Cellular localization of CoPK12, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea, is regulated by N-myristoylation and limited proteolysis.
Cellular localization of CoPK12, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea, is regulated by N-myristoylation and limited proteolysis.
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CoPK12 是蘑菇灰霉中的一种 Ca2/钙调蛋白依赖性蛋白激酶，其细胞定位受 N-肉豆蔻酰化和有限的蛋白水解调节。
DOI：
10.1093/jb/mvu018
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发表时间：
2014
影响因子：
2.7
通讯作者：
Kameshita I
中科院分区：
文献类型：
--
作者：
Kaneko K;Tabuchi M;Sueyoshi N;Ishida A;Utsumi T;Kameshita I
多功能Ca2+/钙调素依赖性蛋白激酶（CaMKs）在哺乳动物中已经被广泛研究，而真菌CaMKs仍然在很大程度上未被表征。我们之前获得了CaMK同源物inoprinopsis cinerea，命名为CoPK12，并与哺乳动物CaMK进行比较，揭示了其独特的催化性能。为了进一步阐明CoPK12的调控机制，本研究对CoPK12的翻译后修饰和亚细胞定位进行了研究。在cinerea中，CoPK12全长（65 kDa）在膜组分中分离，而CoPK12的催化活性片段（46 kDa）通过差速离心在可溶性组分中检测。在酵母细胞中，表达的CoPK12-GFP定位在细胞质和液泡膜上，通过绿色荧光可见。体外肉豆蔻酰化实验显示，CoPK12在n端Gly-2处被n -肉豆蔻酰化。此外，钙调素不仅可以结合到cam结合域，还可以结合到CoPK12的n端肉豆蔻酰基部分。综上所述，这些结果表明，CoPK12的细胞定位和功能受蛋白n -肉豆蔻酰化和有限蛋白水解的调节。
Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog inCoprinopsis cinerea, designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. InC. cinerea, full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells.In vitroN-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis.