Chay Kee-Oh, Park Sung Sup, Mushinski J Frederic
Laboratory of Genetics, NCI, National Institutes of Health, Bethesda, Maryland 20852, USA.
J Biol Chem. 2002 Apr 26;277(17):14521-9. doi: 10.1074/jbc.M111639200. Epub 2002 Feb 1.
We have cloned the complete cDNA from mouse paxillin, a 68-kDa adapter protein found in focal adhesions. We found that paxillin was degraded by caspases in Ba/F3 cell apoptosis induced by withdrawal of interleukin-3 (IL-3), a survival factor for this cell, and by ionizing radiation. Also, paxillin was degraded in vitro by incubation with recombinant caspase-3. Western blot analyses of degradation products of overexpressed green fluorescence protein-tagged paxillin and site-specific mutants demonstrated that Asp-102 and Asp-301 were early caspase cleavage sites, and Asp-5, Asp-146, Asp-165, and Asp-222 were late cleavage sites. Overexpression of paxillin delayed apoptosis of Ba/F3 after IL-3 withdrawal. Furthermore, this anti-apoptotic effect of paxillin was augmented by a triple mutation in aspartic acids at caspase cleavage sites. These results suggest that paxillin plays a critical role in cell survival signaling and that the cleavage of paxillin by caspases might be an important event for focal adhesion disassembly during cell apoptosis, contributing to detachment, rounding, and death.
我们从小鼠桩蛋白中克隆出了完整的cDNA，桩蛋白是一种在粘着斑中发现的68 kDa衔接蛋白。我们发现，在白细胞介素-3（IL-3，该细胞的一种生存因子）撤除诱导的Ba/F3细胞凋亡以及电离辐射过程中，桩蛋白会被半胱天冬酶降解。此外，桩蛋白在体外与重组半胱天冬酶-3孵育时也会被降解。对过表达绿色荧光蛋白标记的桩蛋白及其位点特异性突变体的降解产物进行蛋白质印迹分析表明，天冬氨酸-102和天冬氨酸-301是半胱天冬酶的早期切割位点，而天冬氨酸-5、天冬氨酸-146、天冬氨酸-165和天冬氨酸-222是晚期切割位点。桩蛋白的过表达延迟了IL-3撤除后Ba/F3细胞的凋亡。此外，桩蛋白的这种抗凋亡作用在半胱天冬酶切割位点的天冬氨酸三联突变后增强。这些结果表明，桩蛋白在细胞生存信号传导中起关键作用，并且半胱天冬酶对桩蛋白的切割可能是细胞凋亡过程中粘着斑解体的一个重要事件，有助于细胞脱离、变圆和死亡。
