U.S. patent application number 10/054435 was filed with the patent office on 2003-10-30 for methods and compositions for treating abnormal cell growth related to unwanted guanine nucleotide exchange factor activity.
Invention is credited to Bollag, Gideon, Crompton, Anne, North, Anne, Roscoe, William, Sharma, Sanju.
Application Number | 20030203464 10/054435 |
Document ID | / |
Family ID | 21962222 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203464 |
Kind Code |
A1 |
Bollag, Gideon ; et
al. |
October 30, 2003 |
Methods and compositions for treating abnormal cell growth related
to unwanted guanine nucleotide exchange factor activity
Abstract
Methods and compositions are described that affect the GTPase
activity of members of the Ras superfamily, preferably Rac, such
compositions include guanine nucleotide exchange factors that
modulate the GTPase activity, preferably in the presence of GEF
enhancers, exemplary guanine nucleotide exchange factors being
Rac-GEF and Tiam-1, which are encoded by certain nucleic acid
sequences that are herein described, along with uses for the
guanine nucleotide exchange factors and the nucleic acid sequences
including screening for ligands which recognize Rac-GEF, regulators
of Rac-GEF activity, and methods of treating pathological
conditions associated or related to a Ras superfamily GTPase,
including Rac.
Inventors: |
Bollag, Gideon; (Hercules,
CA) ; Crompton, Anne; (San Francisco, CA) ;
North, Anne; (Pleasant Hill, CA) ; Roscoe,
William; (San Francisco, CA) ; Sharma, Sanju;
(Berkeley, CA) |
Correspondence
Address: |
Gregory Giotta, Ph.D.
Vice President and Chief Legal Counsel
ONYX Pharmaceuticals, Inc.
3031 Research Drive
Richmond
CA
94806
US
|
Family ID: |
21962222 |
Appl. No.: |
10/054435 |
Filed: |
January 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10054435 |
Jan 18, 2002 |
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09079812 |
May 15, 1998 |
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6340575 |
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60049879 |
Jun 17, 1997 |
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Current U.S.
Class: |
435/199 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/82 20130101 |
Class at
Publication: |
435/199 ;
435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/22; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed:
1. An isolated Rac-GEF polypeptide or a biologically-active
fragment thereof.
2. An isolated Rac-GEF, or a biologically-active fragment thereof,
of claim 1, wherein said polypeptide has a guanine nucleotide
exchange activity, a specific binding affinity for a guanine
nucleotide depleted Rac, or a cellular oncogenic transforming
activity.
3. An isolated Rac-GEF or a biologically-active fragment thereof of
claim 1 which is of human.
4. An isolated Rac-GEF of claim 1 comprising amino acid 1 to amino
acid 711, as set forth in FIG. 1 (SEQ. ID NO:2).
5. An isolated biologically-active fragment of Rac-GEF of claim 4
which comprises amino acids 273-605.
6. An isolated Rac-GEF, or a biologically-active fragment thereof,
of claim 1, which is substantially purified.
7. An isolated nucleic acid comprising a nucleotide sequence coding
for a Rac-GEF polypeptide.
8. An isolated nucleic acid of claim 7, wherein said coded for
polypeptide has a guanine nucleotide exchange activity, a specific
binding affinity for a guanine nucleotide depleted Rac, or a
cellular oncogenic transforming activity.
9. An isolated nucleic acid of claim 7 which is human.
10. An isolated nucleic acid of claim 7, wherein the nucleic acid
sequence codes for amino acid 1 to amino acid 711, as set forth in
FIG. 1 (SEQ. ID NO:2).
11. An isolated nucleic acid of claim 7, wherein the nucleotide
sequence is operably linked to an expression control sequence.
12. An isolated nucleic acid of claim 7, wherein the nucleic acid
comprises a naturally-occurring nucleotide sequence.
13. An isolated nucleic acid of claim 7, wherein the nucleic acid
codes for said polypeptide without interruption.
14. An isolated nucleic acid of claim 7, wherein the nucleic acid
is DNA or RNA.
15. An isolated nucleic acid of claim 7, wherein the nucleic acid
further comprises a detectable label.
16. An isolated nucleic acid of claim 7, except where one or more
amino acid positions are substituted or deleted, or both, and the
polypeptide coded for by the nucleic acid is
biologically-active.
17. An isolated nucleic acid of claim 16, wherein the biological
activity is a guanine nucleotide exchange activity, a specific
binding affinity for a guanine nucleotide depleted G-protein, or a
cellular oncogenic transforming activity.
18. An isolated nucleic acid of claim 16, wherein the one or more
substituted amino acid positions are substituted by conservative
amino acids.
19. An isolated nucleic acid of claim 16, wherein the one or more
substituted amino acid positions is in the Dbl homology domain or
the pleckstrin homology domain.
20. An isolated nucleic acid comprising a nucleotide sequence which
hybridizes, or whose nucleic acid complement hybridizes, under
stringent conditions to base pairs of nucleotide sequence 900-1482
as set forth in FIG. 1 (SEQ. ID NO:1).
21. An isolated nucleic acid claim 20 comprising at least 95%
nucleotide sequence identity to the nucleotide sequence set forth
in claim 20.
22. An isolated nucleic acid of claim 20, wherein said nucleic acid
codes for a polypeptide having a guanine nucleotide exchange
activity, a specific binding affinity for a guanine nucleotide
depleted Rac, or a cellular oncogenic transforming activity.
23. An isolated nucleic acid comprising a nucleotide sequence which
is unique to Rac-GEF.
24. An isolated nucleic acid comprising a nucleotide sequence which
hybridizes, or whose nucleic acid complement hybridizes, under
stringent conditions to the unique nucleotide sequence of claim
23.
25. An isolated nucleic acid of claim 24 which codes for a
polypeptide having a guanine nucleotide exchange activity, a
specific binding affinity for a guanine nucleotide depleted Rac, or
a cellular oncogenic transforming activity.
26. A method of expressing in transformed host cells, a Rac-GEF
polypeptide coded for by a nucleic acid, comprising culturing
transformed host cells containing a nucleic acid according to claim
7 under conditions effective to express the polypeptide.
27. A method of expressing, in transformed host cells, a
polypeptide coded for by a nucleic acid, comprising culturing
transformed host cells containing a nucleic acid according to claim
20 under conditions effective to express the polypeptide.
28. A method of claim 26, further comprising isolating the
polypeptide.
29. A method of claim 26, further comprising modulating expression
of the polypeptide.
30. An isolated polypeptide produced by a method of claim 26.
31. An isolated polypeptide produced by a method of claim 27.
32. A transformed host cell containing a nucleic acid of claim
7.
33. A transformed host cell containing a nucleic acid of claim
20.
34. A vector comprising a nucleic acid of claim 7.
35. A vector comprising a nucleic acid of claim 20.
36. A method of modulating an activity of a Rac polypeptide
comprising, administering an effective amount of a Rac-GEF
polypeptide or a biologically-active fragment thereof, or an
effective amount of a compound which modulates the activity of the
Rac-GEF.
37. A method of claim 36, wherein the Rac-GEF, or
biologically-active fragment thereof, comprises an amino acid
sequence which has a specific binding activity for a guanine
nucleotide depleted state of said Rac.
38. A method of modulating an activity of a Rac polypeptide
comprising; introducing a nucleic acid of claim 21 into said cell
under conditions whereby said nucleic acid is expressed in an
effective amount to modulate said activity of Rac in said cell.
39. A method of claim 38 wherein said nucleic acid oncogenically
transforms said cell.
40. A method of isolating a molecule that binds to a guanine
nucleotide-depleted state of a Rac polypeptide comprising;
contacting a Rac polypeptide with a medium comprising said molecule
under conditions effective for said molecule to bind to said Rac
polypeptide; and separating said Rac polypeptide to which said
molecule has bound from said medium.
41. A method of claim 40, wherein said molecule is Rac-GEF.
42. A method of claim 40, wherein said molecule has a molecular
weight of about 82.5 kilodaltons.
43. A method of claim 40, further comprising separating said
molecule from said Rac polypeptide.
44. A method of modulating an activity of a GTPase comprising,
administering an effective amount of a guanine nucleotide exchange
factor or a biologically-active fragment thereof, or an effective
amount of a compound which modulates the activity of the guanine
nucleotide exchange factor.
45. A method of claim 44, wherein the guanine nucleotide exchange
factor, or biologically-active fragment thereof, comprises an amino
acid sequence which has a specific binding activity for a guanine
nucleotide depleted state of said GTPase.
46. A method of testing for an agent which modulates the guanine
nucleotide exchange activity of a guanine nucleotide exchange
factor comprising: contacting a mixture of (a) a polypeptide
comprising a guanine nucleotide exchange factor, or a
biologically-active fragment thereof, and (b) a polypeptide
comprising a GTPase, or a biologically-active fragment thereof, to
which the exchange factor can bind, with an agent; and assaying for
the presence or amount of guanine nucleotide exchange activity in
the presence or absence of a GEF enhancer.
47. A method of claim 46, wherein the GTPase is Rac.
48. A method of claim 47, wherein the guanine nucleotide exchange
factor is Rac GEF and paid GEF enhancer is ascorbyl stearate.
49. An agent identified by the method of claim 48.
50. A method of testing for an agent which modulates the binding
between a guanine nucleotide exchange factor and a GTPase
comprising: contacting a mixture of (a) a polypeptide comprising a
guanine nucleotide exchange factor, or a biologically-active
fragment thereof, and (b) a polypeptide comprising a GTPase, or a
biologically-active fragment thereof, to which the exchange factor
can bind, with an agent; and detecting the presence or amount of
binding between the guanine nucleotide exchange factor polypeptide,
or the biologically-active fragment thereof, and the GTPase.
51. A method of claim 50, wherein the GTPase is Rac.
52. A method of claim 50, wherein the guanine nucleotide exchange
factor is Rac-GEF.
53. An isolated agent identified by the method of claim 50.
54. An isolated antibody which is specific for a Rac-GEF or a
peptide comprising a sequence present therein.
55. An isolated antibody of claim 54, which binds to an amino acid
sequence selected from the group consisting of
2 H.sub.2NAFRELIAOLELDPKCOOH H.sub.2NYQERTYKLPFSSFLCOOH
H.sub.2NPQRSQNKDRRKLGSRNRQCO- OH
56. A method of increasing the guanine nucleotide exchange activity
of a guanine nucleotide exchange factor, or a biologically-active
fragment thereof, said factor capable of acting on a member of the
Ras superfamily of GTPases, comprising the steps of: contacting
said guanine nucleotide exchange factor, or a biologically-active
fragment thereof, with said member of the Ras superfamily of
GTPases, or a biologically-active fragment thereof; and assaying
for guanine nucleotide exchange activity under appropriate
conditions in the presence of a guanine nucleotide exchange factor
enhancer.
57. A method as described in claim 56 wherein said member of the
Ras superfamily of GTPases is Rac and said guanine nucleotide
exchange factor is Tiam-1.
58. A method as described in claim 57 wherein said GEF enhancer is
selected from the group consisting of ascorbyl stearate, ascorbyl
palmitate, and phosphoinositol.
59. A method as described in claim 58 wherein said phosphoinositol
is selected from the group consisting of PI3,4,5P.sub.3,
PI4,5P.sub.2 and PI4P.
60. A method of assaying for a compound to treat disease resulting
from increased guanine nucleotide exchange activity of a guanine
nucleotide exchange factor, or a biologically-active fragment
thereof, said factor acting on a member of the Ras superfamily of
GTPases, comprising the steps of: contacting said guanine
nucleotide exchange factor, or a biologically-active fragment
thereof, with said member of the Ras superfamily of GTPases, or a
biologically-active fragment thereof; assaying for guanine
nucleotide exchange activity under appropriate conditions in the
presence of a guanine nucleotide exchange factor enhancer, and in
the presence and absence of said compound; and determining if said
compound decreases said guanine nucleotide exchange activity.
61. A method as described in claim 60 wherein said member of the
Ras superfamily of GTPases is Rac.
62. A method as described in claim 61 wherein said guanine
nucleotide exchange factor is Tiam-1.
63. A method as described in claim 62 wherein said GEF enhancer is
selected from the group consisting of ascorbyl stearate, ascorbyl
palmitate, and phosphoinositol.
64. A method as described in claim 63 wherein said phosphoinositol
is selected from the group consisting of PI3,4,5P.sub.3,
PI4,5P.sub.2 and PI4P.
65. Compounds of claim 60 that decrease said guanine nucleotide
exchange activity.
66. Ligands that bind to the Src homology 3 domain on Rac-GEF.
67. Ligands that bind to the Src homology 3 domain on Rac-GEF
identified by the methods of claims 46 or 50.
Description
FIELD OF THE INVENTION
[0001] This invention is in the field of molecular biology, and
involves methods and compositions for regulating unwanted cell
growth through the regulation of the activity of certain guanine
nucleotide exchange factors.
BACKGROUND OF THE INVENTION
[0002] Ras is a member of a superfamily of GTPases that regulate
diverse signaling pathways. Ras itself has been shown to be
involved in regulating cell growth and differentiation (See,
Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654). A
subfamily of Ras consists of Rho, Rac, and Cdc42. These GTPase have
also been shown to be involved in regulating cell growth,
particularly as relating to cellular transformation, as well as
controlling the formation of focal contacts and alterations in the
actin cytoskeleton which occur upon growth factor stimulation (See,
Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P.,
Xu, N., Miki, T. and Gutkind, J. S. (1995) Cell 81, 1137-1146;
Hill, C. S., Wynne, J. and Treisman, R. (1995) Cell 81, 1159-1170;
Kozma, R., Ahmed, S., Best, A. and Lim, L. (1995) Mol. Cell. Biol.
15, 1942-1952; Minden, A., Lin, A., Claret, F.-X., Abo, A. and
Karin, M. (1995) Cell 81, 1147-1157; Nobes, C. D. and Hall, A.
(1995) Cell 81, 53-62; Olson, M. F., Ashworth, A. and Hall, A.
(1995) Science 269, 1270-1272). Common to all Ras family members is
their ability to cycle between inactive (GDP bound) and active (GTP
bound) states. In this regard, these GTPases act as molecular
switches, capable of processing information and then disseminating
that information to control a specific pathway.
[0003] This property of cycling between GTP and GDP states has
provided a means to identify and purify proteins which regulate the
nucleotide state of Ras and Ras related GTPases. See, Boguski, M.
S. and McCormick, F. (1993) Nature 366, 643-654.
[0004] By monitoring the hydrolysis of GTP to GDP, GTPase
activating proteins (GAPs) have been characterized for many members
of the Ras family. See, Boguski, M. S. and McCormick, F. (1993)
Nature 366, 643-654; Barfod, E. T., Zheng, Y., Kuang, W.-J., Hart,
M. J., Evans, T., Cerione, R. A. and Ashkenaz, A. (1993) J. Biol.
Chem. 268, 26059-26062; Lamarche, N. and Hall, A. (1994)Trends
Genet. 10, 436-440; Cerione, R. A. and Zheng, Y. (1996) Current
Opinion in Cell Biology 8, 216-222. The latter reference provides a
good discussion of the properties of those proteins that affect the
guanine nucleotide state of Ras and Ras related proteins. Guanine
nucleotide dissociation inhibitors (GDIs) were identified based on
their ability to inhibit the dissociation of GDP. It has
subsequently been determined that they also bind to the GTP state,
inhibiting the intrinsic and GAP stimulated GTP hydrolysis. See,
Boguski, M. S. and McCormick, F. (1993) Nature 366, 643-654. In
general, GAPs and effectors have a high affinity for the GTP-bound
state, while GDI proteins bind most tightly to the GDP-bound state.
These properties have been exploited to purify effectors for
Cdc42Hs ( See, Bagrodia, S., Taylor, S. J., Creasy, C. L.,
Chernoff, J. and Cerione, R. A. (1995) J. Biol. Chem. 270,
22731-22737; Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-s. and
Lim, L. (1994)Nature 367,40-46; Martin, G. A., Bollag, G.,
McCormick, F. and Abo, A. (1995) EMBO J. 14, 1970-1978), Ras (See,
Moodie, S. A., Willumsen, B. M., Weber, M. J. and Wolfman, A.
(1993) Science 260, 1658-1661; Rodriguez-Viciana, P., Warne, P. H.,
Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M.
D. and Downward, J. (1994) Nature 370, 527-532) and Rho (See,
Leung, T., Manser, E., Tan, L. and Lim, L. (1995) J. Biol. Chem.
270, 29051-29054; Watanabe, G., Saito, Y., Madaule, P., Ishizaki,
T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuki, A. and
Narumiya, S. (1996) Science 271, 645-648). An affinity approach has
also been employed with Cdc42Hs-GTP and has led to the
characterization of IQGAP1, a potential mediator for observed
cytoskeletal events induced by Cdc42. See, Hart, M. J., Callow, M.,
Souza, B. and Polakis, P. (1996) EMBO J. 15, 2997-3005.
[0005] A modification of this affinity approach can also be used to
identify and purify guanine nucleotide exchange factors (GEFs).
GEFs can be distinguished from other regulatory proteins by their
ability to interact preferentially with the nucleotide-depleted
state of G-proteins. See, Hart, M. J., Eva, A., Zangrilli, D.,
Aaronson, S. A., Evans, T., Cerione, R. A. and Zheng, Y. (1994) J.
Biol. Chem. 269, 62-65; Mosteller, R. D., Han, J. and Broek, D.
(1994) Mol. Cell. Biol. 14, 1104-1112. By stimulating the
dissociation of GDP and subsequent binding of GTP, GEFs play an
important role in the activation of Ras-like proteins. For example,
Ras is converted to its GTP-bound form by the growth-factor
stimulated translocation of Sos, a Ras-specific GEF. See, Buday, L.
and Downward, J. (1993)Cell 73, 611-620.
[0006] The characterization of GEFs that specifically activate Rac
family members will help elucidate signalling pathways in which
these GTPases participate, and thus lead to a better understanding
of the molecular basis of cell growth. This, in turn, will enable
the identification of drugs for preventing or treating diseases
where uncontrolled cell growth is the cause. Because Rac plays a
key role in signal transduction and cell growth, the identification
and properties of Rac GEFs is presently receiving considerable
scientific attention. One such Rac GEF is known, Tiam-1. See,
Michiels, F., Habets, G. G., Stam, J. C., van der Kammen, R. A.,
and Collard, J. G. (1995) Nature 375, 338-340. See also, Eva, A.
and Aaronson, S. A. (1985) Nature 316, 273-275; Toksoz, D. and
Williams, D. A. (1994) Oncogene 9, 621-628.
DESCRIPTION OF THE INVENTION
[0007] The present invention relates to all aspects of a guanine
exchange factor (GEF), in particular, a Rac-GEF. A GEF modulates
cell signaling pathways, both in vitro and in vivo, by modulating
the activity of a GTPase. By way of illustration, a Rac-GEF, which
modulates the activity of a Rac GTPase, is described. However, the
present invention relates to other GEFs, especially other
Rac-GEFs.
[0008] The present invention preferably relates to an isolated
Rac-GEF polypeptide characterized by having a Src homology, Dbl
homology and pleckstrin homology domains, and variants thereof, or
fragments of such polypeptides, nucleic acids coding for such
Rac-GEFs or nucleic acid fragments, and derivatives of the
polypeptides and nucleic acids.
[0009] The invention also relates to methods of using such
polypeptides, nucleic acids, or derivatives thereof, e.g., in
therapeutics, diagnostics, and as research tools.
[0010] Another aspect of the present invention involves antibodies
and other ligands which recognize the invention Rac-GEF, regulators
of Rac-GEF activity and other GEFs, and methods of treating
pathological conditions associated or related to such Rac
GTPase.
[0011] The invention also relates to methods of testing for and/or
identifying agents which regulate GEF by measuring their effect on
GEF activity, e.g., in binding to a GTPase and/or nucleotide
exchange activity.
[0012] The invention also relates to methods of assaying for GEF
activity, preferrably using activators of GEF activity.
[0013] These and other aspects of the invention will become
apparent upon a full considertion of the following disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the complete nucleotide sequence (SEQ ID NO:1)
and deduced amino acid sequence (SEQ ID NO:2) for a polypeptide
encoded for by a human GEF-Rac gene.
[0015] FIG. 2 shows the brain specific nucleotide sequence for a
Rac-GEF.
[0016] FIG. 3 shows the domain structures of full length Tiam-1,
and truncations of the molecule.
[0017] FIG. 4 shows the stimulatory effect of ascorbyl stearate on
Rac exchange activity by various forms of truncated Tiam-1, the 85
kd and 135 kD molecules.
[0018] FIG. 5 shows the effects of certain ascorbyl compounds,
inositol lipids and phospholipids on Tiam-1 stimulated Rac-GEF
activity.
[0019] FIG. 6 shows the effects of ascorbyl sterate on Tiam-1
constructs that have PH and DH domains.
DETAILED DESCRIPTION OF THE INVENTION
[0020] In accordance with the present invention, a novel
polypeptide and nucleic acid coding for a Rac-GEF has been
identified and isolated. Alternate variants of the molecule have
also beeen identified. As used herein, Rac-GEF means a polypeptide,
or a nucleic acid coding for a Rac-GEF polypeptide, which
polypeptide has a specific binding affinity for a guanine
nucleotide-depleted state of G-proteins (in particular Rac), a
guanine nucleotide exchange activity, an oncogenic transforming
activity, and an immunogenic activity. By specific binding
affinity, it is meant that the polypeptide has a binding preference
for the nucleotide-depleted state of the G-protein, in contrast,
e.g., to the GDP- or GTP-bound state of the G-protein which is
preferentially bound by other regulatory proteins. By guanine
nucleotide exchange activity, it is meant that the polypeptide
stimulates or catalyzes the dissociation of GDP from a G-protein,
such as Rac, and subsequent binding of GTP. By cellular oncogenic
transforming activity, it is meant that introduction of a nucleic
acid coding for Rac-GEF into a cell line, e.g., NIH 3T3 cells,
confers a transformed phenotype on such cells. A transformed
phenotype can be measured by foci formation, e.g., as characterized
and described by Eva and Aaronson, Nature, 316:273-276, 1985.
Immunogenic activity means that the polypeptide binds to Rac-GEF
specific antibodies or is capable of eliciting an immune response
specific for a Rac-GEF. Immunogenic activities are discussed below.
The above-mentioned activities of a Rac-GEF polypeptide can be
assayed, e.g., as described below in the examples or according to
methods which the skilled worker would know. A Rac-GEF polypeptide,
or corresponding nucleic acid coding for it, means a polypeptide
which can be isolated from a natural source. It therefore includes
naturally-occurring normal and mutant alleles. Natural sources
include, e.g., living cells obtained from tissues and whole
organisms, and cultured cell lines.
[0021] To identify a human gene that encodes a Rac-GEF, we
performed a search of the EST data base for Dlb homologs. The
search was performed using an amino acid sequence (residues 1-519)
encoded by the human TIM protein (Chan et al., 1994, Ocogene, Vol.
9, pages 1057-1063). A single clone was identified, and the plasmid
encoding this insert was purchased via the I.M.A.G.E. Consortium
(Research Genetics). Using this cDNA as template, a 511-bp
.sup.32P-labelled PCR product was produced using oligos
5'-GGAGGCCATGTTCGAGCTGG-3' and 5'- GCTGATCATCTGTTCCGTGC-3'(5' and
3' primers, respectively) and .sup.32P labelled nucleotides. This
labeled PCR fragment was used as a probe to screen approximately
4.times.10.sup.5 clones of a human fetal brain Lambda ZAP cDNA
library (Stratagene). A clone with an insert of 2.6-kb was
isolated, and the complete DNA sequence of this clone was
determined and shown to have a single open reading frame of 1950-bp
that is predicted to encode a 650-amino acids protein with a
calculated molecular mass of 74.7 kDa. A comparision of the DNA
sequences of the EST insert to the fetal brain cDNA revealed a 72
base pair insert in the fetal brain sequence. The insert is in the
DH domain.
[0022] As discussed more in the Examples, Northern analysis
revealed a 3.5 kb transcript in brain tissue and a 4 kb transcript
in liver. Consequently, using the additional sequence identified
from the 2.6-kb sequence that was not present in EST #167059 we
identified another EST (#109922) that had been isolated from a
human cDNA liver library. The plasmid containing this insert was
also obtained, and the insert sequenced which revealed an
initiating methionine.
[0023] FIG. 1 (SEQ ID NO(s):1 and 2) show the alignment of the full
length liver nucleotide cDNA sequence, with its deduced amino acid
sequence, respectively. It is note worthy that this sequence has an
additional 126 amino acids which differ from the amino-terminal 66
amino acids of the 2.6 kb brain cloned (FIG. 2). Also shown in the
figure are various domains, including the Src homology 3, Dbl
homology and pleckstrin homology domains. It, or its corresponding
gene, can be isolated from natural sources. Characterization of a
human Rac-GEF is described below and in the examples.
[0024] It is noteworthy that because of the protein-protein
interactive properties of the Src homology 3 domain, ligands that
bind to this domain may be identified, for example, by screening an
expression library, that affect Rac-GEF activity. Such ligands
would have medical applications.
[0025] The present invention also relates to polypeptide fragments
of Rac-GEF. The fragments are preferably biologically-active. By
biologically-active, it is meant that the polypeptide fragment
possesses an activity in a living system or with components of a
living system. Biological-activities include: a specific binding
affinity for a guanine nucleotide-depleted state of G-proteins, in
particular Rac, a guanine nucleotide exchange activity, an
oncogenic transforming activity, an immunogenic activity,
modulating the binding between a Rac-GEF and a Rac GTPase, or
acting as an agonist or antagonist of Rac GTPase activity. Such
activities can be assayed routinely, e.g., according to the methods
described above and below. Various fragments can be prepared. See
the examples below for further discussion. Fragments can also be
selected in which one or more of the mentioned activities are
eliminated or altered when compared to Rac-GEF. As described in the
examples, such fragments can be prepared routinely, e.g., by
recombinant means or by proteolytic cleavage of isolated
polypeptides, and then assayed for a desired activity.
[0026] The present invention also relates to a human Rac-GEF
specific amino acid sequence as set forth in FIG. 1 (SEQ ID NO:2):
A clone encoding such sequence, 128 to 711 amino acids and also
containing 66 divergent amino acids as shown in FIG. 2, has been
deposited on Dec. 11, 1996 with the American Type Culture
Collection with Accession No. 98273. A Rac-GEF specific amino acid
sequence means a defined amino acid sequence. A specific amino acid
sequence can be found routinely, e.g., by searching a gene/protein
database using the BLAST set of computer programs. A Rac-GEF
specific amino acid sequence can be useful to produce peptides as
antigens to generate an immune response specific for Rac-GEF.
Antibodies obtained by such immunization can be used as a specific
probe for the Rac-GEF protein for diagnostic or research purposes.
Such peptides can also be used to inhibit the Rac-GEF binding to
Rac to modulate pathological conditions in cells.
[0027] A polypeptide of the invention, e.g., having a polypeptide
sequence as shown in FIG. 1 (SEQ ID NO:2), can by analyzed by
available methods to identify structural and/or functional domains
in the polypeptide. For example, when the polypeptide coding
sequence set forth in FIG. 1 (SEQ ID NO:2) is analyzed by computer
algorithms, a continuous coding sequence comprising the following
domains is identified: Src homology, Dbl homology and pleckstrin
homology domains. Various programs can be employed to analyze
structure of the polypeptide, including, EMBL Protein Predict; Rost
and Sander, Proteins, 19:55-72, 1994; Kyte and Doolittle, J. Mol.
Bio.: 157:105,1982.
[0028] A polypeptide of the present invention can also have 100% or
less amino acid sequence identity to the amino acid sequence set
forth in FIG. 1. (SEQ ID NO:2). For the purposes of the following
discussion: Sequence identity means that the same nucleotide or
amino acid which is found in the sequence set forth in FIG. 1. (SEQ
ID NO:1 and SEQ ID NO:2) is found at the corresponding position of
the compared sequence(s). A polypeptide having less than 100%
sequence identify to the amino acid sequence set forth in FIG. 1
(SEQ. ID NO:2) can be substituted in various ways, e.g., by a
conservative amino acid. See below for examples of conservative
amino acid substitution. The sum of the identical and conserved
residues divided by the total number of residues in the sequence
over which the Rac-GEF polypeptide is compared is equal to the
percent sequence similarity. For purposes of calculating sequence
identity and similarity, the compared sequences can be aligned and
calculated according to any desired method, algorithm, computer
program, etc., including, e.g., FASTA, BLASTA. A polypeptide having
less than 100% amino acid sequence identity to the amino acid
sequence of FIG. 1 (SEQ ID NO:2) can comprise e.g., about 60, 65,
more preferably, 67, 70, 78, 80, 90, 92, 96, 99, etc.
[0029] A Rac GEF polypeptide, fragment, or substituted GEF
polypeptide can also comprise various modifications, where such
modifications include glycosylation, covalent modifications (e.g.,
of an R-group of an amino acid), amino acid substitution, amino
acid deletion, or amino acid addition. Modifications to the
polypeptide can be accomplished according to various methods,
including recombinant, synthetic, chemical, etc.
[0030] A mutation to a Rac-GEF polypeptide can be selected to have
a biological activity of Rac-GEF, e.g., a specific binding affinity
for a guanine nucleotide-depleted state of G-proteins, in
particular Rac, a guanine nucleotide exchange activity, an
oncogenic transforming activity, and an immunogenic activity. The
selection and preparation of mutations of Rac-GEF is discussed
below.
[0031] Polypeptides of the present invention (e.g., Rac-GEF,
fragments thereto, mutations thereof) can be used in various ways,
e.g., as immunogens for antibodies as described below, as
biologically-active agents (e.g., having one or more of the
activities associated with Rac-GEF), as inhibitors of Rac-GEF. For
example, upon binding of Rac-GEF to Rac, a cascade of events is
initiated in the cell, e.g., promoting cell proliferation and/or
cytoskeletal rearrangements. The interaction between Rac-GEF and
Rac can be modulated by using a peptide fragment of Rac-GEF, e.g.,
a peptide fragment which is an inhibitor at the site where Rac-GEF
interacts (e.g., binds) to Rac. Such a fragment can be useful for
modulating pathological conditions associated with the Rac
signaling pathway. A useful fragment can be identified routinely by
testing the ability of overlapping fragments of the entire length
of Rac-GEF to inhibit a Rac-GEF activity, such as guanine
nucleotide exchange activity, binding to a guanine nucleotide
depleted state of Rac, and oncogenic transforming activity. The
measurement of certain of these activities is described below, and
in the examples. These peptides can also be identified and prepared
as described in EP 496 162. Peptides can be chemically-modified,
etc.
[0032] A polypeptide coding for a Rac-GEF polypeptide, or a
derivative or fragment thereof, can be combined with one or more
structural domains, functional domains, detectable domains,
antigenic domains, and/or a desired polypeptides of interest, in an
arrangement which does not occur in nature, i.e., not
naturally-occurring, e.g., as in a normal Rac-GEF gene, a genomic
fragment prepared from the genome of a living organism, e.g., an
animal, preferably a mammal, such as human, mouse, or cell lines
thereof. A polypeptide comprising such features is a chimeric or
fusion polypeptide. Such a chimeric polypeptide can be prepared
according to various methods, including, chemical, synthetic,
quasi-synthetic, and/or recombinant methods. A chimeric nucleic
acid coding for a chimeric polypeptide can contain the various
domains or desired polypeptides in a continuous or interrupted open
reading frame, e.g., containing introns, splice sites, enhancers,
etc. The chimeric nucleic acid can be produced according to various
methods. See, e.g., U.S. Pat. No. 5,439,819. A domain or desired
polypeptide can possess any desired property, including, a
biological function such as catalytic, signalling, growth
promoting, cellular targeting, etc., a structural function such as
hydrophobic, hydrophilic, membrane-spanning, etc., receptor-ligand
functions, and/or detectable functions, e.g., combined with enzyme,
fluorescent polypeptide, green fluorescent protein GFP (Chalfie et
al., 1994, Science, 263:802; Cheng etal., 1996, Nature
Biotechnology, 14:606; Levy et al., 1996, Nature Biotechnology,
14:610, etc. In addition, a Rac-GEF nucleic acid, or a part of it,
can be used as selectable marker when introduced into a host cell.
For example, a nucleic acid coding for an amino acid sequence
according to the present invention can be fused in-frame to a
desired coding sequence and act as a tag for purification,
selection, or marking purposes. The region of fusion encodes a
cleavage site.
[0033] A polypeptide according to the present invention can be
produced in an expression system, e.g., in vivo, in vitro,
cell-free, recombinant, cell fusion, etc., according to the present
invention. Modifications to the polypeptide imparted by such system
include, glycosylation, amino acid substitution (e.g., by differing
codon usage), polypeptide processing such as digestion, cleavage,
endopeptidase or exopeptidase activity, attachment of chemical
moieties, including lipids, phosphates, etc. For example, some cell
lines can remove the terminal methionine from an expressed
polypeptide.
[0034] A polypeptide according to the present invention can be
recovered from natural sources, transformed host cells (culture
medium or cells) according to the usual methods, including,
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, hydroxyapatite
chromatography and lectin chromatography. It may be useful to have
low concentrations (approximately 0.1-5 mM) of calcium ion present
during purification (Price, et al., J. Biol. Chem., 244:917
(1969)). Protein refolding steps can be used, as necessary, in
completing the configuration of the mature protein. Finally, high
performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0035] In accordance with the present invention, a nucleic acid
coding for a Rac-GEF can comprise, e.g., the complete coding
sequence as set forth in FIG. 1 (SEQ ID NO:1). A nucleic acid
according to the present invention can also comprise a nucleotide
sequence which is 100% complementary, e.g., an anti-sense, to any
nucleotide sequence mentioned above and below.
[0036] A Rac GEF encoding nucleic acid according to the present
invention can be obtained from a variety of different sources. It
can be obtained from DNA or RNA, such as polyadenylated mRNA, e.g.,
isolated from tissues, cells, or whole organism. The nucleic acid
can be obtained directly from DNA or RNA, or from a cDNA library.
The nucleic acid can be obtained from a cell at a particular stage
of development, having a desired genotype, phenotype (e.g., an
oncogenically transformed cell or a cancerous cell), etc.
[0037] A nucleic acid comprising a nucleotide sequence coding for a
polypeptide according to the present invention can include only
coding sequence of Rac-GEF; coding sequence of Rac-GEF and
additional coding sequence (e.g., sequences coding for leader,
secretory, targeting, enzymatic, fluorescent or other diagnostic
peptides), coding sequence of Rac-GEF and non-coding sequences,
e.g., untranslated sequences at either a 5' or 3' end, or dispersed
in the coding sequence, e.g., introns. A nucleic acid comprising a
nucleotide sequence coding without interruption for a Rac-GEF
polypeptide means that the nucleotide sequence contains an amino
acid coding sequence for a Rac-GEF polypeptide, with no non-coding
nucleotides interrupting or intervening in the coding sequence,
e.g., absent intron(s). Such a nucleotide sequence can also be
described as contiguous.
[0038] A nucleic acid according to the present invention also can
comprise an expression control sequence operably linked to a
nucleic acid as described above. The phrase "expression control
sequence" means a nucleic acid sequence which regulates expression
of a polypeptide coded for by a nucleic acid to which it is
operably linked. Expression can be regulated at the level of the
mRNA or polypeptide. Thus, the expression control sequence includes
mRNA-related elements and protein-related elements. Such elements
include promoters, enhancers (viral or cellular), ribosome binding
sequences, transcriptional terminators, etc. An expression control
sequence is operably linked to a nucleotide coding sequence when
the expression control sequence is positioned in such a manner to
effect or achieve expression of the coding sequence. For example,
when a promoter is operably linked 5' to a coding sequence,
expression of the coding sequence is driven by the promoter.
Expression control sequences can be heterologous or endogenous to
the normal gene.
[0039] A nucleic acid in accordance with the present invention can
be selected on the basis of nucleic acid hybridization. The ability
of two single-stranded nucleic acid preparations to hybridize
together is a measure of their nucleotide sequence complementarity,
e.g., base-pairing between nucleotides, such as A-T, G-C, etc. The
invention thus also relates to nucleic acids which hybridize to a
nucleic acid comprising a nucleotide sequence as set forth in FIG.
1 (SEQ ID NO:1). A nucleotide sequence hybridizing to the latter
sequence will have a complementary nucleic acid strand, or act as a
template for one in the presence of a polymerase (i.e., an
appropriate nucleic acid synthesizing enzyme). The present
invention includes both strands of nucleic acid, e.g., a sense
strand and an anti-sense strand.
[0040] Hybridization conditions can be chosen to select nucleic
acids which have a desired amount of nucleotide complementarity
with the nucleotide sequence set forth in FIG. 1 (SEQ ID NO:1). A
nucleic acid capable of hybridizing to such sequence, preferably,
possesses 50%, more preferably, 70% complementarity, between the
sequences. The present invention particularly relates to DNA
sequences which hybridize to the nucleotide sequence set forth in
FIG. 1 (SEQ ID NO:1) under stringent conditions. As used here,
"stringent conditions" means any conditions in which hybridization
will occur where there is at least about 95%, preferably 97%,
nucleotide complementarity between the nucleic acids. Such
conditions include, e.g., hybridization for Northern: 5.times.
SSPE, 10.times. Denhardts solution, 100 .mu.g/ml freshly denatured
and sheared salmon sperm DNA, 50% formamide, 2% SDS at 42.degree.
C.; hybridization for cloning from cDNA library: 1.times. PAM, 0.1%
SDS, 50% formamide at 42.degree. C.
[0041] According to the present invention, a nucleic acid or
polypeptide can comprise one or more differences in the nucleotide
or amino acid sequence set forth in FIG. 1 (SEQ ID NO:1 and SEQ ID
NO:2). Changes or modifications to the nucleotide and/or amino acid
sequence can be accomplished by any method available, including
directed or random mutagenesis.
[0042] A nucleic acid coding for a Rac-GEF according to the
invention can comprise nucleotides which occur in a
naturally-occurring Rac-GEF gene e.g., naturally-occurring
polymorphisms, normal or mutant alleles (nucleotide or amino acid),
mutations which are discovered in a natural population of mammals,
such as humans, monkeys, pigs, mice, rats, or rabbits. By the term
naturally-occurring, it is meant that the nucleic acid is obtained
from a natural source, e.g., animal tissue and cells, body fluids,
tissue culture cells, forensic samples. Naturally-occurring
mutations to Rac-GEF can include deletions (e.g., a truncated
amino- or carboxy-terminus), substitutions, or additions of
nucleotide sequence. These genes can be detected and isolated by
nucleic acid hybridization according to methods which one skilled
in the art would know. It is recognized that, in analogy to other
oncogenes, naturally-occurring variants of Rac-GEF include
deletions, substitutions, and additions which produce pathological
conditions in the host cell and organism.
[0043] A nucleotide sequence coding for a Rac-GEF polypeptide of
the invention can contain codons found in a naturally-occurring
gene, transcript, or cDNA, for example, e.g., as set forth in FIG.
1 (SEQ ID NO:1), or it can contain degenerate codons coding for the
same amino acid sequences.
[0044] In addition, a nucleic acid or polypeptide of the present
invention can be obtained from any desired mammalian organism, but
also non-mammalian organisms. Homologs from mammalian and
non-mammalian organisms can be obtained according to various
methods. For example, hybridization with an appropriate
oligonucleotide selective for Rac-GEF can be employed to select
such homologs, e.g., as described in Sambrook et al., Molecular
Cloning, 1989, Chapter 11.
[0045] Such homologs can have varying amounts of nucleotide and
amino acid sequence identity and similarity to Rac-GEF.
Non-mammalian organisms include, e.g., vertebrates, invertebrates,
chicken, Drosophila, yeasts (such as Saccharomyces cerevisiae), C.
elegans, roundworms, prokaryotes, plants, Arabidopsis, viruses,
etc.
[0046] Modifications to a Rac-GEF sequence, e.g., mutations, can
also be prepared based on homology searching from gene data banks,
e.g., Genbank, EMBL. Sequence homology searching can be
accomplished using various methods, including algorithms described
in the BLAST family of computer programs, the Smith-Waterman
algorithm, etc. For example, conserved amino acids can be
identified between various sequences, Dbl, lbc, Ost, lsc, CDC24,
etc. See, e.g., Touhara et al., J. Biol. Chem., 269:10217-10220,
1994; Toksoz and Williams, Oncogene, 9:621-628, 1994; Whitehead et
al., J. Biol. Chem., 271:18643-18650, 1996. A mutation(s) can then
be introduced into a Rac-GEF sequence by identifying and aligning
amino acids conserved between the polypeptides and then modifying
an amino acid in a conserved or non-conserved position. A mutated
Rac-GEF gene can comprise conserved or nonconserved amino acids,
e.g., between corresponding regions of homologous nucleic acids,
especially between Dbl homology (DH) domains, etc. For example, a
mutated sequence can comprise conserved or non-conserved residues
from any number of homologous sequences as mentioned-above and/or
determined from an appropriate searching algorithm.
[0047] Mutations can be made in specific regions of nucleic acid
coding for the Rac-GEF polypeptide, e.g., in the Dbl homology
domain, such as replacing it, changing amino acid sequences within
it, etc., to analyze a function (e.g., oncogenic transformation,
binding to a G-protein, guanine nucleotide exchange) of the
polypeptide coded for by the nucleic acid. For example, deletion of
the pleckstrin domain would result in the loss of oncogenic
transforming activity. The pleckstrin domain can also be involved
with lipid (e.g., phosphoinositides) binding, binding to Rac,
activation of the guanine nucleotide exchange activity, and
localization of the polypeptide in the cell. Thus, this region can
be mutagenized according to various methods and then assayed for
loss or gain of the mentioned functions. The DH domain is involved
with promoting GDP dissociation from the Rac GTPase. Thus,
substitutions or deletions within this region can be prepared and
assayed routinely for loss or gain of function. A mutation can be
made in these or other regions of Rac-GEF which affect its
phosphorylation or protein/lipid interaction leading to its
modulation of the growth signaling pathway. Such a mutated gene can
be useful in various ways: for diagnosis in patients having such a
mutation, to introduce into cells or animals (transgenic) as a
model for a pathological condition. Mutations which affect both GEF
activity and transforming activity can be analogous to those made
in the DH domain of the Dbl oncogene as described in Hart et al.,
J. Biol. Chem., 269:62-65.
[0048] A nucleic acid and corresponding polypeptide of the present
invention include sequences which differ from the nucleotide
sequence of FIG. 1 (SEQ ID NO:1) but which are phenotypically
silent. These sequence modifications include, e.g., nucleotide
substitution which do not affect the amino acid sequence (e.g.,
different codons for the same amino acid), replacing
naturally-occurring amino acids with homologous or conservative
amino acids, e.g., (based on the size of the side chain and degree
of polarization) small nonpolar: cysteine, proline, alanine,
threonine; small polar: serine, glycine, aspartate, asparagine;
large polar: glutamate, glutamine, lysine, arginine; intermediate
polarity: tyrosine, histidine, tryptophan; large nonpolar:
phenylalanine, methionine, leucine, isoleucine, valine. Such
conservative substitutions also include those described by Dayhoff
in the Atlas of Protein Sequence and Structure 5 (1978), and by
Argos in EMBO J., 8, 779-785 (1989).
[0049] A nucleic acid can comprise a nucleotide sequence coding for
a polypeptide having an amino acid sequence as set forth in FIG. 1.
(SEQ ID NO:2) except where one or more positions are substituted by
conservative amino acids; or a nucleotide sequence coding for a
polypeptide having an amino acid sequence as set forth in FIG. 1.
(SEQ ID NO:2). The invention also relates to polypeptides coded for
by such nucleic acids. In addition, it may be desired to change the
codons in the sequence to optimize the sequence for expression in a
desired host.
[0050] A nucleic acid according to the present invention can
comprise, e.g., DNA, RNA, synthetic nucleic acid, peptide nucleic
acid, modified nucleotides, or mixtures. A DNA can be double- or
single-stranded. Nucleotides comprising a nucleic acid can be
joined via various known linkages, e.g., ester, sulfamate,
sulfamide, phosphorothioate, phosphoramidate, methylphosphonate,
carbamate, etc., depending on the desired purpose, e.g., resistance
to nucleases, such as RNase H, improved in vivo stability, etc.
See, e.g., U.S. Pat. Nos. 5,378,825.
[0051] Various modifications can be made to the nucleic acids, such
as attaching detectable markers (avidin, biotin, radioactive
elements), moieties which improve hybridization, detection, or
stability. The nucleic acids can also be attached to solid
supports, e.g., nitrocellulose, nylon, agarose, diazotized
cellulose, latex solid microspheres, polyacrylamides, etc.,
according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967,
5,476,925, 5,478,893.
[0052] Another aspect of the present invention relates to
oligonucleotides and nucleic acid probes. Such oligonucleotides or
nucleic acid probes can be used, e.g., to detect, quantitate, or
isolate a Rac-GEF nucleic acid in a test sample. Detection can be
desirable for a variety of different purposes, including research,
diagnostic, and forensic. For diagnostic purposes, it may be
desirable to identify the presence or quantity of a Rac-GEF nucleic
acid sequence in a sample, where the sample is obtained from
tissue, cells, body fluids, etc. In a preferred method, the present
invention relates to a method of detecting a Rac-GEF nucleic acid
comprising, contacting a target nucleic acid in a test sample with
an oligonucleotide under conditions effective to achieve
hybridization between the target and oligonucleotide; and detecting
hybridization. An oligonucleotide in accordance with the invention
can also be used in synthetic nucleic acid amplification such as
PCR, e.g., Saiki et al., 1988, Science, 241:53; U.S. Pat. No.
4,683,202.
[0053] Another aspect of the present invention is a nucleotide
sequence which is unique to Rac-GEF. By a unique sequence to
Rac-GEF, it is meant a defined order of nucleotides which occurs in
Rac-GEF, e.g., in the nucleotide sequence of FIG. 1 (SEQ ID NO:1),
but rarely or infrequently in other nucleic acids, especially not
in an animal nucleic acid, preferably mammal, such as human, rat,
mouse, etc. Both sense and antisense nucleotide sequences are
included. A unique nucleic acid according to the present invention
can be determined routinely. A nucleic acid comprising a unique
sequence of Rac-GEF can be used as a hybridization probe to
identify the presence of Rac-GEF in a sample comprising a mixture
of nucleic acids, e.g., on a Northern blot. Hybridization can be
performed under stringent conditions to select nucleic acids having
at least 95% identity (i.e., complementarity) to the probe, but
less stringent conditions can also be used. A unique Rac-GEF
nucleotide sequence can also be fused in-frame, at either its 5' or
3' end, to various nucleotide sequences as mentioned throughout the
patent, including coding sequences for other parts of Rac-GEF,
enzymes, GFP, etc, expression control sequences, etc.
[0054] Hybridization can be performed under different conditions,
depending on the desired selectivity, e.g., as described in
Sambrook et al., Molecular Cloning, 1989. For example, to
specifically detect Rac-GEF, an oligonucleotide can be hybridized
to a target nucleic acid under conditions in which the
oligonucleotide only hybridizes to Rac-GEF, e.g., where the
oligonucleotide is 100% complementary to the target. Different
conditions can be used if it is desired to select target nucleic
acids which have less than 100% nucleotide complementarity, at
least about, e.g., 99%, 97%, 95%, 90%, 70%, 67%. Since a mutation
in a Rac-GEF gene can cause diseases or pathological conditions,
e.g., cancer, benign tumors, an oligonucleotide according to the
present invention can be used diagnostically. For example, a
patient having symptoms of a cancer or other condition associated
with the Rac signaling pathway (see below) can be diagnosed with
the disease by using an oligonucleotide according to the present
invention, in polymerase chain reaction followed by DNA sequencing
to identify whether the sequence is normal, in combination with
other oncogene oligonucleotides, etc., e.g., p53, Rb, p21, Dbl,
MTS1, Wt1, Bcl-1, Bcl-2, MDM2, etc. In a preferred method, the
present invention relates to a method of diagnosing a cancer
comprising contacting a sample comprising a target nucleic acid
with an oligonucleotide under conditions effective to permit
hybridization between the target and oligonucleotide; detecting
hybridization, wherein the oligonucleotide comprises a sequence of
Rac-GEF, preferably a unique sequence of Rac-GEF; and determining
the nucleotide sequence of the target nucleic acid to which the
oligonucleotide is hybridized. The sequence can be determined
according to various methods, including isolating the target
nucleic acid, or a cDNA thereof, and determining its sequence
according to a desired method.
[0055] Oligonucleotides according to the present invention can be
of any desired size, preferably 14-16 oligonucleotides in length,
or more. Such oligonucleotides can have non-naturally-occurring
nucleotides, e.g., inosine. In accordance with the present
invention, the oligonucleotide can comprise a kit, where the kit
includes a desired buffer (e.g., phosphate, tris, etc.), detection
compositions, etc. The oligonucleotide can be labeled or unlabeled,
with radioactive or non-radioactive labels as known in the art.
[0056] Anti-sense nucleic acid can also be prepared from a nucleic
acid according to the present, preferably an anti-sense to a coding
sequence of FIG. 1 (SEQ ID NO:1). Antisense nucleic acid can be
used in various ways, such as to regulate or modulate expression of
Rac-GEF, e.g., inhibit it, to detect its expression, or for in situ
hybridization. For the purposes of regulating or modulating
expression of Rac-GEF, an anti-sense oligonucleotide can be
operably linked to an expression control sequence.
[0057] The nucleic acid according to the present invention can be
labelled according to any desired method. The nucleic acid can be
labeled using radioactive tracers such as .sup.32p, .sup.35S,
.sup.125I, .sup.3H, or .sup.14C, to mention only the most commonly
used tracers. The radioactive labelling can be carried out
according to any method such as, for example, terminal labeling at
the 3' or 5' end using a radiolabeled nucleotide, polynucleotide
kinase (with or without dephosphorylation with a phosphatase) or a
ligase (depending on the end to be labelled). A non-radioactive
labeling can also be used, combining a nucleic acid of the present
invention with residues having immunological properties (antigens,
haptens), a specific affinity for certain reagents (ligands),
properties enabling detectable enzyme reactions to be completed
(enzymes or coenzymes, enzyme substrates, or other substances
involved in an enzymatic reaction), or characteristic physical
properties, such as fluorescence or the emission or absorption of
light at a desired wavelength, etc.
[0058] A nucleic acid according to the present invention, including
oligonucleotides, anti-sense nucleic acid, etc., can be used to
detect expression of Rac-GEF in whole organs, tissues, cells, etc.,
by various techniques, including Northern blot, PCR, in situ
hybridization, etc. Such nucleic acids can be particularly useful
to detect disturbed expression, e.g., cell-specific and/or
subcellular alterations, of Rac-GEF. The levels of Rac-GEF can be
determined alone or in combination with other genes products
(oncogenes such as p53, Rb, Wt1, etc.), transcripts, etc.
[0059] A nucleic acid according to the present invention can be
expressed in a variety of different systems, in vitro and in vivo,
according to the desired purpose. For example, a nucleic acid can
be inserted into an expression vector, introduced into a desired
host, and cultured under conditions effective to achieve expression
of a polypeptide coded for the nucleic acid. Effective conditions
includes any culture conditions which are suitable for achieving
production of the polypeptide by the host cell, including effective
temperatures, pH, medias, additives to the media in which the host
cell is cultured (e.g., additives which amplify or induce
expression such as butyrate, or methotrexate if the coding nucleic
acid is adjacent to a dhfr gene), cyclohexamide, cell densities,
culture dishes, etc. A nucleic acid can be introduced into the cell
by any effective method including, e.g., calcium phosphate
precipitation, electroporation, injection, DEAE-Dextran mediated
transfection, fusion with liposomes, and viral transfection. A cell
into which a nucleic acid of the present invention has been
introduced is a transformed host cell. The nucleic acid can be
extrachromosomal or integrated into a chromosome(s) of the host
cell. It can be stable or transient. An expression vector is
selected for its compatibility with the host cell. Host cells
include, mammalian cells, e.g., COS-7, CHO, HeLa, LTK, NIH 3T3, Rat
1 fibroblasts, yeast, insect cells, such as Sf9 (S. frugipeda) and
Drosophila, bacteria, such as E. coli, Streptococcus, bacillus,
yeast, fungal cells, plants, embryonic stem cells (e.g., mammalian,
such as mouse or human), cancer or tumor cells. Sf9 expression can
be accomplished in analogy to Graziani et al., Oncogene, 7:229-235,
1992. Expression control sequences are similarly selected for host
compatibility and a desired purpose, e.g., high copy number, high
amounts, induction, amplification, controlled expression. Other
sequences which can be employed include enhancers such as from
SV40, CMV, inducible promoters, cell-type specific elements, or
sequences which allow selective or specific cell expression.
[0060] In addition to a Rac-GEF nucleic acid, another gene of
interest can be introduced into the same host for purposes of,
e.g., modulating expression Rac-GEF, elucidating Rac-GEF function
or that of the gene of interest. Genes of interest include other
oncogenes, genes involved in the cell cycle, etc. Such genes can be
the normal gene, or a variation, e.g., a mutation, chimera,
polymorphism, etc.
[0061] A nucleic acid or polypeptide of the present invention can
be used as a size marker in nucleic acid or protein
electrophoresis, chromatography, etc. Defined restriction fragments
can be determined by scanning the sequence for restriction sites,
calculating the size, and performing the corresponding restriction
digest. For example, the Rac-GEF polypeptide from fetal brain can
also be used as a molecular weight marker of about 74.7 kDa for a
protein gel.
[0062] Another aspect of the present invention relates to the
regulation of biological pathways in which a GTPase is involved,
particularly pathological conditions, e.g., cell proliferation
(e.g., cancer), growth control, morphogenesis, stress fiber
formation, and integrin-mediated interactions, such as embryonic
development, tumor cell growth and metastasis, programmed cell
death, hemostasis, leucocyte homing and activation, bone
resorption, clot retraction, and the response of cells to
mechanical stress. See, e.g., Clark and Brugge, Science,
268:233-239, 1995; Bussey, Science, 272:225-226, 1996. Thus, the
invention relates to all aspects of a method of modulating an
activity of a Rac polypeptide comprising, administering an
effective amount of a Rac-GEF polypeptide or a biologically-active
fragment thereof, an effective amount of a compound which modulates
the activity of a Rac polypeptide, or an effective amount of a
nucleic acid which codes for a Rac-GEF polypeptide or a
biologically-active fragment thereof. The activity of Rac which is
modulated can include: GTP binding, GDP binding, GTPase activity,
integrin binding, coupling or binding of Rac to receptor or
effector-like molecules (such as integrins, growth factor
receptors, tyrosine kinases, PI-3K, PIP-5K, etc.). See, e.g., Clark
and Brugge, Science, 268:233-239, 1995. The activity can be
modulated by increasing, reducing, antagonizing, promoting, etc. of
Rac. The modulation of Rac can be measured by assay for GTP
hydrolysis, binding to Rac-GEF, etc. An effective amount is any
amount which, when administered, modulates the Rac activity. The
activity can be modulated in a cell, a tissue, a whole organism, in
situ, in vitro (test tube, a solid support, etc.), in vivo, or in
any desired environment.
[0063] Compounds that regulate the interaction between a GEF, such
Rac-GEF, and a GTPase can be identified using an assay for a GEF
activity, such as guanine nucleotide exchange activity, binding to
a guanine nucleotide-depleted site of a GTPase, or oncogenic
transforming activity, or a GTPase activity such as GTP hydrolysis.
In general, a compound having such an in vitro activity will be
useful in vivo to modulate a biological pathway associated with a
GTPase, e.g., to treat a pathological condition associated with the
biological and cellular activities mentioned above. By way of
illustration, the ways in which GEF regulators can be identified
are described above and below in terms of Rac and Rac-GEF. However,
it is to be understood that such methods can be applied generally
to other GEFs.
[0064] A guanine nucleotide exchange assay, e.g., as described in
Hart et al., Nature, 354:311-314,28 Nov. 1991 (see, especially,
FIG. 2 legend therein), can be used to assay for the ability of a
compound to regulate the interaction between Rac and Rac-GEF. For
example, Rac protein (recombinant, recombinant fusion protein, or
isolated from natural sources) is labeled with tritiated-GDP. The
tritiated-GDP-labeled Rac is then incubated with Rac-GEF and GTP
under conditions in which nucleotide exchange occurs. The amount of
tritiated-GDP that is retained by Rac is determined by separating
bound GDP from free GDP, e.g., using a BA85 filter. The ability of
a compound to regulate the interaction can be determined by adding
the compound at a desired time to the incubation (e.g., before
addition of a Rac-GEF, after addition of a Rac-GEF) and determining
its effect on nucleotide exchange. Various agonist and antagonists
of the interaction can be identified in this manner. For instance,
an aspect of the instant invention is the discovery that certain
compounds greatly enhance the activity of Rac-GEFs, and preferrably
of the Rac-GEF, Tiam-1. Such compounds are hereinafter termed "GEF
enhancers." Such compounds have certain similar chemical features
including a hydrocarbon arm, preferrably consisting of
substantially saturated bonds that link the carbon residues
together, and also preferrably the number of carbon atoms should be
between 12-22. A second feature of such compounds is the
association of the hydrocarbon arm to either a 5 or 6 membered ring
structure. Preferred 5 or 6 membered compounds include ascorbate
and certain cyclohexanes, respectively. The more preferred 5
membered compounds are derivatives of ascorbate, while the more
preferred cyclohexanes include insoitol.
[0065] Binding to a guanine nucleotide-depleted site of Rac can be
determined in various ways, e.g., as described in Hart et al., J.
Biol. Chem., 269:62-65, 1994. Briefly, a Rac protein can be coupled
to a solid support using various methods that one skilled in the
art would know, e.g., using an antibody to Rac, a fusion protein
between Rac and a marker protein, such as glutathione protein
(GST), wherein the fusion is coupled to a solid support via the
marker protein (such as glutathionine beads when GST is used), etc.
The Rac protein is converted to a guanine nucleotide depleted state
(for effective conditions, see, e.g., Hart et al., J. Biol. Chem.,
269:62-65, 1994) and incubated with, e.g., GDP, GTP.gamma.S, and a
GEF such as Rac-GEF. The solid support is then separated and any
protein on it run on a gel. A compound can be added at any time
during the incubation (as described above) to determine its effect
on the binding of the GEF to Rac.
[0066] The modulation of oncogenic transforming activity by a
Rac-GEF, or derivatives thereof, can be measured according to
various known procedures, e.g., Eva and Aaronson, Nature,
316:273-275, 1985; Hart et al., J. Biol. Chem., 269:62-65, 1994. A
compound can be added at any time during the method (e.g.,
pretreatment of cells; after addition of GEF, etc.) to determine
its effect on the oncogenic transforming activity of Rac-GEF.
Various cell lines can also be used.
[0067] Other assays for Rac-mediated signal transduction can be
accomplished according to procedures known in the art, e.g., as
described in U.S. Pat. Nos. 5,141,851; 5,420,334; 5,436,128; and
5,482,954; WO94/16069; WO93/16179; WO91/15582; WO90/00607. In
addition, peptides which inhibit the interaction, e.g., binding,
between Rac-GEF and a G-protein, such as Rac, can be identified and
prepared according to EP 496 162.
[0068] The present invention also relates to a method of testing
for and identifying an agent which modulates the guanine nucleotide
exchange activity of a guanine nucleotide exchange factor, or a
biologically-active fragment thereof, or which modulates the
binding between a Rac-GEF, or a biologically-active fragment
thereof, and a GTPase, or a biologically-active fragment thereof,
to which it binds. The method comprises contacting the GEF and
GTPase with an agent to be tested and then detecting the presence
or amount of binding between the GEF and GTPase, or an activity of
the GEF such as guanine nucleotide exchange activity. By
modulating, it is meant that addition of the agent affects the
activity or binding. The binding or activity modulation can be
affected in various ways, including inhibiting, blocking,
preventing, increasing, enhancing, or promoting it. The binding or
activity affect does not have to be achieved in a specific way,
e.g., it can be competitive, noncompetitive, allosteric, sterically
hindered, via cross-linking between the agent and the GEF or
GTPase, etc. The agent can act on either the GEF or GTPase. The
agent can be an agonist, an antagonist, or a partial agonist or
antagonist. The presence or amount of binding can be determined in
various ways, e.g., directly or indirectly by assaying for an
activity promoted or inhibited by the GEF, such as guanine
nucleotide exchange, GTP hydrolysis, oncogenic transformation, etc.
Such assays are described above and below, and are also known in
the art. The agent can be obtained and/or prepared from a variety
of sources, including natural and synthetic. It can comprise, e.g.,
amino acids, lipids, carbohydrates, organic molecules, nucleic
acids, inorganic molecules, or mixtures thereof. See, e.g.,
Hoeprich, Nature Biotechnology, 14:1311-1312, 1996, which describes
an example of automated synthesis of organic molecules. The agent
can be added simultaneously or sequentially. For example, the agent
can be added to the GEF and then the resultant mixture can be
further combined with the GTPase. The method can be carried out in
liquid on isolated components, on a matrix (e.g., filter paper,
nitrocellulose, agarose), in cells, on tissue sections, etc. In
accordance with the method, a GEF can bind to the GTPase, which
binding will modulate some GTPase activity. For example, as
discussed above and below, a Rac-GEF binds to Rac, causing guanine
nucleotide dissociation. The effect can be directly on the binding
site between the GEF and GTPase, or it can be allosteric, or it can
be on only one component (e.g., on the GEF only). Assays for
guanine nucleotide dissociation can be readily adapted to identify
agents which regulate the activity of a GTPase. The method further
relates to obtaining or producing agents which have been identified
according to the above-described method.
[0069] The present invention also relates to products identified in
accordance with such methods. Various GEFs and GTPases can be
employed, including, -Rac-GEF, mSOS, SOS, C3G, lsc, Dbl,
Dbl-related proteins, polypeptides comprising one or more DH
domains, CDC24, Tiam-1, Ost, Lbc, Vav, Ect2, Bcr, Abr, Rho (A, B,
and C), Rac, Ras, CDC42, chimeras thereof, biologically-active
fragments thereof, muteins thereof, etc.
[0070] The present invention thus also relates to the treatment and
prevention of diseases and pathological conditions associated with
Rac-mediated signal transduction, e.g., cancer, diseases associated
with abnormal cell proliferation. For example, the invention
relates to a method of treating cancer comprising administering, to
a subject in need of treatment, an amount of a compound effective
to treat the disease, where the compound is a regulator of Rac-GEF
gene or polypeptide expression. Treating the disease can mean,
delaying its onset, delaying the progression of the disease,
improving or delaying clinical and pathological signs of disease.
Similarly, the method also relates to treating diseases associated
with inflammation, and/or the chemotactic ability of neutrophils. A
regulator compound, or mixture of compounds, can be synthetic,
naturally-occurring, or a combination. A regulator compound can
comprise amino acids, nucleotides, hydrocarbons, lipids,
polysaccharides, etc. A regulator compound is preferably a
regulator of Rac-GEF, e.g., inhibiting or increasing its mRNA,
protein expression, or processing, or its interaction with Rac,
e.g., guanine nucleotide exchange. Additionally, cells can be
supplemented with Rac-GEF, or derivatives thereof. To treat the
disease, the compound, or mixture, can be formulated into
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and other excipients as apparent to the skilled worker.
See, e.g., Remington's Pharmaceutical Sciences, Eighteenth Edition,
Mack Publishing Company, 1990. Such composition can additionally
contain effective amounts of other compounds, especially for
treatment of cancer.
[0071] The present invention also relates to antibodies which
specifically recognize a Rac-GEF polypeptide. Antibodies, e.g.,
polyclonal, monoclonal, recombinant, chimeric, can be prepared
according to any desired method. For example, for the production of
monoclonal antibodies, a polypeptide according to FIG. 1 (SEQ ID
NO:2), can be administered to mice, goats, or rabbit subcutaneously
and/or intraperitoneally, with or without adjuvant, in an amount
effective to elicit an immune response. The antibodies can also be
single chain or FAb. The antibodies can be IgG, subtypes, IgG2a,
IgG1, etc.
[0072] An antibody specific for Rac-GEF means that the antibody
recognizes a defined sequence of amino acids within or including
the Rac-GEF amino acid sequence of FIG. 1 (SEQ ID NO:2). Thus, a
specific antibody will bind with higher affinity to an amino acid
sequence, i.e., an epitope, found in FIG. 1 (SEQ ID NO:2) than to a
different epitope(s), e.g., as detected and/or measured by an
immunoblot assay. Thus, an antibody which is specific for an
epitope of Rac-GEF is useful to detect the presence of the epitope
in a sample, e.g., a sample of tissue containing Rac-GEF gene
product, distinguishing it from samples in which the epitope is
absent. Such antibodies are useful as described in Santa Cruz
Biotechnology, Inc., Research Product Catalog, can be formulated
accordingly, e.g., 100 .mu.g/ml.
[0073] In addition, ligands which bind to a Rac-GEF polypeptide
according to the present invention, or a derivative thereof, can
also be prepared, e.g., using synthetic peptide libraries, or
nucleic acid ligands (e.g., Pitrung et al., U.S. Pat. No.
5,143,854; Geysen et al., 1987, J. Immunol. Methods, 102:259-274;
Scott et al., 1990, Science, 249:386; Blackwell et al., 1990,
Science, 250:1104; Tuerk et al., 1990, Science, 249:505.
[0074] Antibodies and other ligands which bind Rac-GEF can be used
in various ways, including as therapeutic, diagnostic, and
commercial research tools, e.g, to quantitate the levels of Rac-GEF
polypeptide in animals, tissues, cells, etc., to identify the
cellular localization and/or distribution of Rac-GEF, to purify
Rac-GEF or a polypeptide comprising a part of Rac-GEF, to modulate
the function of Rac-GEF, etc. Antibodies to Rac-GEF, or a
derivative thereof, can be used in Western blots, ELISA,
immunoprecipitation, RIA, etc. The present invention relates to
such assays, compositions and kits for performing them, etc.
[0075] An antibody according to the present invention can be used
to detect Rac-GEF polypeptide or fragments thereof in various
samples, including tissue, cells, body fluid, blood, urine,
cerebrospinal fluid. A method of the present invention comprises
contacting a ligand which binds to a peptide of FIG. 1. (SEQ ID
NO:2) under conditions effective, as known in the art, to achieve
binding, detecting specific binding between the ligand and peptide.
By specific binding, it is meant that the ligand attaches to a
defined sequence of amino acids, e.g., within or including the
amino acid sequence of FIG. 1. (SEQ ID NO:2) or derivatives
thereof. The antibodies or derivatives thereof can also be used to
inhibit expression of Rac-GEF or a fragment thereof. The levels of
Rac-GEF polypeptide can be determined alone or in combination with
other gene products. In particular, the amount (e.g., its
expression level) of Rac-GEF polypeptide can be compared (e.g., as
a ratio) to the amounts of other polypeptides in the same or
different sample, e.g., p21, p53, Rb, WT1, etc.
[0076] A ligand for Rac-GEF can be used in combination with other
antibodies, e.g., antibodies that recognize oncological markers of
cancer, including, Rb, p53, c-erbB-2, oncogene products, etc. In
general, reagents which are specific for Rac-GEF can be used in
diagnostic and/or forensic studies according to any desired method,
e.g., as U.S. Pat. Nos. 5,397,712; 5,434,050; 5,429,947.
[0077] The present invention also relates to a labelled Rac-GEF
polypeptide, prepared according to a desired method, e.g., as
disclosed in U.S. Pat. No. 5,434,050. A labelled polypeptide can be
used, e.g., in binding assays, such as to identify substances that
bind or attach to Rac-GEF, to track the movement of Rac-GEF in a
cell, in an in vitro, in vivo, or in situ system, etc.
[0078] A nucleic acid, polypeptide, antibody, Rac-GEF ligand etc.,
according to the present invention can be isolated. The term
"isolated" means that the material is in a form in which it is not
found in its original environment, e.g., more concentrated, more
purified, separated from component, etc. An isolated nucleic acid
includes, e.g., a nucleic acid having the sequence of Rac-GEF
separated from the chromosomal DNA found in a living animal. This
nucleic acid can be part of a vector or inserted into a chromosome
(by specific gene-targeting or by random integration at a position
other than its normal position) and still be isolated in that it is
not in a form which it is found in its natural environment. A
nucleic acid or polypeptide of the present invention can also be
substantially purified. By substantially purified, it is meant that
nucleic acid or polypeptide is separated and is essentially free
from other nucleic acids or polypeptides, i.e., the nucleic acid or
polypeptide is the primary and active constituent.
[0079] The present invention also relates to a transgenic animal,
e.g., a non-human-mammal, such as a mouse, comprising a Rac-GEF
nucleic acid. Transgenic animals can be prepared according to known
methods, including, e.g., by pronuclear injection of recombinant
genes into pronuclei of 1-cell embryos, incorporating an artificial
yeast chromosome into embryonic stem cells, gene targeting methods,
embryonic stem cell methodology. See, e.g., U.S. Pat. Nos.
4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986;
5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad.
Sci., 77:7380-7384 (1980); Palmiter et al., Cell, 41:343-345
(1985); Palmiter et al., Ann. Rev. Genet., 20:465-499 (1986); Askew
et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature,
373:523-527, 1995; Valancius and Smithies, Mol. Cell. Bio.,
11:1402-1408, 1991; Stacey et al., Mol. Cell. Bio., 14:1009-1016,
1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al.,
Nucl. Acid Res., 21:2613-2617,1993. A nucleic acid according to the
present invention can be introduced into any non-human mammal,
including a mouse (Hogan et al., 1986, in Manipulating the Mouse
Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.), pig (Hammer et al., Nature, 315:343-345,
1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle,
rat, or primate. See also, e.g., Church, 1987, Trends in Biotech.
5:13-19; Clark et al., 1987, Trends in Biotech. 5:20-24; and
DePamphilis et al., 1988, BioTechniques, 6:662-680. In addition,
e.g., custom transgenic rat and mouse production is commercially
available. These transgenic animals are useful as a cancer model,
e.g., to test drugs.
[0080] Generally, the nucleic acids, polypeptides, antibodies, etc.
of the present invention can be prepared and used as described in
U.S. Pat. Nos. 5,501,969; 5,506,133; 5,441,870; WO 90/00607; and WO
91/15582.
[0081] For other aspects of the nucleic acids, polypeptides,
antibodies, etc., reference is made to standard textbooks of
molecular biology, protein science, and immunology. See, e.g.,
Davis et al. (1986), Basic Methods in Molecular Biology, Elsevir
Sciences Publishing, Inc., New York; Hames et al. (1985), Nucleic
Acid Hybridization, IL Press, Molecular Cloning, Sambrook et al.;
Current Protocols in Molecular Biology, Edited by F. M. Ausubel et
al., John Wiley & Sons, Inc; Current Protocols in Human
Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley &
Sons, Inc.; Current Protocols in Protein Science; Edited by John E.
Coligan et al., John Wiley & Sons, Inc.; Current Protocols in
Immunology; Edited by John E. Coligan et al., John Wiley &
Sons, Inc.
EXAMPLES
Example 1
[0082] Cloning of cDNA encoding Rac GEF
[0083] A Dbl-homology domain containing protein was identified in a
human fetal brain cDNA library as follows. A TBLASTN search of the
dbEST database was performed using the amino acid sequence
(residues 1-519) encoded by the human TIM protein (Chan et al.,
1994, Oncogene, Vol. 9, pages 1057-1063). One EST clone, # 167059
was identified with high sequence homology to the TIM cDNA. The
plasmid encoding this insert was purchased via the I.M.A.G.E.
Consortium (Research Genetics). Using this cDNA as template, a
511-bp .sup.32P-labelled PCR product was produced using oligos
5'-GGAGGCCATGTTCGAGCTGG-3' and 5'-GCTGATCATCTGTTCCGTGC-3' (5' and
3' primers, respectively) and .sup.32P labelled nucleotides. This
labeled PCR fragment was used as a probe to screen approximately
4.times.10.sup.5 clones of a human fetal brain Lambda ZAP cDNA
library (Stratagene). A clone with an insert of 2.6-kb was
isolated, and the complete DNA sequence of this clone was
determined using an ABI sequencer. This 2.6-kb clone harbored a
single open reading frame of 1950-bp that is predicted to encode a
650-amino acids protein with a calculated molecular mass of 74.7
kDa. However, this open reading frame is not full-length, as the
initiating methionine is missing. This cDNA is on deposit with the
American Type Culture Collection, Dec. 11, 1996, with the Accession
No.98273, and is denoted p67 Rac-GEF.
[0084] Northern analysis using the probe described above was
conducted. The results revealed a 3.5 kb transcript specific to
brain tissue and an additional 4 kb transcript of lower abundance
specific to liver tissue. Other normal tissues tested, including
heart, placenta, lung, muscle, kidney, pancreas, spleen, thymus,
prostate, testis, ovary, intestine, colon and peripheral blood
lymphocytes were also essentially negative. In a preliminary screen
of human tumor cell lines, abundant 3.5 kb mRNA levels were
detected in the lung carcinoma cell line A549 and the colon
carcinoma cell line SW480. Other tumor cell lines were negative,
including HL-60, HeLa, K-562, Molt-4, Raji and G-36. Further
screening of a number of primary tumor samples revealed
over-expression in liver, lung and colon tumors.
[0085] Using the additional sequence identified in the 2.6-kb
clone, further analysis of the dbEST database using the Blastn
program identified an additional clone, # 109922, which had been
isolated from a liver library. The plasmid encoding this insert was
purchased via the I.M.A.G.E. Consortium (Genome Systems), and the
sequence of the insert was determined. This sequence revealed an
initiating methionine and 126 additional amino acids which differed
from the amino-terminal 66 amino acids of the 2.6 kb brain clone
described above. This new sequence most likely encodes the
liver-specific alternatively spliced form which had been identified
by Northern analysis. Pieced together with the previously
determined sequence, this liver-derived sequence reveals an open
reading frame of 2133-bp predicted to encode a 710-amino acid
protein.
[0086] In addition to the alternatively-spliced brain/liver
isoforms, another putative splice variant was identified: an
insertion of 72-bp coding for 24-amino acids within the Dbl
homology region is encoded by the 2.6-kb brain clone. The sequence
encoded by these 24-amino acids is conserved among other exchange
factors including Tim (Chan et al., 1994, Oncogene, Vol. 9, pages
1057-1063) and Vav2 (Henske et al., 1995, Ann Hum Genet 59, Pt. 1,
pages 25-37).
Example 2
[0087] Properties of Rac GEF
[0088] Two Rac-GEFs were tested for guanine nucleotide activity.
Firstly, a Glu-epitope tag (MEYMPMEIRHD) was engineered onto the
carboxy-terminal 423 amino acids of the Rac GEF encoded by EST No.
167059 by introducing the oligos
5'-TCGAGGAGGTTATAAATATGGAATACATGCCAATGGA-3' and the complementary
5'-AATTTCCATTGGCATGTATTCCATATTTATAACCTCC-3' into the XhoI/EcoRI
sites of the clone. The protein encoded by this construct is
referred to as Type I Rac-GEF.
[0089] Next, the sequence encoding the insertion in the Dbl
homology region, as described in Example 1, was engineered into the
open reading frame in the expression plasmid pET21a (Novagen). The
protein encoded by this construct is referred to as Type II
Rac-GEF. The resulting expression plasmids were introduced into E.
coli strain BL21(DE3)pLysS (Novagen), and the epitope tagged
protein expression was induced with IPTG. The expressed proteins
were purified using a resin with the antibody to the Glu-epitope
covalently attached. The resulting proteins were partially pure and
were assayed for exchange activity on Rac1, RhoA and Cdc42. See,
Hart, in U.S. Ser. No. 60/029,979, filed Nov. 6, 1996. The results
showed that Rac GEF is primarily selective for Rac1, but also
displays activity against both RhoA and Cdc42. Furthermore, the
Type I form lacking the Dbl insert region is unaffected by the
addition of the PH domain ligand ascorbyl stearate, while the Type
II form containing the Dbl insert region is strongly stimulated by
ascorbyl stearate.
Example 3
[0090] Immunochemical detection
[0091] Antibody specific to Rac-GEF was raised in rabbits against
three fragments of the purified recombinant molecule. The fragments
correspond to amino acids 385-398, Type II and amino acids, 372-386
of Type I Rac-GEF referred to in Example 2, and 693-710 amino acids
of Type II. The peptides were coupled to KLH, and antibody raised
in rabbits using standard procedures.
Example 4
[0092] Cloning and Expression of Tiam-1 and Truncations Thereof
[0093] Cloning and expression of Tiam-1, and various Tiam-1
truncations, is described below. This work, and that shown in
Examples 5 and 6, was undertaken to determine those regions of
Tiam-1 that realize GEF enhancer stimulation of Rac GEF
activity.
[0094] cDNA Cloning Of Human Tiam-1 and Tiam-1 Truncations: Primers
designed against published mouse Tiam-1 cDNA sequence (See, Habets,
G. G, Scholtes, E. H., Zuydgeest, D., van der Kammen, R. A., Stam,
J. C., Berns, A., and Collard, J. G. (1994) Cell 77,537-549; NCBI
Gen Bank Accession #U05245) were used in PCR reactions using a
human fetal brain library (Stratagene #936206) as template to
obtain fragments of the human Tiam-1 gene which were radiolabled
and used as probes in Southern hybridizations of the same library.
Primer pairs used were both
1 5'-CCATAAAACCATGGGAAACGC-3' and 5'-GGTTCCGCGGAAGAGAAGGAT-3' with
5'-GACTGGCCCGGGGAACTGAGG-3'; and 5'-TCGGATGCGGATAAGCTGCGC-3'
with
[0095] 5'-GTGACTGGCGACCTTGTTCAT-3'. Two partial clones of human
Tiam-1 cDNA were retrieved, one contained nucleotides (nt) 1-2972
and the other (nt) 2972-4657 (numbering throughout corresponds to
previously published Tiam-1 cDNA (See, Habets, G. G., van der
Kammen, R. A., Stam, J. C., Michiels, F., and Collard, J. G. (1995)
Oncogene 10, 1371-1376; NCBI Gen Bank Accession #U16296). To obtain
missing C-terminal sequences, a PCR reaction employing
oligonucleotides designed against the human Tiam-1 cDNA (See,
Habets, G. G., van der Kammen, R. A., Stam, J. C., Michiels, F.,
and Collard, J. G. (1995) Oncogene 10, 1371-1376; NCBI Gen Bank
Accession #U16296) 5'-CGGAATTCAGATTTCGACACATGATC-3' (sense) and
5'-TCGCCCGGGGCAGGTGACGCAGTCAGA-3' (antisense, contains SmaI site
downstream of stop codon) as primers and a human hippocampal
library (Clontech #HL3023b) as template produced a fragment
containing nt 4458-5366 which was added to existing clones using
the internal Eco47III (4487) site. A similar strategy using the
antisense primer
5'-GATCCCGGGTCATGTTTCTGGTTCTGGGATCTCAGTGTTCAGTTTCCTG-3' was used to
add the KT3 epitope tag "PEPET," a stop codon, and a SmaI site to
the end of Tiam-1. To splice the two partial clones together, a PCR
reaction using 5'-CGGAATTCCATGGGCCGCCTTGGAATCT-3' (sense) and
5'-TCGCCCGGGCGTCAGCAGCACGA- TTAT-3' (antisense) as primers, and a
human fetal brain cDNA library (Clontech #HL50156) as template
produced a product spanning nt 2422-3189 which was cloned into pBS
SK+ (Stratagaene #212201) using EcoRI and SmaI NcoI (472)-NcoI
(2422) and StuI (3134) -SmaI fragments were ligated into this
vector, creating full-length clones, with and without the KT3
tag.
[0096] It is note worthy that the isolated Tiam-1 sequence was
altered from the published sequence. The 5' clone obtained from the
library contained an insert of sequence
5'-GGTGAGCAGTTTACACTTTCATATACTCCCTGTCATGT- GCTTTGAAGGACTT
TCTAGGGGCATCAAG-3' in the upstream non-coding region at nt 105.
Original clones from the Stratagene fetal brain library as well as
all PCR products from Clontech hippocampal and brain libraries
contained a difference in sequence from the published Tiam-1 cDNA
((See, Habets, G. G., van der Kammen, R. A., Stam, J. C., Michiels,
F., and Collard, J. G. (1995) Oncogene 10, 1371-1376; NCBI Gen Bank
Accession #U16296); a G at nt 3005 instead of a C, which therefore
encodes a Gln in position 844 instead of a His. In addition, PCR
introduced silent mutations G4739A and G5153A.
Example 5
[0097] Expression of Tiam-1 and Truncations
[0098] The following expression vectors were constructed and used
to express the appropriate Tiam-1 constructs.
[0099] Full-length (178 kD) Tiam-1: A KT3-tagged 4792 basepair (bp)
NcoI (472)-SmaI fragment was ligated into NcoI-SmaI-digested pAcC4
(See, Rubinfeld, B., et al. Cell 65, 1033-1042 (1991)):
Bio/Technology 6:47-55 (derived from pAc436)).
[0100] 135 kD Tiam-1: The 5' phosphorylated oligonucleotides
5'-GTCATGATGG-3' and 5'-TCCATCATGACGGCC-3' were used as linkers to
recircularize ApaI-EcoNI (1673)-digested pBS SK+-based full-length
Tiam-1. The linker-created BspHI site and the vector-derived SpeI
site were used to clone the 4006 bp fragment into NcoI-X
bal-digested pAcC4 (See, Rubinfeld, B., et al. Cell 65, 1033-1042
(1991)).
[0101] 106 kD Tiam-1: The NcoI (472)-NcoI (2422) fragment was
removed from the full-length pAcC4-based expression vector.
[0102] 85 kD Tiam-1: PCR using
5'-CTTGAATTCCACCATGGAAATCTGTCCAAAAGTCACT-3' (sense) and
5'-TCGCCCGGGCGTCAGCAGCACGATTAT-3' (antisense) as primers and the
Stratagene Tiam-1 nt 2972-4657 clone as template was used to create
an NcoI-StuI (3134) fragment that placed an ATG before nt 2972. The
2297 bp NcoI-SmaI was ligated into NcoI-SmaI-digested pAcC4 (See,
Rubinfeld, B., et al. Cell 65, 1033-1042 (1991)).
[0103] 66 kD Tiam-1: The 5' phosphorylated oligonucleotides
5'-CATGGACCAGAACCCATCTCC-3' and 5'-TGAGGAGATGGGTTCTGGTC-3' were
used as linkers to recircularize NcoI (472)-Bsu36I (3534)-digested
pBS SK+-based full-length Tiam-1. The linker-regenerated NcoI site
and the vector-derived SpeI site were used to clone the 1761 bp
fragment into NcoI-XbaI-digested pAcC4 (See, Rubinfeld, B., et al.
Cell 65, 1033-1042 (1991)).
[0104] .DELTA.PH versions of Tiam-1: The oligonucleotides
5'-GCCAGAACCAGAAACATGAC-3' and 5'-CCGGGTCATGTTTCTGGTTCTGGC-3' were
used as linkers to recircularize Eco47III (4487) and XmaI-digested
pAcC4-based expression vectors containing the 135 kD, 106 kD, 85
kD, and 66 kD versions of Tiam-1. These primers also restored the
KT3 tag.
[0105] GST-PH domain fusion proteins: Products from PCR reactions
using Tiam-1 cDNA as template and
5'-GAGGAATTCGATCTGAGCATGGGAGACCTG-3' and
5'-CTGCTCGAGCTACTTATCACGCAGGATTGAATG-3' (C-terminal PH domain) or
5'-CAGGAATTCGTGCGCAAGGCCGGCGCCCTG-3' and
5'-GTGCTCGAGCTACGCAGTGGCGCAGGCAG- AGTG-3' (N-terminal PH domain) as
primers were cloned into pGEX20T (See, Helin, K., Harlow, E. and
Fattaey, A. (1993) Mol. Cell. Biol. 13, 6501-6508) using EcoRI and
XhoI.
[0106] All Tiam-1 constructs, except for the GST fusions, were
produced in baculoviurs-infected S. frugiperda-9 cells and were
purified using KT3-mAb immunoaffinity chromatography. See,
Schreurs, J., Yamamoto, R., Lyons, J., Munemitsu, S., Conroy, L.,
Clark, R., Takeda, Y., Krause, J. E., and Innis, M. (1995) J.
Neurochem. 64, 1622-1631. GST fusion proteins were produced in E.
coli and purified using glutathione-agarose. See, Smith, D. B. and
Johnson, K. S, (1988) Gene 67,31-40.
Example 6
[0107] Stimulators of Tiam-1 Rac GEF Activity
[0108] The GEF activity of the various Tiam-1 constructs described
above was determined in the presence and absence of certain
compounds. The following assay was utilized. Reactions were
conducted at room temperature in Buffer A (20mM Hepes pH7.3, 50mM
NaCl, 2mM DTT, 2mM MgCl2). All proteins and compounds were diluted
to 4.times. their final concentrations in Buffer A (GTPases were
diluted in Buffer A containing 1 .mu.M GDP). To dilute Ascorbyl
Stearate, Ascorbyl Palmitate, and Stearic Acid, 25mM EtOH solutions
were slowly added to Buffer A while vortexing vigorously. Other
lipids were resuspended in aqueous solution with vortexing and bath
sonication and then diluted into Buffer A. Reactions were prepared
and at time 0,.lambda.-.sup.32P-GTP (DuPont NEN #NEG006H) was added
to 4.5nM, and after 10 minutes reactions were stopped by filtering
onto nitrocellulose filters (Millipore #HAWP02500) and immediately
washed with wash buffer (25mM Tris 7.5, 100mM NaCl, 30mM MgCl2).
Bound .lambda.-.sup.32P-GTP was measured, using standard
techniques.
[0109] The 85 kD portion of human Tiam-1 protein was produced in
insect cells and purified by affinity chromatography, as described
above. This protein contained an intact PDZ domain, Dbl-homology
(DH) domain and adjacent pleckstrin homology (PH) domain (FIG. 3).
Using the above described assay, this truncation alone, at various
concentrations, exhibited no GEF activity towards Rac 1 (FIG. 4).
In contrast, ascorbyl stearate (AS) stimulated the rate of
Tiam-1-mediated nucleotide exchange on Rac 1 (FIG. 4). Because AS
has the potential to act as a detergent or a reducing agent, other
detergents (nOG, Trition X-100, NP40) and reducing agents (DTT,
TCEP, or Tris (2-carboxyethyl)phosphine) were tested and shown not
to significantly stimulate Tiam-1 GEF activity. Several other
lipids were tested to determine the specificity of activation.
Ascorbyl palmitate (AP), phosphatidylinositol-4-phosphate (PI(4)P),
and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) significantly
enhanced Tiam-1 activity; phosphatidylinositol-3,4,5-trisph-
osphate (PI(3,4,5)P3) and phosphatidylserine had weak effects; and
phosphatidylglycerol, phosphatidylinositol and phosphatidylcholine
had little or no effect (FIG. 5). As a control, experiments were
run to determine if IP3, IP2, ascorbic acid, stearic acid and
ascorbic acid with stearic acid were sufficient to activate the GEF
activity of Tiam-1. The results showed that these reagents were
incapable of stimulating GEF activity (FIG. 5).
[0110] It has been previously reported that Tiam-1 has GEF activity
in the absence absence of lipids. See, Michiels, F., Habets, G. G.,
Stam, J. C., van der Kammen, R. A., and Collard, J. G. (1995)
Nature 375, 338-340. Those studies used a mouse version containing
additional upstream sequences, including the N-terminal PH domain
and the coiled-coil region. To determine if upstream regions are
necessary for expression of the DH domain GEF activity, the
corresponding human construct was prepared (FIG. 3). This 135 kD
Tiam-1 truncation shows weak GEF activity towards Rac in the
absence of AS, but is still greatly stimulated by AS (FIG. 4).
Other truncations of Tiam-1, all containing the DH and PH domains
(FIG. 3), also exhibited AS stimulated GEF activity on Rac 1 (FIG.
6).
[0111] To determine if AS binding to the PH domain was responsible
for activation of Tiam-1 GEF activity, sequences 3' of the Eco47III
site were deleted, removing half of the C-terminal PH domain as
well as the rest of the C-terminus (FIG. 3). These truncations of
Tiam-1 were not activated by AS, including one that contained the
N-terminal PH domain (FIG. 4). While it is possible that deleting
the PH domain destroys activity of the DH domain altogether,
similar truncations of the PH domain of the Dbl protein do not
affect its GEF activity (See, Zheng, Y., Zangrilli, D., Cerione, R.
A., and Eva, A. (1996). J. Biol. Chem. 271, 19017-19020). To
further determine if the PH domains could bind to AS, GST-PH fusion
proteins were included in the reaction. While GST alone did not
affect AS-stimulated Tiam-1 exchange activity, both of the Tiam-1
GST-PH domain fusions reduced the effectiveness of AS (FIG. 6).
[0112] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The preceding preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever.
[0113] The entire disclosure of all patents/patent applications and
publications, cited above and in the figures are hereby
incorporated by reference in the entirety.
[0114] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
Sequence CWU 1
1
33 1 3171 DNA Liver Rac GEF CDS Complement (76)..(2208) 1
tcactcaaac cagtgaagct tgggaaagtc attgacctcc agtcgttctg ctgagaaaca
60 tctggctcta tttcc atg gag acc agg gaa tct gaa gat ttg gaa aag acc
111 Met Glu Thr Arg Glu Ser Glu Asp Leu Glu Lys Thr 1 5 10 cgg agg
aaa tca gca agt gat caa tgg aac act gat aat gaa cca gcc 159 Arg Arg
Lys Ser Ala Ser Asp Gln Trp Asn Thr Asp Asn Glu Pro Ala 15 20 25
aag gtg aaa cct gag tta ctc cca gaa aaa gag gag act tct caa gct 207
Lys Val Lys Pro Glu Leu Leu Pro Glu Lys Glu Glu Thr Ser Gln Ala 30
35 40 gac cag gat atc caa gac aaa gag cct cat tgc cac atc cca att
aag 255 Asp Gln Asp Ile Gln Asp Lys Glu Pro His Cys His Ile Pro Ile
Lys 45 50 55 60 aga aat tcc atc ttc aat cgc tcc ata aga cgc aaa agc
aaa gcc aag 303 Arg Asn Ser Ile Phe Asn Arg Ser Ile Arg Arg Lys Ser
Lys Ala Lys 65 70 75 gcc aga gac aac ccc gaa cgg aac gcc agc tgc
ctg gca gat tca cag 351 Ala Arg Asp Asn Pro Glu Arg Asn Ala Ser Cys
Leu Ala Asp Ser Gln 80 85 90 gac aat gga aaa tct gta aat gag ccc
ctg acc ttg aat atc ccc tgg 399 Asp Asn Gly Lys Ser Val Asn Glu Pro
Leu Thr Leu Asn Ile Pro Trp 95 100 105 agc aga atg cct cct tgc aga
aca gca atg cag aca gac cca gga gcc 447 Ser Arg Met Pro Pro Cys Arg
Thr Ala Met Gln Thr Asp Pro Gly Ala 110 115 120 cag gaa atg agt gag
tcg tcc tcc acc ccg gga aat ggg gcc acg ccc 495 Gln Glu Met Ser Glu
Ser Ser Ser Thr Pro Gly Asn Gly Ala Thr Pro 125 130 135 140 gag gag
tgg ccg gcc ctg gcc gac agc ccc acc acg ctc acc gag gcc 543 Glu Glu
Trp Pro Ala Leu Ala Asp Ser Pro Thr Thr Leu Thr Glu Ala 145 150 155
ctg cgg atg atc cac ccc att ccc gcc gac tcc tgg aga aac ctc att 591
Leu Arg Met Ile His Pro Ile Pro Ala Asp Ser Trp Arg Asn Leu Ile 160
165 170 gaa caa ata ggg ctc ctg tat cag gaa tac cga gat aaa tcg act
ctc 639 Glu Gln Ile Gly Leu Leu Tyr Gln Glu Tyr Arg Asp Lys Ser Thr
Leu 175 180 185 caa gaa atc gaa acc agg agg caa cag gat gca gaa ata
gaa gac aat 687 Gln Glu Ile Glu Thr Arg Arg Gln Gln Asp Ala Glu Ile
Glu Asp Asn 190 195 200 acc aat ggg tcc ccg gcc agt gag gac acc ccg
gag gag gaa gaa gaa 735 Thr Asn Gly Ser Pro Ala Ser Glu Asp Thr Pro
Glu Glu Glu Glu Glu 205 210 215 220 gag gag gag gag gag gag ccg gcc
agc cca cca gag agg aag act ctg 783 Glu Glu Glu Glu Glu Glu Pro Ala
Ser Pro Pro Glu Arg Lys Thr Leu 225 230 235 ccc cag atc tgc ctg ctc
agt aac ccc cac tca agg ttc aac ctc tgg 831 Pro Gln Ile Cys Leu Leu
Ser Asn Pro His Ser Arg Phe Asn Leu Trp 240 245 250 cag gat ctt ccc
gag atc cgg agc agc ggg gtg ctt gag atc cta cag 879 Gln Asp Leu Pro
Glu Ile Arg Ser Ser Gly Val Leu Glu Ile Leu Gln 255 260 265 cct gag
gag att aag ctg cag gag gcc atg ttc gag ctg gtc act tcc 927 Pro Glu
Glu Ile Lys Leu Gln Glu Ala Met Phe Glu Leu Val Thr Ser 270 275 280
gag gcg tcc tac tac aag agt ctg aac ctg ctc gtg tcc cac ttc atg 975
Glu Ala Ser Tyr Tyr Lys Ser Leu Asn Leu Leu Val Ser His Phe Met 285
290 295 300 gag aac gag cgg ata agg aag atc ctg cac ccg tcc gag gcg
cac atc 1023 Glu Asn Glu Arg Ile Arg Lys Ile Leu His Pro Ser Glu
Ala His Ile 305 310 315 ctc ttc tcc aac gtc ctg gac gtg ctg gct gtc
agt gag cgg ttc ctc 1071 Leu Phe Ser Asn Val Leu Asp Val Leu Ala
Val Ser Glu Arg Phe Leu 320 325 330 ctg gag ctg gag cac cgg atg gag
gag aac atc gtc atc tct gac gtg 1119 Leu Glu Leu Glu His Arg Met
Glu Glu Asn Ile Val Ile Ser Asp Val 335 340 345 tgt gac atc gtg tac
cgt tat gcg gcc gac cac ttc tct gtc tac atc 1167 Cys Asp Ile Val
Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr Ile 350 355 360 acc tac
gtc agc aat cag acc tac cag gag cgg acc tat aag cag ctg 1215 Thr
Tyr Val Ser Asn Gln Thr Tyr Gln Glu Arg Thr Tyr Lys Gln Leu 365 370
375 380 ctc cag gag aag gca gct ttc cgg gag ctg atc gcg cag cta gag
ctc 1263 Leu Gln Glu Lys Ala Ala Phe Arg Glu Leu Ile Ala Gln Leu
Glu Leu 385 390 395 gac ccc aag tgc agg ggg ctg ccc ttc tcc tcc ttc
ctc atc ctg cct 1311 Asp Pro Lys Cys Arg Gly Leu Pro Phe Ser Ser
Phe Leu Ile Leu Pro 400 405 410 ttc cag agg atc aca cgc ctc aag ctg
ttg gtc cag aac atc ctg aag 1359 Phe Gln Arg Ile Thr Arg Leu Lys
Leu Leu Val Gln Asn Ile Leu Lys 415 420 425 agg gta gaa gag agg tct
gag cgg gag tgc act gct ttg gat gct cac 1407 Arg Val Glu Glu Arg
Ser Glu Arg Glu Cys Thr Ala Leu Asp Ala His 430 435 440 aag gag ctg
gaa atg gtg gtg aag gca tgc aac gag ggc gtc agg aaa 1455 Lys Glu
Leu Glu Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys 445 450 455
460 atg agc cgc acg gaa cag atg atc agc att cag aag aag atg gag ttc
1503 Met Ser Arg Thr Glu Gln Met Ile Ser Ile Gln Lys Lys Met Glu
Phe 465 470 475 aag atc aag tcg gtg ccc atc atc tcc cac tcc cgc tgg
ctg ctg aag 1551 Lys Ile Lys Ser Val Pro Ile Ile Ser His Ser Arg
Trp Leu Leu Lys 480 485 490 cag ggt gag ctg cag cag atg tca ggc ccc
aag acc tcc cgg acc ctg 1599 Gln Gly Glu Leu Gln Gln Met Ser Gly
Pro Lys Thr Ser Arg Thr Leu 495 500 505 agg acc aag aag ctc ttc cac
gaa att tac ctc ttc ctg ttc aac gac 1647 Arg Thr Lys Lys Leu Phe
His Glu Ile Tyr Leu Phe Leu Phe Asn Asp 510 515 520 ctg ctg gtg atc
tgc cgg cag att cca gga gac aag tac cag gta ttt 1695 Leu Leu Val
Ile Cys Arg Gln Ile Pro Gly Asp Lys Tyr Gln Val Phe 525 530 535 540
gac tca gct ccg cgg gga ctg ctg cgt gtg gag gag ctg gag gac cag
1743 Asp Ser Ala Pro Arg Gly Leu Leu Arg Val Glu Glu Leu Glu Asp
Gln 545 550 555 ggc cag acg ctg gcc aac gtg ttc atc ctg cgg ctg ctg
gag aac gca 1791 Gly Gln Thr Leu Ala Asn Val Phe Ile Leu Arg Leu
Leu Glu Asn Ala 560 565 570 gat gac cgg gag gcc acc tac atg cta aag
gcg tcc tct cag agt gag 1839 Asp Asp Arg Glu Ala Thr Tyr Met Leu
Lys Ala Ser Ser Gln Ser Glu 575 580 585 atg aag cgt tgg atg acc tca
ctg gcc ccc aac agg agg acc aag ttt 1887 Met Lys Arg Trp Met Thr
Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe 590 595 600 gtt tcg ttc aca
tcc cgg ctg ctg gac tgc ccc cag gtc cag tgc gtg 1935 Val Ser Phe
Thr Ser Arg Leu Leu Asp Cys Pro Gln Val Gln Cys Val 605 610 615 620
cac cca tac gtg gct cag cag cca gac gag ctg acg ctg gag ctc gcc
1983 His Pro Tyr Val Ala Gln Gln Pro Asp Glu Leu Thr Leu Glu Leu
Ala 625 630 635 gac atc ctc aac atc ctg gac aag act gac gac ggg tgg
atc ttt ggc 2031 Asp Ile Leu Asn Ile Leu Asp Lys Thr Asp Asp Gly
Trp Ile Phe Gly 640 645 650 gag cgt ctg cac gac cag gag aga ggc tgg
ttc ccc agc tcc atg act 2079 Glu Arg Leu His Asp Gln Glu Arg Gly
Trp Phe Pro Ser Ser Met Thr 655 660 665 gag gag atc ttg aat ccc aag
atc cgg tcc cag aac ctc aag gaa tgt 2127 Glu Glu Ile Leu Asn Pro
Lys Ile Arg Ser Gln Asn Leu Lys Glu Cys 670 675 680 ttc cgt gtc cac
aag atg gat gac cct cag cgc agc cag aac aag gac 2175 Phe Arg Val
His Lys Met Asp Asp Pro Gln Arg Ser Gln Asn Lys Asp 685 690 695 700
cgc agg aag ctg ggc agc cgg aat cgg caa tga cccccaccca gggggccagc
2228 Arg Arg Lys Leu Gly Ser Arg Asn Arg Gln 705 710 gggagcaggg
cctgcatgag accccgacag aaggtggggg gggggggggg ggctctggga 2288
agcacaggcc agcacctccc caggtggcag gatctggctt ggggtgcccg gccctcatcc
2348 ctgcccacgc agtgagtgct catgtgtctt ggccccttgc tcgcaaactg
gataaagggt 2408 gcccaagcct ctcctgatgc atttgtaaac aagaaggttt
cagcagtatt acaccacctc 2468 cctcatgcct ccgagggggt ggaagggggt
gggcacactc cagggccccc catgcccctg 2528 gcccccaggg attggaagag
gctcccaacc cagagtgtcc ctgtgggagg caggcagaag 2588 gtgacaattg
acacgatttc ctgcacgcgt cttcttttac cttggaagca gttagaattt 2648
accaggcaca gatgaggccg cccttgcctg acggagcttg atgagcagcc cttggtctcc
2708 ggttccagga ctgagagccc agctgcctct gcccaccctt ccccaggcct
ctgccagcct 2768 ctggctgcac ggtcaggccc tgccccatgg caggcctgcc
agagcttggc tggggacccc 2828 tcccgcctct ggctccctga tgggctggat
gtaacttgtg tcttctagcc ccttaaggag 2888 cccaggtgtt ttaaggaatg
aattggtcac tgcatcttgt atcgattatg gttctgagaa 2948 aagcaaatat
cggaattcct gcagcccggg aaatggggcc acgcccgagg agtggccggc 3008
cctggccgac agccccacca cgctcaccga ggccctgcgg atgatccacc ccattcccgc
3068 cgactcctgg agaaacctca ttgaacaaat agggctcctg tatcaggaat
accgagataa 3128 atcgactctc caaaaaaaaa aaaaaaaaaa gatctttaat taa
3171 2 710 PRT Liver Rac GEF 2 Met Glu Thr Arg Glu Ser Glu Asp Leu
Glu Lys Thr Arg Arg Lys Ser 1 5 10 15 Ala Ser Asp Gln Trp Asn Thr
Asp Asn Glu Pro Ala Lys Val Lys Pro 20 25 30 Glu Leu Leu Pro Glu
Lys Glu Glu Thr Ser Gln Ala Asp Gln Asp Ile 35 40 45 Gln Asp Lys
Glu Pro His Cys His Ile Pro Ile Lys Arg Asn Ser Ile 50 55 60 Phe
Asn Arg Ser Ile Arg Arg Lys Ser Lys Ala Lys Ala Arg Asp Asn 65 70
75 80 Pro Glu Arg Asn Ala Ser Cys Leu Ala Asp Ser Gln Asp Asn Gly
Lys 85 90 95 Ser Val Asn Glu Pro Leu Thr Leu Asn Ile Pro Trp Ser
Arg Met Pro 100 105 110 Pro Cys Arg Thr Ala Met Gln Thr Asp Pro Gly
Ala Gln Glu Met Ser 115 120 125 Glu Ser Ser Ser Thr Pro Gly Asn Gly
Ala Thr Pro Glu Glu Trp Pro 130 135 140 Ala Leu Ala Asp Ser Pro Thr
Thr Leu Thr Glu Ala Leu Arg Met Ile 145 150 155 160 His Pro Ile Pro
Ala Asp Ser Trp Arg Asn Leu Ile Glu Gln Ile Gly 165 170 175 Leu Leu
Tyr Gln Glu Tyr Arg Asp Lys Ser Thr Leu Gln Glu Ile Glu 180 185 190
Thr Arg Arg Gln Gln Asp Ala Glu Ile Glu Asp Asn Thr Asn Gly Ser 195
200 205 Pro Ala Ser Glu Asp Thr Pro Glu Glu Glu Glu Glu Glu Glu Glu
Glu 210 215 220 Glu Glu Pro Ala Ser Pro Pro Glu Arg Lys Thr Leu Pro
Gln Ile Cys 225 230 235 240 Leu Leu Ser Asn Pro His Ser Arg Phe Asn
Leu Trp Gln Asp Leu Pro 245 250 255 Glu Ile Arg Ser Ser Gly Val Leu
Glu Ile Leu Gln Pro Glu Glu Ile 260 265 270 Lys Leu Gln Glu Ala Met
Phe Glu Leu Val Thr Ser Glu Ala Ser Tyr 275 280 285 Tyr Lys Ser Leu
Asn Leu Leu Val Ser His Phe Met Glu Asn Glu Arg 290 295 300 Ile Arg
Lys Ile Leu His Pro Ser Glu Ala His Ile Leu Phe Ser Asn 305 310 315
320 Val Leu Asp Val Leu Ala Val Ser Glu Arg Phe Leu Leu Glu Leu Glu
325 330 335 His Arg Met Glu Glu Asn Ile Val Ile Ser Asp Val Cys Asp
Ile Val 340 345 350 Tyr Arg Tyr Ala Ala Asp His Phe Ser Val Tyr Ile
Thr Tyr Val Ser 355 360 365 Asn Gln Thr Tyr Gln Glu Arg Thr Tyr Lys
Gln Leu Leu Gln Glu Lys 370 375 380 Ala Ala Phe Arg Glu Leu Ile Ala
Gln Leu Glu Leu Asp Pro Lys Cys 385 390 395 400 Arg Gly Leu Pro Phe
Ser Ser Phe Leu Ile Leu Pro Phe Gln Arg Ile 405 410 415 Thr Arg Leu
Lys Leu Leu Val Gln Asn Ile Leu Lys Arg Val Glu Glu 420 425 430 Arg
Ser Glu Arg Glu Cys Thr Ala Leu Asp Ala His Lys Glu Leu Glu 435 440
445 Met Val Val Lys Ala Cys Asn Glu Gly Val Arg Lys Met Ser Arg Thr
450 455 460 Glu Gln Met Ile Ser Ile Gln Lys Lys Met Glu Phe Lys Ile
Lys Ser 465 470 475 480 Val Pro Ile Ile Ser His Ser Arg Trp Leu Leu
Lys Gln Gly Glu Leu 485 490 495 Gln Gln Met Ser Gly Pro Lys Thr Ser
Arg Thr Leu Arg Thr Lys Lys 500 505 510 Leu Phe His Glu Ile Tyr Leu
Phe Leu Phe Asn Asp Leu Leu Val Ile 515 520 525 Cys Arg Gln Ile Pro
Gly Asp Lys Tyr Gln Val Phe Asp Ser Ala Pro 530 535 540 Arg Gly Leu
Leu Arg Val Glu Glu Leu Glu Asp Gln Gly Gln Thr Leu 545 550 555 560
Ala Asn Val Phe Ile Leu Arg Leu Leu Glu Asn Ala Asp Asp Arg Glu 565
570 575 Ala Thr Tyr Met Leu Lys Ala Ser Ser Gln Ser Glu Met Lys Arg
Trp 580 585 590 Met Thr Ser Leu Ala Pro Asn Arg Arg Thr Lys Phe Val
Ser Phe Thr 595 600 605 Ser Arg Leu Leu Asp Cys Pro Gln Val Gln Cys
Val His Pro Tyr Val 610 615 620 Ala Gln Gln Pro Asp Glu Leu Thr Leu
Glu Leu Ala Asp Ile Leu Asn 625 630 635 640 Ile Leu Asp Lys Thr Asp
Asp Gly Trp Ile Phe Gly Glu Arg Leu His 645 650 655 Asp Gln Glu Arg
Gly Trp Phe Pro Ser Ser Met Thr Glu Glu Ile Leu 660 665 670 Asn Pro
Lys Ile Arg Ser Gln Asn Leu Lys Glu Cys Phe Arg Val His 675 680 685
Lys Met Asp Asp Pro Gln Arg Ser Gln Asn Lys Asp Arg Arg Lys Leu 690
695 700 Gly Ser Arg Asn Arg Gln 705 710 3 20 DNA Oligo 3 ggaggccatg
ttcgagctgg 20 4 20 DNA Oligonucleotide 4 gctgatcatc tgttccgtgc 20 5
37 DNA Oligonucleotide 5 tcgaggaggt tataaatatg gaatacatgc caatgga
37 6 37 DNA Oligonucleotide 6 aatttccatt ggcatgtatt ccatatttat
aacctcc 37 7 21 DNA Oligonucleotide 7 ccataaaacc atgggaaacg c 21 8
21 DNA Oligonucleotide 8 ggttccgcgg aagagaagga t 21 9 21 DNA
Oligonucleotide 9 gactggcccg gggaactgag g 21 10 21 DNA
Oligonucleotide 10 tcggatgcgg ataagctgcg c 21 11 21 DNA
Oligonucleotide 11 gtgactggcg accttgttca t 21 12 26 DNA
Oligonucleotide 12 cggaattcag atttcgacac atgatc 26 13 27 DNA
Oligonucleotide 13 tcgcccgggg caggtgacgc agtcaga 27 14 49 DNA
Oligonucleotide 14 gatcccgggt catgtttctg gttctgggat ctcagtgttc
agtttcctg 49 15 28 DNA Oligonucleotide 15 cggaattcca tgggccgcct
tggaatct 28 16 27 DNA Oligonucleotide 16 tcgcccgggc gtcagcagca
cgattat 27 17 67 DNA Oligonucleotide 17 ggtgagcagt ttacactttc
atatactccc tgtcatgtgc tttgaaggac tttctagggg 60 catcaag 67 18 10 DNA
Oligonucleotide 18 gtcatgatgg 10 19 15 DNA Oligonucleotide 19
tccatcatga cggcc 15 20 37 DNA Oligonucleotide 20 cttgaattcc
accatggaaa tctgtccaaa agtcact 37 21 27 DNA Oligonucleotide 21
tcgcccgggc gtcagcagca cgattat 27 22 21 DNA Oligonucleotide 22
catggaccag aacccatctc c 21 23 20 DNA Oligonucleotide 23 tgaggagatg
ggttctggtc 20 24 20 DNA Oligonucleotide 24 gccagaacca gaaacatgac 20
25 24 DNA Oligonucleotide 25 ccgggtcatg tttctggttc tggc 24 26 30
DNA Oligonucleotide 26 gaggaattcg atctgagcat gggagacctg 30 27 34
DNA Oligonucleotide 27 ctgctcgagc tacttatcac gcaggattga aatg 34 28
30 DNA Oligonucleotide 28 caggaattcg tgcgcaaggc cggcgccctg 30 29 33
DNA Brain Specific Nucleotide for Rac-GEF 29 gtgctcgagc tacgcagtgg
cgcaggcaga gtg 33 30 198 DNA Glu-Epitope Tag CDS Complement
(1)..(198) 30 gaa ttc ccg cag ccc gtt agt cgc ccc cga ccc agc cca
ggg ccc cgg 48 Glu Phe Pro Gln
Pro Val Ser Arg Pro Arg Pro Ser Pro Gly Pro Arg 1 5 10 15 cgt ggc
ccc aga ccc ggc ccc agc acc cgc ccc gcc gca gac cct atg 96 Arg Gly
Pro Arg Pro Gly Pro Ser Thr Arg Pro Ala Ala Asp Pro Met 20 25 30
gag ctg ctg gcc gct gcc ttc agc gcc gcc tgc gcc gtg gac cac gac 144
Glu Leu Leu Ala Ala Ala Phe Ser Ala Ala Cys Ala Val Asp His Asp 35
40 45 agt tcc acc tcg gaa agc gac gcg cgc gac tcg gcg gcg gga cac
ctg 192 Ser Ser Thr Ser Glu Ser Asp Ala Arg Asp Ser Ala Ala Gly His
Leu 50 55 60 ccc ggc 198 Pro Gly 65 31 66 PRT Glu-Epitope Tag 31
Glu Phe Pro Gln Pro Val Ser Arg Pro Arg Pro Ser Pro Gly Pro Arg 1 5
10 15 Arg Gly Pro Arg Pro Gly Pro Ser Thr Arg Pro Ala Ala Asp Pro
Met 20 25 30 Glu Leu Leu Ala Ala Ala Phe Ser Ala Ala Cys Ala Val
Asp His Asp 35 40 45 Ser Ser Thr Ser Glu Ser Asp Ala Arg Asp Ser
Ala Ala Gly His Leu 50 55 60 Pro Gly 65 32 11 PRT Glu-Epitope Tag
32 Met Glu Tyr Met Pro Met Glu Ile Arg His Asp 1 5 10 33 5 PRT KT3
EPITOPE TAG 33 Pro Glu Pro Glu Thr 1 5
* * * * *