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.
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Department of Pathology, University of Pittsburgh, PA 15261, USA.
Kucik
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Wu
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques; D020744:rac GTP-Binding Proteins; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D002352:Carrier Proteins; C099818:DOCK1 protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020744:rac GTP-Binding Proteins