Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-07
2002-08-20
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S021000, C435S196000, C424S094600
Reexamination Certificate
active
06436637
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a new human protein phosphatase and to the use of these sequences in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
BACKGROUND OF THE INVENTION
The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. It is estimated that more than 10% of the active proteins in a typical mammalian cell are phosphorylated. During protein phosphorylation/dephosphorylation, phosphate groups are transferred from adenosine triphosphate molecules to a protein by protein kinases and are removed from the protein by protein phosphatases.
Protein phosphatases function in cellular signaling events that regulate cell growth and differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis. Three protein phosphatase families have been identified as evolutionarily-distinct. These include the serine/threonine phosphatases, the protein tyrosine phosphatases, and the acid/alkaline phosphatases (Carbonneau H. and Tonks N. K. (1992) Annu. Rev. Cell Biol. 8:463-93).
The serine/threonine phosphatases are either cytosolic or associated with a receptor. On the basis of their sensitivity to two thermostable proteins, inhibitors 1 and 2, and their divalent cation requirements, the serine/threonine phosphatases can be separated into four distinct groups, PP-I, PP-IIA, PP-IIB, and PP-IIC.
PP-I dephosphorylates many of the proteins phosphorylated by cylic AMP-dependent protein kinase and is therefore an important regulator of many cyclic AMP mediated, hormone responses in cells. PP-IIA has broad specificity for control of cell cycle, growth and proliferation, and DNA replication and is the main phosphatase responsible for reversing the phosphorylations of serine/threonine kinases. PP-IIB, or calcineurin (Cn), is a Ca
+2
-activated phosphatase; it is involved in the regulation of such diverse cellular functions as ion channel regulation, neuronal transmission, gene transcription, muscle glycogen metabolism, and lymphocyte activation.
PP-IIC is a Mg
++
-dependent phosphatase which participates in a wide variety of functions including regulating cyclic AMP-activated protein-kinase activity, Ca
++
-dependent signal transduction, tRNA splicing, and signal transmission related to heat shock responses. PP-IIC is a monomeric protein with a molecular mass of about 40-45 kDa. One &agr; and several &bgr; isoforms of PP-IIC have been identified (Wenk, J. et al. (1992) FEBS Lett. 297: 135-138; Terasawa, T. et al. (1993) Arch. Biochem. Biophys. 307: 342-349; and Kato, S. et al. (1995) Arch. Biochem. Biophys. 318: 387-393).
The discovery of a new human protein phosphatase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of inflammation and disorders associated with cell proliferation and apoptosis.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, human protein phosphatase (PROPHO), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding PROPHO under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified PROPHO having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist which decreases the effect of the polypeptide of SEQ ID NO:1. In one aspect, the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO:1.
The invention also provides a method for stimulating cell proliferation comprising administering to a cell an effective amount of purified PROPHO.
The invention also provides a method for treating a disorder associated with increased apoptosis comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition comprising purified PROPHO.
The invention also provides a method for treating a disorder associated with cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the effect of PROPHO.
The invention also provides a method for treating inflammation comprising administering to a subject in need of such treatment an effective amount of an antagonist which decreases the effect of PROPHO.
The invention also provides a method for detecting a polynucleotide which encodes PROPHO in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to PROPHO (SEQ ID NO:1) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding PROPHO in the biological sample. In a preferred embodiment, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
REFERENCES:
patent: 5853997 (1998-12-01), Bandman et al.
patent: WO9710796 (1997-03-01), None
Charbonneau, H. et al., “1002 Protein Phosphatases?”,Annu. Rev. Cell Biol., 8: 463-493 (1992).
Wenk, J. et al., “Molecular cloning and primary structure of a protein phosphatase 2C isoform”,FEBS Lett., 297: 135-138 (1992).
Terasawa, T. et al., “Molecular Cloning of a Novel Isotype of Mg2+-Dependent Protein Phosphatase &bgr; (Type 2C&bgr;) Enriched in Brain and Heart”,Arch. Biochem. Biophys., 307: 342-349 (1993).
Kato, S. et al., “Molecular Cloning and Expression of Mouse Mg2+-Dependent Protein Phosphatase &bgr;-4 (Type 2C&bgr;-4)”,Arch. Biochem. Biophys., 318: 387-393 (1995).
Wenk, J., et al., (Direct Submission), GenBank Sequence Database (Accession 247927), National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland,
Bandman Olga
Corley Neil C.
Goli Surya K.
Lal Preeti
Zhang Hong
Incyte Genomics Inc.
Incyte Genomics, Inc.
Prouty Rebecca E.
Steadman David J.
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