Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-05-19
2001-06-12
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S012200
Reexamination Certificate
active
06245888
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to certain proteins which may be involved in regulating physiological changes (e.g. changes in cell-cell adhesion) and to uses thereof.
Cell adhesion is important for a wide variety of regulatory and developmental processes. The cadherins comprise a family of transmembrane, cell surface glycoproteins that mediate Ca
2+
-dependent cell-cell adhesion in a homotypic manner (Takeichi, 1991). In cells with well developed intercellular junctions, the cadherins are localized to the adherens junction (Boller et al., 1985) but appear to influence other intercellular junctions such as gap junctions (Matsuzaki et al., 1990; Musil et al., 1990) and tight junctions (Gumbiner and Simons, 1986; Gumbiner et al., 1988). The adherens junction also plays a crucial role in the development and maintenance of cell polarity (see Nelson, 1992) and its dysfunction has been strongly implicated in the invasiveness and carcinogenesis of tumour cells (see e.g. Behrens et al., 1989; Frixen et al., 1991; Vleminckx et al., 1991; Shimoyama et al., 1992; and Hedrick et al., 1993; Tsukita et al., 1993; Birchmeier and Behrens, 1994).
The conserved cytoplasmic domain of cadherins is known to associate with three proteins, termed &agr;-&bgr;-and &ggr;-catenin (Pzawa et al., 1989), which serve to link cadherins to the actin-based cortical cytoskeleton (Hirano et al., 1987). The association of cadherins with catenins is essential for intercellular Ca
2+
-dependent adhesiveness (Nagafuchi and Takeichi, 1988; Ozawa et al., 1990; Kintner, 1992). &agr;-catenin is homologous to vinculin (Herrenknecht et al., 1991; Nagafuchi et al., 1991), making it a good candidate for interaction with the actin-based cytoskeleton (see Ozawa et al., 1990; Hirano et al., 1992). &bgr;-catenin is homologous to the Drosophilia segment polarity gene
armadillo,
suggesting a role in developmental signalling in vertebrates (McCrea et al., 1991). &ggr;-catenin is probably identical to plakoglobin (Knudsen and Wheelock, 1992; but see Piepenhagen and Nelson, 1993), which again is homologous to
armadillo
(see Franke et al., 1989; Peifer and Wieschaus, 1990). Indeed, &bgr;-catenin and plakoglobin appear to form a multigene family (Peifer et al., 1992)
A repeating 42 amino acid motif that was originally identified in
armadillo
(Riggleman et al., 1989) has also been found in several other proteins, including &bgr;-catenin and plakoglobin, with a variety of functions (Peifer et al, 1994). These include the APC gene product, a tumor suppressor protein (Kinzler et al., 1991), p120, a pp60
STC
substrate (Reynolds et al., 1992), smgGDS, an exchange factor for ras-related G proteins (Kikuchi et al., 1992), a suppressor of RNA polymerase I mutations in yeast (Yano et al., 1992; 1994) and band 6 protein, a major desmosomal constituent (Hatzfeld et al., 1994). The function of the repeats in these arm proteins is unknown. Interestingly, the APC gene product associates with &bgr;-catenin (Rubinfeld et al., 1993; Su et al., 1993), supporting an important role for catenins in intracellular processes that regulate cell growth. Furthermore, this illustrates that cadherins are not exclusive cellular partners of catenins, raising the possibility of other interactions among catenins, cadherins and arm proteins, important in a variety of biological processes.
p120 was initially identified as one of several substrates of the tyrosine kinase pp60
src
(Reynolds et al., 1989; Kanner et al., 1990). It is membrane-associated and can be myristoylated, but does not appear to be glycosylated (Kanner et al., 1991). Mutational analysis suggested that tyrosine phosphorylation of p120 is necessary for of pp60
STC
-mediated cellular transformation (Linder and Burr, 1998/ Reynolds et al., 1989). Tyrosine phosphorylation of p120 was also observed in response to epidermal growth factor, platelet-derived growth factor, colony-stimulating factor 1 and in polyoma virus middle T antigen-transformed cells (Dowing and Reynolds, 1991; Kanner et al., 1991), but the exact role of p120 in cellular physiology and pathology was not clear.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a protein having a molecular weight of about 100 kDa which is associated in vivo with the cadherin/catenin complex of epithelial or endothelial cells (e.g. by binding). This protein is referred to herein as “p100”. p100 is immunologically related to p120 by virtue of cross-reactivity of antibody to p120 with p100.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Partial amino acid sequence data of human p100 is given in
FIG. 9
(SEQ ID NOS.: 1 through 5) where it is compared with sequence data already available for mouse p120 (SEQ ID NOS.: 1 and 6 through 9). The present invention covers the specific protein of molecular weight of about 100 kDa described in the present examples and other proteins having substantial amino-acid homology therewith (excluding p120, and optionally also excluding proteins having greater amino acid homology with p120 than with p100). Human p100 is particularly preferred.
The term “substantial amino acid homology” is used herein to cover proteins having at least 50%, preferably more than 90% or more than 95% amino acid homology with another protein. The present invention also covers fragments of these proteins or of p100 itself. Preferably these fragments are at least twenty amino acids long, more preferably they are at least fifty or at least one hundred amino acids long. These proteins or fragments may be partially or totally tyrosine phosphorylated. They may be provided in glycosylated or non-glycosylated form. Desirably they are provided in substantially pure form. One definition of “substantially pure form” is a form which is substantially free of other proteins.
The present invention also includes nucleic acid sequences (preferably DNA sequences) coding for the above mentioned proteins or fragments. These sequences may be in isolated form. They may be incorporated as part of a recombinant nucleic acid molecule e.g. as part of a vector. The vector may be incorporated into a host cell and used for expression of p100.
Nucleic acid sequences complementary to the aforesaid sequences may be useful in antisense studies to alter the expression of gene products. Such sequences are therefore also within the scope of the present invention.
In view of the data provided herein, it is believed that the protein p100 may be involved in the regulation of cell tight junction permeability. Phosphorylation of one or more tyrosine residues of p100 may be involved in increasing tight junctions permeability and dephosphorylation of one or more tyrosine residues may be involved in decreasing tight junction permeability. The above comments also apply in respect of p120, which the present inventors have shown to be immunologically closely related to p100.
p100 and p120 are therefore useful for studying cell-cell adhesion generally and tight junction permeability in particular. They may therefore be used, for example, to investigate the effect of tyrosine kinase and/or of tyrosine phosphatase on tight junction permeability. Variants of these proteins may be prepared in order to investigate which regions of p100 or p120 are important in regulating tight junction permeability.
Our data indicate that p120 and p100 associate with the cadherin/catenin complex. Furthermore, since p120 and p100 are substrates for tyrosine kinases, it follows that p120 and p100 proteins per se and the phosphorylation of these proteins may influence cellular functions directly, and indirectly, regulated by cadherins and catenins. Such an influence could be mediated by the physical association between p120, p100 and the cadherin/catenin complex, in which case modulation could be achieved by enhancing or blocking the expression of p120 and p100. Disruption of the association could also be achieved, for example, by small molecule mimetics of the site of binding. As indicated above, the function of p120 and p100
Eisai Co. Ltd.
Harris Alana M.
Huff Sheela
Sterne Kessler Goldstein & Fox P.L.L.C.
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