Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-10-29
2002-04-16
Murthy, Ponnathapuachuta (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000
Reexamination Certificate
active
06372467
ABSTRACT:
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made in part with Government funding, and the Government therefore has certain rights in the invention.
BACKGROUND OF THE INVENTION
The invention relates to the diagnosis and treatment of conditions associated with cell proliferation.
The control of cell proliferation is a fundamental issue in medicine. Cell proliferation is regulated by the formation and dissociation of multiple protein complexes, the components of which often use post-translational modifications as an additional control mechanism. Many diseases result from inappropriate cell proliferation, including cancer. Methods and reagents to modulate cell proliferation would have the potential to yield new treatments for cancer, major opportunistic infections, immune disorders, certain cardiovascular diseases, and inflammatory disorders.
SUMMARY OF THE INVENTION
In general, the invention features a substantially pure nucleic acid (for example, genomic DNA, cDNA, synthetic DNA, mRNA, or antisense RNA) encoding a p54
S6K
or p85
S6K
polypeptide, as defined below.
In a preferred embodiment, the substantially pure nucleic acid encoding a p54
S6K
or p85
S6K
polypeptide is mammalian DNA. More preferably, the substantially pure nucleic acid encoding a p54
S6K
or p85
S6K
polypeptide is human DNA. In another embodiment, the invention features a DNA sequence substantially identical to, or which hybridizes with high stringency to, the DNA sequence shown in
FIG. 1A
or
5
(SEQ ID NO: 1).
In another embodiment, the invention features DNA encoding fragments of p54
S6K
or p85
S6K
polypeptides. In preferred embodiments, the fragments include the C-terminal and N-terminal regions of p54
S6K or p
85
S6K
that are distinct from p70
S6k
, the catalytic domain of p54
S6K
or p85
S6K
, the linker domain, the proline rich domain, or the acidic region of p54
S6K
or p85
S6K
. In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54
S6K
(SEQ ID NOs: 4-10). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85
S6K
(SEQ ID NOs: 10-15). In another embodiment, the invention features nucleic acids that bind with high stringency to SEQ ID NOs: 1 or 2.
In another aspect, the invention features a substantially pure polypeptide having a sequence substantially identical to SEQ ID NOs: 2 or 3. In a preferred embodiment, the substantially pure polypeptide has an acidic region at its N-terminus and a central catalytic region. In another the preferred embodiment, the substantially pure polypeptide has a post-translational modification
In other aspects of the invention, a substantially pure p54
S6K
or p85
S6K
polypeptide has one or more amino acids changed by natural or artificial means, the p54
S6K
or p85
S6K
polypeptide is p54
S6K
KR or p85
S6K
KR, or the p54
S6K
or p85
S6K
polypeptide is a fusion with a heterologous polypeptide, for example, glutathione-S-transferase (GST) or influenza hemagglutinin (HA), relative to the wild-type p54
S6K
or p85
S6K
sequence shown in
FIG. 1A
or FIG.
5
.
In another aspect, the invention features fragments of p54
S6K
or p85
S6K
polypeptides. In a preferred embodiment, the fragment is a peptide comprising a C-terminal sequence of p54
S6K
or p85
S6K
that is distinct from p70
S6k
or p85
S6K
. In another embodiment, the fragment comprises a N-terminal sequence of p54
S6K
or p85
S6K
that is distinct from p70
S6k
. In another embodiment, the fragment comprises the catalytic domain of p54
S6K
or p85
S6K
. In another embodiment, the fragment comprises the acidic region of p54
S6K
or p85
S6K
In another embodiment, the fragment comprises the linker domain of p54
S6K
or p85
S6K
. In another embodiment, the fragment comprises the proline rich region of p54
S6K
or p85
S6K
. In another embodiment, the fragment contains a post-translational modification of p54
S6K
or p85
S6K
. In yet another embodiment, the fragment is a fragment of p54
S6K
KR or p85
S6K
KR. In still another embodiment, the fragment is fused to a heterologous polypeptide. In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54
S6K
(SEQ ID NOs: 4-9). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85
S6K
(SEQ ID NOs: 10-15).
In another aspect, the invention features an antibody that specifically binds to a p54
S6K
or p85
S6K
polypeptide, a p54
S6K
or p85
S6K
polypeptide fragment, or a post-translational modification of a p54
S6K
or p85
S6K
polypeptide. In a preferred embodiment, the antibody is Ab#167.
In another aspect, the invention features a cell line having a genetically engineered null mutation in a p54
S6K
or p85
S6K
gene. In a preferred embodiment, the cell line is an embryonic stem cell line. If the cell is in a mammal, the mammal is preferably a non-human.
In another aspect, the invention features a cell line, genetically engineered to overexpress a p54
S6K
or p85
S6K
polypeptide. In a preferred embodiment, the cell line is a tumor cell line.
In another aspect, the invention features a non-human transgenic animal, or embryo thereof, with a knockout mutation in the p54
S6K
or p85
S6K
gene. In a related aspect, the invention features a non-human transgenic animal with additional copies of p54
S6K
or p85
S6K
nucleic acids added to its genome. In a preferred embodiment of these aspects, the non-human transgenic animal is a rodent, more preferably a mouse. In a preferred embodiment the animal has a knockout mutation in both genes.
In other aspects, the invention features methods of identifying a compound which modulates, or whose activity is modulated by, p54
S6K
or p85
S6K
biological activity involving: a) providing a cell expressing a p54
S6K
or p85
S6K
polypeptide, or a lysate from the cell expressing a p54
S6K
or p85
S6K
polypeptide, or a transgenic animal expressing a p54
S6K
or p85
S6K
polypeptide; b) exposing the cell, lysate, or transgenic animal to the test compound; c) assaying for a modulation in p54
S6K
or p85
S6K
biological activity; and d) comparing the modulation in p54
S6K
or p85
S6K
biological activity to that of a cell, lysate, or transgenic animal which did not receive the test compound, wherein a modulation of p54
S6K
or p85
S6K
biological activity identifies a test compound. In a preferred embodiment the biological activity is assayed by measuring p54
S6K
or p85
S6K
phosphorylation, p54
S6K
or p85
S6K
kinase activity, p54
S6K
or p85
S6K
polypeptide or nucleic acid levels, or cell proliferation. In another preferred embodiment, p54 or p85
S6K
biological activity is measured by using S6 or BRCA1 as a substrate. In other embodiments, the cell is induced or genetically engineered to express the p54
S6K
or p85
S6K
polypeptide. In a preferred embodiment the cell is a tumor cell and/or the p54
S6K
or p85
S6K
polypeptide is p54
S6K
KR or p85
S6K
KR. In another preferred embodiment, the test compound is BRCA1. In a related aspect, the cell may express an altered p54
S6K
or p85
S6K
, as described herein before (e.g., a fusion, fragment, etc.),
In other embodiments, the cell is exposed to a stimulus which could include serum or insulin. In other embodiments the cell is exposed to an inhibitor which could include rapamycin or wortmannin.
In another aspect, the invention features a method of diagnosing an increased likelihood of a cell proliferative disease in a subject. The method includes detecting the level
Blenis John
Kuo Calvin J.
Lee-Fruman Kay K.
Bieker-Brady Kristina
Clark & Elbing LLP
Murthy Ponnathapuachuta
President and Fellows of Harvard College
Rao Manjunath N.
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