Syndecan-4 binding protein (S4BP) and uses thereof

Details Syndecan-4 binding protein (S4BP) and uses thereof Syndecan-4 binding protein (S4BP) and uses thereof

2000-03-15

2003-05-20

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C530S326000, C530S350000

Reexamination Certificate

active

06566489

ABSTRACT:

BACKGROUND OF THE INVENTION
Cell morphology, migration, growth and differentiation result from both adhesion dependent and growth factor receptor mediated signaling events. Adhesion dependent signaling events link the extracellular matrix with the intracellular cytoskeleton both structurally and biochemically through the formation of macromolecular complexes at sites of cell matrix interactions. In cell culture, these macromolecular complexes are referred to as focal adhesions. Burridge and Chrzanowska-Wodnicka (1996)
Ann. Rev. Cell. Dev. Biol
. 12:463-518. The ability of adhesion receptors to mediate both signaling and structural events occurs, in part, through the association of their cytoplasmic domains with cytoskeletal components. The cytoskeletal components in turn provide structural and adapter functions for the assembly of the intracellular signaling complexes. Cytoskeletal proteins that provide structural and/or adapter functions include actin, alpha-actinin, paxillin, talin, tensin and vinculin. Associated signaling proteins include tyrosine kinases (focal adhesion kinase, src, csk and fyn), the serine-threonine kinase families of PKC and MAPK and members of the RAS family of small GTP binding proteins. Clark and Brugge (1996)
Science
268:233-239; Burridge and Chrzanowska-Wodnicka (1996)
Ann. Rev. Cell. Dev. Biol
. 12:463-518.
Members of the syndecan family of cell surface heparan sulfate proteoglycans (syndecan-1 through 4) have been implicated in mediating cell adhesion and morphology. Bernfield et al. (1991)
Ann. Rev. Cell Biol
. 8:365-393; Carey (1997)
Biochem. J
. 327:1-16; Liu et al. (1998)
J. Biol. Chem
. 273:22825-22832; Woods and Couchman (1998)
Trends Cell Biol
. 8:189-192. All four syndecan family members contain a high degree of sequence conservation of their cytoplasmic domains, which, based on homology can be divided into conserved membrane proximal, variable central, and conserved C-terminal subdomains. Several cellular components which associate, either directly or indirectly, with the cytoplasmic domain of syndecan family members have been identified. These include: PKC&agr; and phosphatidyl inositol 4,5-biphosphate (PIP2) which interact directly with the variable central region of the cytoplasmic domain of syndecan-4 (Oh et al. (1997)
J. Biol. Chem
. 272:11805-11811; Oh et al. (1997)
J. Biol. Chem
. 272:8133-8136; Oh et al. (1998)
J. Biol. Chem
. 273:10624-10629); the PDZ containing proteins syntenin (Grootjans et al. (1997)
Prot. Natl. Acad. Sci. USA
94:13683-13688) and CASK/LIN-2 (Cohen et al. (1998)
J. Cell Bio
. 142:129-138; Hsueh et al. (1998)
J. Biol. Chem
. 142:139-151) which interact with the cytoplasmic domains of all syndecan family members through the highly conserved C-terminal EFYA sequence, and a Src-cortactin complex which associates with the membrane proximal domain of syndecan-3. Kinnunen et al. (1998)
J. Biol. Chem
. 273:10702-10708.
Syndecan-4 is observed in focal contacts (Woods and Couchmana (1994)
Mol. Biol. Cell
5:183-192; Baciu and Goetinck (1995)
Mol. Biol. Cell
1:1503-1513) and associates with PKC&agr; (Oh et al. (1997)
J. Biol. Chem
. 272:11805-11811; Oh et al. (1997)
J. Biol. Chem
. 272:8133-8136).
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of a gene which encodes a novel cellular protein referred to herein as syndecan-4 binding protein (S4BP). The S4BP protein was found to interact with the cytoplasmic domain of syndecan-4 but not other members of the syndecan family. When S4BP is overexpressed, it mediates cell spreading and actin cytoskeleton organization. S4BP plays a role in linking syndecan-4 to the focal adhesion complex.
Accordingly, in one aspect, the invention features an isolated nucleic acid molecule (e.g., cDNAs) comprising a nucleotide sequence encoding an S4BP protein or a biologically active portion thereof, as well as, nucleic acid fragments suitable as primers or hybridization probes for the detection of S4BP-encoding nucleic acid (e.g., mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 1, or the coding region (SEQ ID NO:3), or a complement of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention includes a nucleotide sequence which hybridizes, preferably under stringent conditions, to or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO:1, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2. The preferred S4BP nucleic acid encodes a protein which also preferably possesses at least one of the S4BP activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2, e.g., sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains an S4BP biological activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to play a role in cell matrix interactions. In one embodiment, the protein encoded by the nucleic acid molecule has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86, 88, 90%, and most preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 (e.g., the entire amino acid sequence of SEQ ID NO:2). In another preferred embodiment, the protein is a full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by the open reading frame shown in SEQ ID NO:3). In another embodiment, the protein is a mammalian protein, e.g., a human protein, which is substantially homologous to the amino acid sequence of SEQ ID NO:2, or a portion thereof.
In yet another embodiment, the isolated nucleic acid molecule encodes a portion of an S4BP protein which includes a sequence encoding a SH3 domain binding site motif. Preferably, the SH3 domain binding site motif encoded by the nucleic acid molecule has at least about 80% or more sequence identity to the SH3 domain binding site motif (i.e., about amino acid residues 24 to 27) of SEQ ID NO:2. Preferably, the SH3 domain binding site motif has a consensus sequence Pro-Xaa-Pro-Pro, where Xaa is any amino acid.
In another preferred embodiment, the isolated nucleic acid molecule encodes an S4BP protein or portion thereof which has at least about 55% or more sequence identity to SEQ ID NO:2 and has one or more of the following activities involved with cell matrix interactions: 1) it interacts, directly or indirectly, with syndecan-4; 2) it interacts, directly or indirectly, with paxillin; 3) it interacts, directly or indirectly, with intracellular signaling proteins (e.g., GTP binding protein, focal adhesion kinase, serine-threonine kinase); 4) it modulates cytoskeletal organization, e.g., it modulates the interaction of a matrix receptor (e.g., syndecan-4) and intracellular proteins associated with cytoskeleton (e.g., actin, vinculin); 5) it interacts, directly or indirectly, with PKC&agr;; 6) it modulates actin stress fiber formation and/or organization; 7) it plays a role in an adhesion formation signaling pathway; 8) it modulates cell attachment; and/or 9) it modulates cell spreading.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic ac

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