Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation
Modulation of erythrocyte membrane mechanical function by protein 4.1 phosphorylation
复制标题
DOI：
10.1074/jbc.m410650200
复制
发表时间：
2005-03-04
影响因子：
4.8
通讯作者：
Mohandas, N
中科院分区：
文献类型：
--
作者：
Manno, S;Takakuwa, Y;Mohandas, N
红细胞膜的机械功能受血影蛋白为基础的膜骨架的调节，膜骨架由α-和β-血影蛋白、肌动蛋白、蛋白4.1R(4.1R)和内收蛋白组成。这些蛋白质的翻译后修饰被认为可以调节膜的机械功能。事实上，酪蛋白激酶I的β-血影蛋白磷酸化已被证明降低了膜的机械稳定性。然而，蛋白激酶C(PKC)是一种丝氨酸/苏氨酸激酶，对骨骼蛋白的磷酸化作用尚不清楚。在本研究中，我们探讨了PKC对4.1R和内收蛋白磷酸化的功能影响。我们确定4.1R中的Ser-312为PKC的磷酸化位点。使用针对4.1R和内收蛋白的磷酸肽的抗体，我们证明了PKC对内收蛋白和4.1R磷酸化的时间过程有显著的差异。尽管通过佛波醇12-肉豆蔻酸酯13-醋酸酯激活膜结合的非典型PKC，内收蛋白被迅速磷酸化，但4.1R的磷酸化有明显的延迟，这是因为传统的PKC从胞浆重新募集到膜上。结合膜机械稳定性的测量，这种4.1R和内收蛋白磷酸化的不同时间过程使我们能够证明，尽管PKC对内收蛋白的磷酸化对膜机械稳定性的影响很小，但额外的4.1R磷酸化会导致膜机械稳定性的显著下降。我们进一步表明，4.1R被PKC磷酸化导致其与血影蛋白和肌动蛋白形成三元复合体的能力降低，并导致血糖蛋白C从膜骨架上解离。这些发现使我们能够确定4.1R磷酸化在红细胞膜特性的动态调节中的调节作用。
Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha-and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.