Tu Y, Kucik D F, Wu C
Department of Pathology, University of Pittsburgh, PA 15261, USA.
FEBS Lett. 2001 Mar 2;491(3):193-9. doi: 10.1016/s0014-5793(01)02195-0.
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.
Nck-2是一种新发现的衔接蛋白，由三个N端SH3结构域和一个C端SH2结构域组成。我们在酵母双杂交筛选中鉴定出DOCK180，一种与膜 ruffling和迁移调节有关的信号蛋白，作为Nck-2的结合蛋白。表面等离子体共振分析表明，第二个和第三个SH3结构域与DOCK180的C端区域相互作用。然而，由单个SH3结构域介导的相互作用比全长Nck-2的相互作用弱得多。此外，使第二个或第三个SH3结构域失活的点突变显著降低了Nck-2与DOCK180的相互作用，表明两个SH3结构域都有助于与DOCK180结合。一个主要的Nck-2结合位点，主要由第三个SH3结构域识别，已定位到DOCK180的1819-1836位残基。另外两个虽然弱得多的Nck-2 SH3结合位点分别位于DOCK180的1793-1810位残基和1835-1852位残基。与突变研究一致，表面等离子体共振的动力学分析表明，两个平衡解离常数分别为4.15±1.9x10(-7) M和3.24±1.9x10(-9) M的结合事件介导了GST-Nck-2与含有DOCK180 C端区域的GST融合蛋白的结合。这些研究确定了Nck-2与DOCK180之间的一种新的相互作用。此外，它们还对由多个SH3结构域介导的蛋白质复合物形成进行了详细分析，揭示了串联SH3结构域显著增强了每个单个SH3结构域介导的弱相互作用。
