Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-06-22
2003-10-21
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S320100, C536S023500, C530S395000
Reexamination Certificate
active
06635446
ABSTRACT:
BACKGROUND OF THE INVENTION
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP). It is characterized by thrombocytopenia, eczema, impaired immunity and a predisposition to develop lymphomas and leukemias (Cooper, M. D., Chase, H. P., Lowman, J. T., Krivit, W. & Good, R. A. (1968)
Am. J. Medicine
, 44: 499-513). The size of platelets and lymphocytes is reduced in WAS and scanning electron microscopy of T lymphocytes shows a relatively smooth surface with decrease in the number and size of microvilli, suggesting a defect in cytoskeletal architecture (Remold-O'Donnell, E. & Rosen, F. S. (1993) in
Sialophorin
(CD43)
and the Wiskott-Aldrich Syndrome
, eds. Rosen, F. S. & Seligmann, M. S. (Harwood Academic Publishers, Chur), pp. 225-241), pp.225-241 (1993)). The WAS gene is located on Xp11.22-Xp11.23 and encodes a 502 amino acid (a.a.) long proline rich protein, WASP (Derry, J. M. J., Ochs, H. D. & Francke, U. (1994)
Cell
, 78. 635-644.). WASP contains an N-terminal pleckstrin homology (PH) domain, which partially overlaps with a WASP homology (WH) domain, WH1, found in several proteins involved in the maintenance of cytoskeletal integrity that include Ena, Mena, Evl and VASP (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehiand, J. & Soriano, P. (1996)
Cell
, 87: 227-239). The WH1 domain in WASP is followed by a GTPase binding domain (GBD/CRIB) (Bunnell, S. C., Henry, P. A., Kolluri, R., Kirchhausen, T., Rickles, R. J. & Berg, L. J. (1996)
J. Biol. Chem
. 271: 25646-25656), a number of proline rich stretches, a second WH domain (WH2), a short verprolin homology sequence, a cofilin homology sequence, and an acidic C-terminal region. Recently, a protein highly homologous to WASP was cloned from bovine brain and was termed N-WASP (Miki, H., Miura, K. & Takenawa, T. (1996)
EMBO J
. 15, 5326-5335). N-WASP has a domain organization similar to that of WASP, and is widely expressed, in contrast to WASP which is expressed only in hematopoietic cells.
WASP binds via its GBD domain to the small molecular weight GTPase Cdc42 and weakly to Rac, but not to Rho (Aspenstrom, P., Lindberg, U. & Hall, A. (1996)
Curr. Biol
. 6: 70-75; Kolluri, R., Tolias, K. F., Carpenter, C. L., Rosen, F. S. & Kirchhausen, T. (1996)
Proc. Natl. Acad. Sci
. (USA) 93: 5615-5618; Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCorrnick, F., Francke, U. & Abo, A. (1996)
Cell
84: 723-734). Cdc42, Rac and Rho regulate cytoskeletal organization (Nobes, C. D. & Hall, A. (1995)
Cell
81: 53-62). Overexpression of WASP induces the formation of actin-containing clusters (Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCorrnick, F., Francke, U. & Abo, A. (1996)
Cell
84. 723-734). This is inhibited by dominant negative mutants of Cdc42, but not of Rac or Rho (Symons, M., Derry, J. M. J., Kariak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. & Abo, A. (1996)
Cell
84: 723-734). These findings suggest that WASP may provide a link between Cdc42, Rac and the cytoskeleton.
WASP interacts with components of signal transduction pathways via their SH3 domains (Src homology 3) which recognize the proline rich domain in WASP (Featherstone, C. (1997)
Science
275: 27-28). WASP associates with the adaptor protein Nck (Rivero-Lezcano, O. M., Marcilla, A., Sameshima, J. H. & Robbins, K. C. (1995)
Mol. Cell Biol
., 15: 5725-5731). Nck can be recruited via its SH2 domain to tyrosine phosphorylated receptors (Galisteo, M. L., Chernoff, J., Su, Y.-C., Skolnich, E. Y. & Schlessinger, J. (1996)
J. Biol. Chem
. 271: 20997-21000). WASP also binds in vivo to fyn (Rivero-Lezcano, O. M., Marcilla, A., Sameshima, J. H. & Robbins, K. C. (1995)
Mol. Cell Biol
. 15: 5725-5731; Banin, S., Truong, O., Katz. D. R., Waterfield, M. D., Brickell, P. M. & Gout, I. (1996)
Curr. Biol
., 6: 981-988) and in vitro to the src kinase fgr, to the tyrosine kinases btk, itk, Abl and to the p85 subunit of PLC-g (Banin, S., Truong, O., Katz. D. R., Waterfield, M. D., Brickell, P. M. & Gout, I. (1996)
Curr. Biol
. 6, 981-988; Molina, I. J., Sancho, J., Terhorst, C., Rosen, F. S. & Remold-O'Donnell, E. (1993)
J. Immunol
., 151. 4383-4390; Finan, P. M., Soames, C. J., Wilson, L., Nelson, D. L., Stewart, D. M., Truong, O., Hsuan, J. J. & Kellie, S. (1996)
J. Biol. Chem
., 271: 26291-26295).
It would be helpful to have a better understanding of the function of WASP.
SUMMARY OF THE INVENTION
Described herein is a novel human gene whose 503 amino acid (a.a.) product interacts with WASP. The protein is referred to as WIP, for WASP-interacting protein. The proline-rich WIP, which co-immunoprecipitated with WASP from lymphocytes, has been shown to bind to WASP at a site distinct from the Cdc42 binding site and to have actin, profilin and Nck binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. Work described herein supports the role of WIP in cortical actin assembly that may be important for lymphocyte function. Overexpression of WIP increases F-actin content and induces actin containing structures in the human B cell line BJAB, suggesting an important role for WIP in the organization of the actin cytoskeleton.
In particular, the present invention relates to isolated (e.g., purified, essentially pure) nucleic acids (oligonucleotides, polynucleotides, nucleotide sequences) which encode mammalian (e.g., human) WIP, and include for example, nucleic acids (DNA, RNA) which are obtained from natural sources, recombinantly produced or chemically synthesized. The nucleic acids of the present invention include nucleic acids encoding human WIP (SEQ ID NO: 1) and characteristic portions of the nucleic acid sequences (e.g., probes, primers). The invention also includes complementary sequences (i.e., a complement) of SEQ ID NO: 1 and characteristic portions thereof. The nucleic acids of the present invention encompass nucleic acids encoding a human WIP amino acid sequence (SEQ ID NO: 2) and characteristic portions thereof.
The present invention further relates to isolated, recombinantly produced or synthetic nucleic acids which hybridize to the nucleic acids described herein (e.g., the complement of SEQ ID NO: 1 or characteristic portions thereof) and encode WIP (a protein having the same amino acid sequence as the amino acid sequences included herein and/or a protein which exhibits the same characteristics as WIP described herein). In particular, the invention relates to nucleic acids which hybridize, under moderate or high stringency conditions, to SEQ ID NO: 1 characteristic portions thereof or other sequences which encode WIP.
Also encompassed by the present invention is a nucleic acid construct comprising nucleic acid which encodes a WIP (e.g., SEQ ID NO: 1 and characteristic portions thereof), wherein the nucleic acid of the construct is expressed when the construct is present in an appropriate host cell. In one embodiment, the nucleic acid construct of the present invention is operably linked to exogenous regulatory sequence(s) such as a promoter and/or enhancer, whereby mammalian WIP is expressed hen the host cell is maintained under conditions suitable for expression. The present invention also relates to a host cell comprising nucleic acid encoding mammalian WIP.
Also encompassed by the present invention is a method for producing a WIP (mammalian, such as human). In one embodiment, a nucleic acid construct comprising a nucleotide sequence (DNA, RNA) which encodes a WIP is introduced into a host cell, resulting in production of a recombinant host cell which contains a WIP coding sequence operably linked to an (i.e., at least one) expression control sequence. The host cells produced are maintained in a suitable medium under conditions appropriate for the nucleotide sequence to be expressed, whereby the encoded WIP is produced.
The present invention a
Anton Ines M.
Geha Raif S.
Hartwig John H.
Ramesh Narayanaswamy
Chan Christina
Haddad Maher
Hamilton Brook Smith & Reynolds P.C.
The Children's Medical Center Corporation
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