亚砷酸盐诱导HSP27的细胞内移位依赖于p38 MAPK介导的磷酸化

为研究HSP27的磷酸化与其细胞内定位之间的关系，利用定点突变和DNA重组技术构建EGFP融合的HSP27野生型和第82位丝氨酸突变体的真核表达载体并转染NIH 3T3细胞，观察两者在静息状态和亚砷酸盐刺激下的细胞内定位情况.利用p38 MAPK特异性抑制剂SB203580预处理细胞后，观察对HSP27磷酸化和细胞内定位的影响.结果发现，野生型HSP27受到NaAsO₂刺激后移位入核，而其突变体HSP27(S82A)不能入核.同时，SB203580的预处理使HSP27的磷酸化和NaAsO₂诱导的移位入核都被阻断.这些结果表明,p38介导的HSP27磷酸化在其细胞内定位中具有重要作用

Translocation of HSP27 Induced by Arsenite Depends on Its Phosphorylation Mediated by p38 MAPK

Small heat shock proteins, such as HSP27, play important roles in cellular response to environmental stresses. To further investigate the connections between the phosphorylation and intracellular localization of HSP27, we constructed an EGFP-tagged wild-type and a mutated HSP27, i.e., EGFP-HSP27 (WT) and EGFP-HSP27(S82A) based on pEGFP-C2 vectors by site-directed mutagenesis and DNA subcloning. When transfected into NIH 3T3 cells with and without arsenite-stimulated, the intracellular localizations of the expressed HSP27 forms were examined. The correlation of HSP27 phosphorylation and its intracellular localization were also studied in NIH 3T3 cells pretreated with the specific inhibitor of p38, SB203580. Our results showed that wild-type HSP27 translocated into the nucleus with NaAsO_2 stimulation, whereas this translocation was abolished for HSP27(S82A). Moreover, both protein phosphorylation and NaAsO_2-induced translocation of HSP27 were blocked by SB203580 pretreatment. These results suggest that p38 mitogen-activated protein kinase-dependent phosphorylation of HSP27 is required in its intracellular localization.