGE (See et al., 1989, in Protein


CA 02238217 1998-OS-22
Structure: A Practical Approach Creighton, T. E., ed, pp. 1-21, IRL Press,
Oxford). Further, the 14.6 kDa polypeptide is recognized by two different
antibodies and shares the characteristic heat-stability of 4E-BP1 and 4E-
BP2 (Pause et al., 1994, supra; Gingras et al., 1997, supra; Lin et al.,
5 1996, supra). Taken together, these data strongly suggest that 4E-BP3
it is the third member of the 4E-BP family.
EXAMPLE 3
Tissue distribution of 4E-BP3
10 The tissue distribution of 4E-BP3 mRNA was analyzed
by Northern blotting (Fig. 2). Two transcripts were detected: a major
transcript of approximately 800 by and a minor transcript of approximately
8.6 kb. The smaller transcript most probably corresponds to the cDNA
characterized in Example 2. Although 4E-BP3 RNA is expressed in
15 almost every tissue, the levels of expression vary dramatically among
tissues. 4E-BP3 expression is highest in skeletal muscle, heart, kidney
and pancreas, whereas there is very little expression in brain and thymus.
The origin of the 8.6 kb transcript is not clear at present; it could
represent
a product of alternative splicing or a stable nuclear RNA containing intron
20 sequences. A similar pattern of expression with two different transcripts
was also observed for 4E-BP2 (Tsukiyama-Kohara et al., 1996, Genomics
38 3 :353-363), possibly pointing to an alternative splicing pattern
conserved in 4E-BP3.
4E-BP3 mRNA tissue expression differs from that of 4E-
25 BP1 and 4E-BP2 (Tsukiyama-Kohara et al., 1996, supra;Hu et al., 1994,
supra). This may suggest that the regulation of eIF4E function by the 4E-
BPs is modulated in a tissue-specific manner, and is dependent upon the


CA 02238217 1998-OS-22
41
relative abundance of each 4E-BP. Since the 4E-BPs share biochemical
and functional characteristics, it is likely that their relative activity in a
given tissue or cell type is regulated post-translationally (see Example 7).
EXAMPLE 4
Identification of 4E-BP3 in cells
To determine whether 4E-BP3 protein is expressed in
cells, we used two different antisera raised in rabbits. The first (#1862)
was raised against a GST-4E-BP3 fusion. The second (#1791 ) was
raised against a peptide sequence derived from amino acids 67 to 80 in
the C-terminal half of 4E-BP3 (bold character in Fig. 1A), which is not
conserved in the other 4E-BPs (Fig. 1 B). Since 4E-BP3 cDNA was cloned
from a cDNA library constructed from an LNCaP human prostate
carcinoma cell line (Webber et al., 1997, Prostate 30(21:58-64), an extract
from this cell line was used to perform Western blotting. Each of the anti-
4E-BP3 antisera detected a single polypeptide of 14.6 kDa (Fig. 3A, lanes
2 and 3). Moreover, the band corresponding to the 14.6 kDa polypeptide
disappeared when the two antibodies were pre-incubated with their
respective antigens (Fig. 3A, lanes 1 and 4). To further substantiate the
authenticity of the 14.6 kDa polypeptide, we expressed 4E-BP3 cDNA in
HeLa cells, which do not express 4E-BP3 at detectable levels (Fig. 3B,
lane 1). A polypeptide which co-migrates with 4E-BP3 from LNCaP was
observed in cells transfected with pcDNA3-4E-BP3 (Fig. 3B, compare
lane 2 to lane 3). Since 4E-BP1 and 4E-BP2 are heat stable proteins
(Pause et al., 1994, supra; Lin et al., 1994, supra), we wished to
determine whether 4E-BP3 is also heat stable. An LNCaP cell extract was
boiled, the soluble fraction subjected to SDS-PAGE and the proteins


CA 02238217 1998-OS-22
42
transferred to a nitrocellulose membrane. Probing with antisera 1791
demonstrated that 4E-BP3 is also heat stable (Fig. 3B, lane 4).
EXAMPLE 5
Interaction of 4E-BP3 with eIF4E
Since the eIF4E binding region in 4E-BPs is highly
conserved in 4E-BP3 (Fig. 1 B, boxed residues), it is most likely that 4E-
BP3 would also interact with eIF4E. Mutants in the predicted eIF4E-
binding site were generated to further characterize this interaction. The
design of the mutants was based on the results of Mader et al. (Mader et
al., 1995, supra), and from analysis of the conserved residues of the
eIF4E binding motif found in all of the eIF4E binding proteins reported so
far (Pause et al., 1994, supra; Gradi et al., 1998, Mol. Cell. Biol.
18 1 :334-342; Altmann et al., 1997, EMBO J. 16(5):1114-1121; Goyer
et al., 1993, Mol. Cell. Biol. 130:4860-4874). I n the first construct, Tyr 40
of 4E-BP3 was mutated to an alanine (4E-BP3-Y40A). An identical
mutation in the eIF4E binding motif of eIF4G abolished its binding to
eIF4E (Mader et al., 1995, supra). The second construct was a mutant in
which Leu 45 was changed to an alanine (4E-BP3-L45A). Such a
mutation was not analyzed by Mader et al., where only the two
consecutive leucines of the motif were mutated to alanines, resulting in
the loss of interaction with eIF4E (Mader et al., 1995, supra).
To demonstrate an interaction between eIF4E and 4E-
BP3, the proteins were synthesized by in vitro translation, and analyzed
for complex formation. Presence of the in vitro translated proteins was
confirmed by SDS-PAGE, followed by fluorography (Fig. 4A). Very little
wild type 4E-BP3 bound to the resin (Fig. 4B, lane 1). However, a


CA 02238217 1998-OS-22
43
significant amount of 4E-BP3 was retained on the resin in the presence
of FLAG-eIF4E (4 fold increase as determined by PhosphorlmagerT""
analysis, compare lane 2 to lane 1 ). This demonstrates that, as with 4E-
BP1 and 4E-BP2 (Pause et al., 1994, supra), association of 4E-BP3 with
eIF4E does not affect the binding of eIF4E to the cap structure. Analysis
of the binding was also performed using 4E-BP3 containing mutations in
the eIF4E binding region. The mutants showed no binding to FLAG-eIF4E
(Fig. 4B, lanes 3 and 4). As expected, FLAG-eIF4E bound to the resin
(Fig. 4B, lane 5). Thus, the mutational analysis of the eIF4E interaction
domain demonstrates that the Y40A mutation and the L45A mutation in
the context of 4E-BP3 abrogate in vitro binding therof to eIF4E.
To confirm the interaction of 4E-BP3 and eIF4E in vivo,
co-immunoprecipitations were performed on extracts from HeLa cells
transiently transfected with HA-tagged 4E-BP3. The light chain of the anti-
HA antibody co-migrates with the endogenous eIF4E on SDS-PAGE,
rendering detection of co-immunoprecipitated endogenous eIF4E difficult.
To circumvent this problem, FLAG-tagged eIF4E, which migrates more
slowly than endogenous eIF4E, was co-expressed with HA-tagged 4E-
BP3, 4E-BP3-Y40A, 4E-BP3-L45A or La, an unrelated RNA binding
protein (Chambers et al., 1988, J. Biol. Chem. 263 34 :18043-18051 ).
Expression of the proteins was confirmed by Western blotting with aHA
and aeIF4E (Fig. 4C). Immunoprecipitations were carried out using a
monoclonal anti-HA antibody and the immunoprecipitates were assayed
by Western blotting for the presence of eIF4E and HA-tagged protein.
FLAG-eIF4E was not co-immunoprecipitated when cells were co-
transfected with the pcDNA3-HA vector (Fig. 4D, lane 1 ), nor in the
presence of HA-La (Fig. 4D, lane 2). However, FLAG-eIF4E was co-


CA 02238217 1998-OS-22
44
immunoprecipitated in the presence of HA-4E-BP3 (Fig 4D, lane 3). 4E-
BP3 mutants Y40A and L45A failed to co-immunoprecipitate FLAG-eIF4E
(Fig. 4D, lanes 4 and 5). The expression of the Y40A and L45A
constructs was approximately 2 to 4 fold less efficient as compared to that
of the wild type construct (Fig. 4C, compare lanes 4 and 5 to lane 3),
which is expected to affect the amount of FLAG-eIF4E brought down in
the assay. However, this cannot explain the complete absence of FLAG-
eIF4E.
Association of endogenous eIF4E with 4E-BP3 was also
examined in LNCaP cells. The presence of eIF4E and 4E-BP3 in LNCaP
cell extract was demonstrated by Western blotting with aeIF4E and a4E-
BP3 (Fig. 4E, lane 1). eIF4E was precipitated from the extract using
m~GDP-agarose resin and the presence of endogenous eIF4E and 4E-
BP3 in the precipitate was observed by Western blotting (Fig. 4E, lane 2).
Interaction of 4E-BP3 with eIF4E was demonstrated by
co-immunoprecipitation and by m~GDP-affinity chromatography, both in
cell extracts and with in vitro translated proteins. The interaction was also
analyzed by generating mutants in the eIF4E binding motif of 4E-BP3.
The two mutants (Y40A and L45A) did not show any detectable
interaction with eIF4E in the assays performed. This result with the Y40A
mutant, in the context of eIF-4G, was suggested by the previously
showing that such a mutation in eIF4G abolishes binding to eIF4E (Mader
et al., 1995, supra). Nevertheless, the instant demonstration with Y40A
shows that this mutation, in the context of 4E-BP3, also abbrogates its
interaction with eIF-4E. In addition, the inability to detect an interaction
of the L45A mutant with eIF4E demonstrates that the first of the two
consecutive leucine residues in the eIF4E binding motif, alone,


CA 02238217 1998-OS-22
dramatically affects the interaction. Together with absolute conservation
of the first leucine in all of the eIF4E binding proteins from yeast to human
(Pause et al., 1994, supra; Gradi et al., 1998, supra; Altmann et al., 1997,
supra; Goyer et al., 1993, supra; Yan et al., 1992, J. Biol. Chem.
5 267 32 :23226-23231 ), this first leucine is therefore demonstrated to be
critical in eIF-4E interaction.
EXAMPLE 6
Inhibition of cap-dependent translation in vivo
10 To determine whether 4E-BP3 inhibits cap dependent
translation, a transient expression assay was used in which HeLa cells
were infected with the recombinant vaccinia virus vTF7-3 (Fuerst et al.,
1986, supra; Belsham et al., 1990, supra), and co-transfected with 4E-
BP3 wild type or mutants constructs and a reporter plasmid. The reporter
15 construct consisted of two cistrons separated by the poliovirus internal
ribosome entry site (POLIRES) (Fig. 5A). Translation of the Renilla
reniformis Luciferase (RLUC) cistron is cap-dependent, whereas that of
the Firefly Luciferase (FLUC) cistron is directed by the poliovirus IRES
and is therefore cap-independent (Pelletier et al., 1988, Nature
20 334 6180 :320-325). Expression of the 4E-BP3 proteins was assayed by
Western blotting and the results show a similar expression level for all the
constructs (Fig. 5B). The level of expression of RLUC and FLUC was set
at 100% when the reporter plasmid was co-transfected with an empty
vector (pcDNA3) (Fig. 5C and 5D, column 1 ). When co-transfected with
25 a vector containing the coding sequence for 4E-BP3, the expression of
RLUC was decreased by 72% (Fig. 5C, column 2), whereas the
expression of FLUC showed a twofold increase (Fig. 5D, column 2). The


CA 02238217 1998-OS-22
46
Y40A mutant 4E-BP3 as well as the L45A mutant lost their capacity to
inhibit cap-dependent translation (Fig. 5C, column 3, 110% of the control;
column 4, 90% of the control, respectively). Of note, the two mutants did
not affect cap-independent translation (Fig. 5D, columns 3 and 4), in
contrast to wild type 4E-BP3.
The effect of the Y40A and L45A mutants on translation
was also assessed in a transient expression assay in which 4E-BP3 was
shown to inhibit eIF4E-dependent translation by 72%. As expected from
mutants having an impaired ability to bind to eIF4E, no effect was
observed on eIF4E-dependent translation. Strikingly however, the
mutants also abolished the stimulation (2 fold) of IRES-mediated
translation by 4E-BP3. Such a stimulation had not been observed in
earlier reports with 4E-BP1 and 4E-BP2 (Pause et al., 1994, supra;
Ohlmann et al., 1996, EMBO J. 15(6):1371-1382). The stimulation is
specific as the mutants which are defective in binding to eIF4E, although
expressed at a level similar to that of wild type 4E-BP3 (Fig 5B, compare
lanes 3 and 4 to lane 2), lost the ability to stimulate IRES-dependent
translation. Since 4E-BPs and eIF4G compete for a limiting amount of
eIF4E, it is possible that the high level of overexpression of 4E-BP3 in this
system (Fig. 5B, lane 2) releases more of eIF4G that can participate in
IRES-mediated translation and thus increase the expression of FLUC. In
view of the structural and functional conservation of the 4E-BP family
members, it is expected that similar cap-independent stimulation results
will be obtained with 4E-BP1 and 4E-BP2.
Taken together, these data demonstrate the novel
feature of 4E-BPs to affect both cap-dependent and cap-independent
translation. With the increasing number of highly regulated mRNAs


CA 02238217 1998-OS-22
47
(Jackson, R., 1996, pp. 71-112, Translation Control, CSH Laboratory
Press CSH, NY) which are translated at least in part via a cap-
independent mechanism, the instant finding is of significant importance.
EXAMPLE 7
Metabolic labelling_of 4E-BP3
To demonstrate that 4E-BP3 is a phosphoprotein,
metabolic labelling of transfected HeLa cells or LNCaP cells was
performed using [3zP] orthophosphate. Analysis of the immunoprecipitated
material shows that 4E-BP3 was labelled with [32P] in HeLa cells
transfected with pcDNA3-4E-BP3 (Fig. 6, lane 2), but not in HeLa cells
transfected with pcDNA3 (Fig. 6, lane 1 ). Moreover, the endogenous 4E-
BP3 was also found to be labelled in LNCaP cells (Fig. 6, lane 3). Taken
together the data indicate that, as with 4E-BP1 and 4E-BP2 (Pause et al.,
1994, supra; Lin et al., 1994, supra; Gingras et al., 1997, supra), 4E-BP3
is also a phosphoprotein.
It is well established that 4E-BP1 activity is regulated via
phosphorylation (Pause et al., 1994, supra; Fleurent et al., 1997, supra;
Gingras et al., 1996, Proc. Natl. Acad. Sci. USA 93 11 :5578-5583; von
Manteuffel et al., 1996, Proc. Natl. Acad. Sci. USA 9:4076-4080).
Since 4E-BP3 is a phosphoprotein, it is highly likely that its activity is
also
regulated by its phosphorylation state. A differential regulation of the
phosphorylation of the 4E-BPs could explain the redundancy of their
function. However, recent reports show that 4E-BP1 and 4E-BP2 behave
similarly in response to insulin and cAMP treatment (Lin et al., 1996,
supra) or to adenovirus infection (Gingras et al., 1997, supra;Feigenblum
et al., 1996, Mol. Cell. Biol. 16 10 :5450-7). Therefore, the overlapping


CA 02238217 1998-OS-22
48
expression of the 4E-BPs may also reflect a safeguard mechanism. In
support of such an hypothesis, a mouse knock-out of 4E-BP1 failed to
show any phenotype2 (Blackshear et al., 1997, J. Biol. Chem.
272 50 :31510-31514), suggesting that the overlapping expression of
other 4E-BPs may compensate for the disrupted 4E-BP1. This
underscores the importance of 4E-BPs in regulating eIF4E-dependent
translation initiation.
Recent data has now shown that Ser37, Ser45 and
especially Ser64 mutations in 4E-BP1 (all of which affect the
phosphorylation state thereof) abrogated their release from eIF-4E.
Although their biological activity has yet to be analyzed, it is expected that
such mutants should be more efficient at reverting, for example the
transformation phenotype of eIF-4E overexpressing cells. Based on the
conservation between 4E-BP1, -BP2 and -BP3, similar mutations in the
other family members, at residues which are phosphorylated, are
expected to show similar results in relation to eIF-4E interaction and
translation control. It will be interesting to verify whether a mutation of
these phosphorylated residues (or others) with one that mimicks the
negative charge of the phosphate group, behaves in the opposite way as
the Ser mutations listed above.
Conclusion
The data presented herein show that 4E-BP3 shares
biochemical and functional characteristics with 4E-BP1 and 4E-BP2. To
examine whether the 4E-BPs are differentially regulated, it will be
important to determine if 4E-BP3 behaves in a manner similar to 4E-BP1
and 4E-BP2 in response to extracellular stimuli. Specifically, analysis of


CA 02238217 1998-OS-22
49
the phosphorylation of the 4E-BPs under various conditions will be
required to ascertain if differences among the 4E-BPs exist.
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified,
without departing from the spirit and nature of the subject invention as
defined in the appended claims.
' The abbreviations used are: eIF, Eukaryotic Initiation Factor; 4E-BP,
eIF4E Binding Protein; EST, Expressed Sequence Tag; RLUC, Renilla
reniformis luciferase; FLUC, Firefly luciferase; IRES, Internal Ribosome
Entry Site; GST, Glutathione S-Transferase; HA, hemagglutinin; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.
2 K. Tsukiyama-Kohara and N. Sonenberg, unpublished data.
The nucleotide sequence of human 4E-BP3 cDNA has been deposited
in the GeneBank database under GeneBank Accession Number
AF038869, but has yet to be released publicly.