title: Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G.
abstract: p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.
authors.affiliation: Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, USA.
authors.forename: K H
authors.initials: KH
authors.lastname: Kirsch
authors.forename: M M
authors.initials: MM
authors.lastname: Georgescu
authors.forename: H
authors.initials: H
authors.lastname: Hanafusa
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D050719:Crk-Associated Substrate Protein; D004252:DNA Mutational Analysis; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D018909:src Homology Domains / Q000502:physiology
chemical_list: C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D050717:Retinoblastoma-Like Protein p130; D011392:Proline