Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-06
2001-05-01
Priebe, Scott D. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S325000, C435S252100
Reexamination Certificate
active
06225456
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to Ras suppressors, in particular the Ras suppressor SUR-5.
BACKGROUND OF THE INVENTION
The Ras family of proteins play critical roles in cell proliferation, differentiation, and cell migration in response to extracellular signals. Ras proteins are small membrane-bound GTPases that act as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. In the most well studied Ras-mediated signal transduction pathways, Ras is activated by receptor tyrosine kinases (RTK) through guanine nucleotide exchange factors that promote GTP binding and a change in Ras conformation to an active state (See e.g., McCormick, Nature 363:15 [1993]). GTP-bound Ras then binds to the serine/threonine kinase Raf and recruits it to plasma membrane where it is activated. Once activated, Raf phosphorylates and activates the dual specific kinase MEK, which in turn phosphorylates and activates MAP kinase. Activated MAP kinase (MAPK) is proposed to regulate the activity of multiple targets including transcription factors for various physiological functions (Marshall, Curr. Opin. Genet. Dev., 4:82 [1994]). Although this model for Ras-dependent signal transduction has been heavily studied, there has been almost no development or identification of effectors that regulate Ras signal transduction or that alter the associated cellular and physiological events stimulated by Ras. Little is known about the nature of Ras effectors or the pathways they control (Rubin et al., WO 97/21820 [1997]).
Recent studies using various model systems including biochemical studies in mammalian tissue culture and genetics in
C elegans
and Drosophila suggest that the RTK-Ras-MAPK-mediated signal transduction pathway is not a simple linear pathway, but is likely part of complicated signal transduction network (Katz, Curr. Opin. Genet. Dev., 7:15 [1997]; Sundaram and Han, Cell 83, 889 [1995]; and Kornfeld, Trends Genet., 13:55 [1997]). Thus, a series of converging and diverging signalling pathways are likely responsible for the diverse cellular responses mediated by Ras. In recent years, several potential Ras effectors in addition to Raf, including PI3 kinase and Ral GDS, have been described (Katz, supra) and are candidates for defining branch points of Ras signalling. However, these effectors cannot account for all of the cellular responses mediated by Ras (See e.g., White et al., Cell 80:533 [1995]) and have not been sufficiently characterized.
Adding to the complexity of the various signaling processes is the collaboratory roles of multiple factors and signaling branches in regulating the output of the signal. The main players of the RTK-Ras-MAPK pathway may be essential elements of a given signaling process, but there are other factors that feed into or out of this pathway that may play important regulatory functions to ensure maximal activity of the pathway and to tighten the regulation of the signal. For example, the ksr genes were identified as suppressors of activated ras in
C. elegans
and Drosophila (Sundaram and Han, Cell 83:889 [1995]; Kornfeld et al., Cell 83:903 [1995]; and Therrien et al., Cell 83:879 [1995]), however, their biochemical relation to the Ras pathway is still not well understood. In
C. elegans
, it has been shown that mutations in the ksr-1 gene do not obviously disrupt vulval signal transduction mediated by ras (i.e., a pathway controlled by ras in
C. elegans
). However, the ksr-1 activity becomes essential when the activity in the main pathway is compromised (Sundaram and Han, 1995, supra; and Kornfeld et al., 1995, supra).
The art is in need of additional regulators of the Ras signal transduction pathways. To gain regulatory control of Ras signaling and its physiological consequences (e.g., effects on cancer), new Ras effectors and their genes need to be identified and isolated. Without such developments, the ability to control Ras-mediated proliferation, differentiation, and cell migration will be severely limited.
SUMMARY OF THE INVENTION
The present invention relates to Ras suppressors, in particular the Ras suppressor SUR-5.
In one embodiment, the present invention provides an isolated nucleotide sequence encoding at least a portion of a SUR-5 protein. In some embodiments, the isolated nucleotide sequence encodes a SUR-5 protein selected from the group consisting of human SUR-5, murine SUR-5, and
C. elegans
SUR-5. In certain embodiments the isolated nucleotide sequence is selected from SEQ ID NO:1 (
C. elegans
), SEQ ID NO:2 (human) and SEQ ID NO:3 (mouse). In another embodiment, the nucleotide sequence further comprises 5′ and 3′ flanking regions. In yet another embodiment, the nucleotide sequence further comprises intervening regions. In alternative embodiments, the nucleotide sequence comprises portions or fragments of the sequences described above.
In another embodiment, the present invention provides vectors comprising a nucleotide sequence encoding at least a portion of SUR-5. In yet another embodiment, the present invention provides a host cell transformed with a vector comprising a nucleotide sequence encoding at least a portion of SUR-5. It is intended that the nucleotides, as well as the vector comprise deoxyribonucleotides and/or ribonucleotides. It is not intended that the vector be limited to any particular nucleotide sequences. It is also not intended that the host cell be limited to any particular cell type. The host cell may be contained within a living animal, as well as in culture (i.e., in cell cultures). In certain embodiments, the host cell is selected from bacteria, yeast, amphibian, and mammalian cells.
In one embodiment, the present invention provides an isolated peptide sequence comprising at least a fragment of SUR-5. In some embodiments, the isolated peptide sequence is selected from SEQ ID NO:4 (
C elegans
), SEQ ID NO:5 (human), and SEQ ID NO:6 (mouse), and fragments thereof. The present invention is not limited to proteins and fragments with identical sequences to those disclosed. Indeed, a variety of proteins with altered functions are contemplated including fusion proteins, variants with altered stability and binding specificity, agonists of SUR-5 and antagonists of SUR-5. In a preferred embodiment, a SUR-5-Green Fluorescent Protein fusion protein is provided.
The present invention also provides antibodies capable of specifically binding to any of the polypeptides described above. It is intended that the antibodies be produced using any suitable method known in the art, including polyclonal, as well as monoclonal antibodies. The present invention is not limited to polyclonal or monoclonal antibodies. Indeed, a variety of antigen binding proteins are contemplated, including, but not limited to single chain antibodies, chimeric antibodies, and Fab fragments.
The present invention also provides a polynucleotide sequence comprising at least fifteen nucleotides, capable of hybridizing under stringent conditions to at least a portion of an isolated nucleotide sequence encoding at least a portion of a SUR-5 protein. In certain embodiments, the polynucleotide sequence is selected from the group consisting of SEQ ID NOS:7 and 8.
The present invention also provides methods for detection of a polynucleotide encoding SUR-5 protein in a biological sample suspected of containing the polynucleotide encoding SUR-5, comprising the step of hybridizing at least a portion of the polynucleotide sequence capable of hybridizing under stringent conditions to at least a portion of an isolated nucleotide sequence encoding at least a portion of a SUR-5 protein, to nucleic acid of said biological sample to produce a hybridization complex. In one embodiment, the method further comprises the step of detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding SUR-5 in the biological sample. In some embodiments, t
Gu Trent
Han Min
Orita Satoshi
Medlen & Carroll LLP
Priebe Scott D.
Shukla Ram R.
University Technololy Corporation
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