Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-23
2003-01-28
Nguyen, Dave T. (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100
Reexamination Certificate
active
06512102
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is modulation of signal transduction using casein kinase I, polynucleotides encoding casein kinase I (CKI), and variants and fragments of CKI or the polynucleotides. The invention is specifically directed to modulating the Wnt pathway using CKI polypeptides or polynucleotides. The invention is particularly directed to using CKI polypeptides or polynucleotides for diagnosis and treatment of disorders mediated by the Wnt signal pathway, especially hyperproliferative disorders, and particularly breast and colon cancer. The invention further relates to assays for screening drugs using the polypeptides and polynucleotides. The invention further relates to methods for producing the polypeptides or polynucleotides, especially by recombinant means. The invention finally relates to pharmaceutical compositions containing CKI polypeptides, polynucleotides, antibodies, variants, and fragments.
BACKGROUND OF THE INVENTION
CKI is a ubiquitous protein kinase that was first described as one of the two protein kinases responsible for the Ser/Thr protein kinase activity on acidic rather than basic polypeptides in total cell extracts (Matsumara,
Biochem. Biophys. Acta
289:237-241, (1972)). Since then, CKI homologs have been identified in eukaryotes from yeast to human. Several isoforms are known. Most organisms contain more than one isoform. In vertebrates seven CKI isoforms have been reported (&agr;, &bgr;, &ggr;1, &ggr;2, &ggr;3, &dgr;and &egr;). They range in size from 34 to 49 kDa (Fish et al.,
J. Biol. Chem
. 270:14875-83 (1995); Graves et al.,
J. Biol. Chem
. 268:6394-6401 (1993); Rowles et al.,
Proc. Natl. Acad. Sci. USA
88:9548-9552 (1991); Zhai et al.,
Biochem. Biophys. Res. Comm
. 189:944-949 (1992)).
Wnt Signaling
Signaling proteins and the hierarchies in which they operate are highly conserved in evolution. This is particularly true of Wnt signaling.
The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in various developmental processes. Cell fate, morphogenesis, and mitogenesis can be affected by changes in Wnt expression. Signal transduction by the Wnt family of ligands has been recently reviewed (McMahon,
Trends in Genetics
8:236-242 (1992); Nusse, et al
Cell
. 69:1073-1087 (1992); Dale,
Biochem. J
. 329:209-223 (1998)). Wnt signaling has also been addressed in relationship to the formation and function of Spemann's organizer (
Ann. Rev. Cell Dev. Biol
. 13:611-667(1997)).
Drosophila gene wingless (wg) is the ortholog of the mouse Wnt-1 gene (Nusse et al.,
Cell
69:1073-1087 (1992); Rijsewijk et al.,
Cell
50:649-657 (1987)). Wg binds to its receptor frizzled on the signal receiving cell. This is believed to result in a signal that is transmitted through the disheveled (dsh) gene product (Klingensmith et al.,
Genes Dev
. 8:118-130 (1994); Noordermeer et al.,
Nature
367:80-83 (1994); Theisen et al.,
Development
121:347-360 (1994)), ultimately resulting in regulation of the zeste white 3 (zw3) serine/threonine kinase (also known as shaggy (sgg)) (Bourouis et al.,
EMBO J
. 9:2877-2884 (1990); Siegfried et al.,
Nature
367:76-80 (1994); Siegfried et al.,
Cell
71:1167-1179 (1992)). Zw3 in turn negatively regulates the protein levels of the armadillo (arm) gene product (Peifer et al.,
Dev. Biol
. 166:543-556 (1994a); Peifer et al.,
Development
111:1029-1043 (1991); Peifer et al.,
Development
120:369-380 (1994b); Riggleman et al.,
Cell
63:549-560 (1990)). All of these proteins have vertebrate counterparts. The Dsh ortholog in Xenopus is referred to as Xdsh and in mouse as Dvl-1 (Sussman et al.,
Dev. Biol
. 166:71-86 (1994)). The Sgg ortholog in mammals is GSK3 (Sutherland et al.,
Biochem. J
. 296:15 (1993)). The Arm ortholog in mammals is &bgr;-catenin (Peifer et al.,
J. Cell. Biol
. 118:681-691 (1992)). Recent biochemical studies indicate that the vertebrate HMG-domain proteins Lef-1 and Xtcf-3 can physically interact with &bgr;-catenin and then regulate transcription of target genes (Behrens et al.,
Nature
382:638-42 (1996); Molenaar et al.,
Cell
86:391-399 (1996)). Genetic studies indicate that pangolin (Pan), a Drosophila homolog of the mammalian Lef-1, binds to Arm protein in vivo (Brunner et al.,
Nature
385:829-33 (1997)). Recently, CKI was shown to associate with and phosphorylate Dsh in Drosophila (Willert et al.,
EMBO J
. 16:3089-3096 (1997)).
Wnt binds to its receptor (a Frizzled ortholog; see below) on the cell surface. This activates Dvl-1 (Disheveled in Drosophila). Activation of disheveled inhibits GSK3 (Sgg in Drosophila) activity. Normally, GSK3 is active and phosphorylates &bgr;-catenin. Phosphorylated &bgr;-catenin is degraded. When GSK3 activity is inhibited, the unphosphorylated &bgr;-catenin level increases, the protein enters the nucleus, binds to Lef-1 and the binary complex activates the Lef-1 enhancer causing transcription of target genes.
The Wnt signaling pathway is involved in mammary tumor and colon cancer. Ectopically-expressed Wnt-1 in mammary epithelium can induce hyperplasia, presumably by interfering with hormone-regulated Wnt pathway of other Wnt family members (Weber-Hall et al.,
Differentiation
57:205-214 (1994); Wong et al.,
Mol. Cell. Biol
. 14:6278-6286 (1994)). &bgr;-catenin, a component in the Wnt signal pathway, is found associated with adenomatous polyposis coli (APC) which is a familial predisposition to colon cancer (Rubinfeld et al.,
Science
262:1731-1734 (1993); Su et al.,
Science
262:1734-1737 (1993)), and the levels of free &bgr;-catenin is regulated by APC together with GSK3 (Rubinfeld et al.,
Science
272:1023-6 (1996)). &bgr;-catenin is identified as an accomplice in causing colon cancer and is strongly implicated in melanoma (Rubinfeld et al.,
Science
275:1790-1792 (1997)).
The Wnt-1 proto-oncogene was originally identified as a common integration site of mouse mammary tumor virus in independently isolated adenocarcinomas of mammary epithelial tissue (Nusse et al.
Cell
. 31:99-109 (1982)). Ectopic expression of the normally silent Wnt-1 locus results from the introduction of transcriptional enhancers contained in the mouse mammary tumor virus long terminal repeats (Nusse et al.
Nature
307:131-136 (1984); Nusse et al.
Cell
. 31:99-109 (1982)). Formal proof of a causative role for Wnt-1 in mammary oncogenesis has come from experiments on gene transfer into mammary epithelial cell lines (Brown et al.
Cell
46:1001-1009 (1986); Rijsewijk et al.
EMBO J
. 6:127-131 (1987)) and transgenic mice (Tsukamoto et al.
Cell
55:619-625 (1988)).
Accordingly, there is a need in the art for agents that can be used to modulate the Wnt pathway and to detect disorders mediated by this pathway.
SUMMARY OF THE INVENTION
The invention is based on the inventor's discovery that CKI can modulate the Wnt pathway. The inventor has found that normally, CKI allows a basal level of transduction in the Wnt pathway, but under-expression of CKI or a CKI variant lacking kinase activity can down-regulate the pathway, and over-expression of CKI can upregulate the pathway. A CKI variant causing over-phosphorylation should increase Wnt signal transduction. Increased signal transduction can result in a Wnt signal transduction mediated disorder, and particularly a hyperproliferative disorder. Hence, CKI or its variants or fragments can be used to modulate the Wnt pathway.
The invention encompasses the entire genus of CKI as well all species that retain the ability to affect Wnt signaling. These include homologs and orthologs from other animals or tissues as well as all isoforms.
The invention therefore provides a pharmaceutical composition comprising The invention also provides a pharmaceutical composition containing nucleic acid molecules encoding CKI.
The invention also provides variant CKI polypeptides containing a mutation in the kinase region that results in over- or under-phosphorylation of the CKI or its substrate.
The invention also provides variant CKI nucleic acid sequences containing a mutation in the kina
Harrison Stephen D.
Williams Lewis T.
Xu Licen
Blackburn Robert P.
Chiron Corporation
Dollard Anne S.
Nguyen Dave T.
Nguyen Quang
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