The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein
The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein
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DOI：
10.1128/mcb.25.9.3519-3534.2005
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发表时间：
2005-05-01
影响因子：
5.3
通讯作者：
Litchfield, DW
中科院分区：
文献类型：
--
作者：
Canton, DA;Olsten, MEK;Litchfield, DW
CKIP-1 是一种含有 pleckstrin 同源结构域的蛋白质，可与蛋白激酶 CK2 相互作用。为了阐明 CKIP-1 的功能，我们生成了具有四环素调节的 Flag-CKIP-1 表达的人骨肉瘤细胞系。 Flag-CKIP-1 表达导致细胞形态的明显变化。因此，我们通过免疫荧光、鬼笔环肽结合的定量测量和免疫印迹分析来检查肌动蛋白谱。这些研究表明 Flag-CKIP-1 表达导致 F-肌动蛋白染色和 P-肌动蛋白蛋白水平增加。为了阐明观察到的表型背后的机制，我们利用串联亲和纯化来分离 CKIP-1 相互作用蛋白。质谱分析确定了肌动蛋白加帽蛋白亚基 CP α 和 CP β 作为新型 CKIP-1 相互作用伙伴。通过免疫共沉淀和共定位证实了相互作用。此外，我们还证明CPα的Ser9在体外被蛋白激酶CK2磷酸化，CPα被磷酸化。在体内，用 CK2 特异性抑制剂治疗会导致 CP α 磷酸化减少。最后，我们证明 CKIP-1 和 CK2 抑制肌动蛋白丝倒刺末端肌动蛋白加帽蛋白的活性。总的来说，我们的结果与 CKIP-1 通过与肌动蛋白加帽蛋白的相互作用在肌动蛋白细胞骨架的调节中发挥作用是一致的。
CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of P-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CP alpha and CP beta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CP alpha is phosphorylated by protein kinase CK2 in vitro, that CP alpha is phosphorylated. in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CP alpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.