Identification and kinetic analysis of the interaction between Nck-2 and DOCK180.
Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain. We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that ... the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Carrier Proteins, Cell Line, Humans, Kidney, Mutagenesis, Site-Directed, Protein Binding, Protein Structure, Tertiary, Proteins, Recombinant Fusion Proteins, Signal Transduction, Surface Plasmon Resonance, Two-Hybrid System Techniques, rac GTP-Binding Proteins, src Homology Domains
Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Carrier Proteins, Cell Line, Humans, Kidney, Mutagenesis, Site-Directed, Protein Binding, Protein Structure, Tertiary, Proteins, Recombinant Fusion Proteins, Signal Transduction, Surface Plasmon Resonance, Two-Hybrid System Techniques, rac GTP-Binding Proteins, src Homology Domains
FEBS Lett.
Date: Mar. 02, 2001
PubMed ID: 11240126
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