Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-12-01
2001-10-30
Prouty, Rebecca E. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S352000, C530S359000, C530S358000, C435S007100, C435S183000
Reexamination Certificate
active
06310181
ABSTRACT:
INTRODUCTION
The present invention relates generally to a newly identified adaptor protein FRS2 and related products and methods. FRS2 links protein kinases to activating partners.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (—OH) of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases are generally divided into two groups—receptor and non-receptor type proteins. Receptor protein kinases straddle the cell membrane and harbor an extracellular region, a transmembrane region, and an intracellular region. Non-receptor protein kinases exist within the cell and harbor a catalytic region attached to other functional regions that can localize the protein kinase to different regions in the cell.
Protein kinases are also typically divided into three classes based upon the amino acids they act upon. Some phosphorylate serine or threonine only, some phosphorylate tyrosine only, and some phosphorylate serine, threonine, and tyrosine.
Many protein kinases, particularly receptor protein kinases, function by binding adaptor proteins. Adaptor proteins link the protein kinase to other proteins that cause a cellular reaction to a protein kinase signal. The epidermal growth factor receptor (EGFR), for example, phosphorylates itself upon binding the EGF ligand. The resulting phosphate moieties on the EGFR intracellular region bind adaptor proteins such as Grb-2. Grb-2 then binds a guanine nucleotide exchange factor protein (Sos), which thereby activates Ras. Ras consequently activates the mitogen activated protein kinase (MAPK) cascade, which causes cellular proliferation or differentiation. Thus, Sos and Ras are direct activating partners of EGFR while members of the MAPK cascade are indirect activating partners of EGFR.
Multiple adaptor proteins harbor domains that directly bind to the phosphate moieties on receptor protein kinase. Grb-2, for example, harbors a Src homology 2 domain (SH2 domain) that tightly binds phosphotyrosine moieties within EGFR and other receptor protein kinases. Pawson and Schlessinger, 1993,
Current Biol.
3:434-442. Other adaptor proteins, such as IRS-1, can bind phosphotyrosine moieties of receptor protein kinases via a phosphoryl tyrosine binding domain (PTB domain). Gustafson et al., 1995,
Mol. Cell. Biol.
15:2500-2508. Adaptors such as Shc harbor both SH2 and PTB domains. Blaikie et al., 1994,
J. Biol. Chem.
269:32031-32034. Some adaptor proteins, such as SNT-like proteins, harbor unidentified phosphotyrosine binding domains because their nucleotide and amino acid sequences are unknown. Wang et al., 1996,
Oncogene
13:721-729.
It has become evident that receptor protein kinases other than EGFR, such as the fibroblast growth factor receptor protein kinase (FGFR), stimulate the MAPK cascade without directly binding Grb-2. Nakafuku et al., 1992,
J. Biol. Chem.
267:22963-22966. Scientists are therefore searching for adaptor proteins that link protein kinases to their activating partners to determine the mechanism of activation for these protein kinases. Adaptor proteins involved in protein kinase activation mechanisms are drug targets as compounds that can enhance or abrogate the interactions between the proteins in a protein kinase activation mechanism could potentially prevent and even treat abnormal conditions in organisms caused by altered protein kinase function. Examples of abnormal conditions caused by altered protein kinase function are cancer and other cell proliferative disorders such as arthritis, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, glomerulopathies, hepatic cirrhosis, ocular diseases such as diabetic retinopathy, and restenosis.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid molecules encoding a newly identified protein kinase adaptor protein named FRS2. The invention also relates to nucleic acid molecules encoding portions of the full length protein, nucleic acid vectors harboring such nucleic acid molecules, cells containing such nucleic acid vectors, purified polypeptides encoded by such nucleic acid molecules, antibodies to such proteins and polypeptides, and methods of identifying compounds that enhance or block interactions of FRS2 with natural binding partners. Also disclosed are methods for diagnosing abnormal conditions in an organism with FRS2 related molecules or compounds. The nucleic acid molecules, nucleic acid vectors, host cells, polypeptides, and antibodies may be produced using the information provided herein in conjunction with well known and standard techniques used currently in the art.
FRS2 (Fibroblast Growth Factor Receptor Protein Kinase substrate 2) regulates growth factor stimulation of cellular differentiation and cellular proliferation by linking stimulated fibroblast growth factor receptor (FGFR) to the Ras/MAPK cascade via the Grb-2/Sos complex. Various treatments of cell proliferative disorders and cell differentiation disorders are therefore provided based on the discovery of FRS2.
The FRS2 adaptor protein of the invention is isolated from human NIH 3T3 cells. The invention also relates to closely related adaptor proteins preferably from mammalian tissue and more preferably from human tissue. The term “closely related” refers to greater than 50% amino acid identity or a similar three dimensional structure. The term “amino acid identity” is described herein.
Thus in a first aspect, the invention features an isolated, enriched, or purified nucleic acid molecule encoding a FRS2 polypeptide.
The term “isolated”, in reference to nucleic acid molecules, indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material such as chromosomal DNA or proteins.
The term “enriched”, in reference to nucleic acid molecules, means that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. A person skilled in the art could enrich a nucleic acid mixture by preferentially reducing the amount of other DNA or RNA present, or preferentially increasing the amount of the specific DNA or RNA, or both. However, nucleic acid molecule enrichment does not imply that there is no other DNA or RNA present, the term only indicates that the relative amount of the sequence of interest has been significantly increased. The term “significantly” qualifies “increased” to indicate that the level of increase is useful to the person performing the recombinant DNA technique, and generally means an increase relative to other nucleic acids of at least 2 fold, o
Kouhara Haruhiko
Lax Irit
Schlessinger Joseph
Spivak-Kroizman Taly
Foley & Lardner
Hutson Richard
New York University Medical Center
Prouty Rebecca E.
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