Hubberstey A, Yu G, Loewith R, Lakusta C, Young D
Department of Medical Biochemistry, University of Calgary Health Science Centre, Alberta, Canada.
J Cell Biochem. 1996 Jun 1;61(3):459-66. doi: 10.1002/(SICI)1097-4644(19960601)61:3%3C459::AID-JCB13%3E3.0.CO;2-E.
We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.
我们之前鉴定出了人源CAP，它是酵母腺苷酸环化酶相关蛋白的同源物。先前的研究表明，CAP的N端和C端结构域具有不同的功能。我们探索了人源CAP与各种蛋白质的相互作用。首先，通过进行酵母双杂交筛选，我们鉴定出了几种蛋白质的肽段，这些肽段与人源CAP的C端和/或N端结构域相互作用。这些肽段包括来自CAP和BAT3（一种功能未知的蛋白质）的区域。我们进一步表明，与这些肽段融合的MBP在体外可与融合到GST的CAP的N端或C端结构域结合。我们的观察结果表明，CAP在N端和C端结构域中都含有能够相互或自身相互作用的区域。此外，我们发现，带有myc表位标签的CAP可与来自酵母或哺乳动物细胞提取物的带有HA表位标签的CAP共同免疫沉淀。类似的结果表明，人源CAP也可与人源CAP2相互作用。我们还表明，通过酵母双杂交试验以及从酵母或哺乳动物细胞提取物中对表位标签化的CAP进行共同免疫沉淀，人源CAP与肌动蛋白相互作用。这种相互作用需要CAP的C端结构域，但不需要N端结构域。因此，CAP似乎能够在体内与其他CAP分子、CAP2和肌动蛋白相互作用。我们还表明，肌动蛋白可与来自哺乳动物细胞提取物的HA-CAP2共同免疫沉淀。
