Kirsch K H, Georgescu M M, Hanafusa H
Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 1998 Oct 2;273(40):25673-9. doi: 10.1074/jbc.273.40.25673.
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.
p130（Cas）（Cas；Crk相关底物）属于一类新的对接分子家族。它在氨基末端区域含有一个Src同源（SH）3结构域，其后是一个包含SH2和SH3结构域结合基序的区域。为了进一步深入了解Cas信号传导，我们在双杂交筛选中使用Cas的SH3结构域，在人胎盘文库中寻找结合伙伴。该筛选证实了其与粘着斑激酶（FAK）结合的先前发现，但也鉴定出鸟嘌呤核苷酸交换因子C3G。我们发现Cas和C3G在体外和体内存在直接相互作用。对C3G缺失突变体的一系列分析揭示了一个富含脯氨酸的Cas结合位点（Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7），位于C3G中先前鉴定的Crk结合基序的氨基末端。诱变研究表明，配体结合位点内的Pro1、Lys3和Pro4对于高亲和力相互作用至关重要。这些结果，结合已知与Cas结合的蛋白质中富含脯氨酸的结合元件的序列比对，确定了被CasSH3结构域识别的共有序列XXPXKPX。Cas显示出对接分子的结构特征，可能有助于将C3G带到细胞内的特定区域。
