Aspenström Pontus, Richnau Ninna, Johansson Ann-Sofi
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala University, Box 595, S-751 24 Uppsala, Sweden.
Exp Cell Res. 2006 Jul 15;312(12):2180-94. doi: 10.1016/j.yexcr.2006.03.013. Epub 2006 Apr 21.
Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.
通过酵母双杂交系统以及在免疫共沉淀实验中检测潜在的CIP4结合蛋白，确定了Cdc42效应蛋白CIP4的结合伙伴。其中一种CIP4结合蛋白DAAM1得到了更详细的表征。DAAM1是哺乳动物中与透明质酸相关的formin家族中广泛表达的成员，该家族包括mDia1和mDia2等蛋白质。DAAM1在体内被证明能与CIP4的SH3结构域结合。异位表达的DAAM1以点状模式定位于转染细胞的背侧，并且该蛋白在微管组织中心附近积累。此外，DAAM1的异位表达诱导了细胞形态的显著改变，表现为转染细胞变圆、形成分支状突起以及应力纤维减少。免疫共沉淀实验表明，DAAM1以GTP依赖的方式与RhoA和Cdc42结合。此外，发现DAAM1在分支状突起的形成中与非受体酪氨酸激酶Src相互作用并协同作用。综上所述，我们的数据表明，DAAM1在协调肌动蛋白丝系统动力学的信号通路调节中与Rho GTP酶、CIP4和Src进行通讯。
