Expression and gene knockdown of zebrafish Ca²⁺/calmodulin-dependent protein kinase I未-LL

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• 作者：Yukako Senga ; Keiko Yoshioka ; Isamu Kameshita ; Noriyuki Sueyoshi
• 关键词：CaMKI未 ; Embryogenesis ; Gene knockdown ; Protein phosphorylation ; Zebrafish
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：December, 2013
• 年：2013
• 卷：540
• 期：1-2
• 页码：41-52
• 全文大小：2635 K

文摘

Ca²⁺/calmodulin-dependent protein kinase I未 (CaMKI未) is expressed ubiquitously, but little is known about its physiological functions. Recently, we cloned and characterized two splice variants of zebrafish (<em>Danio rerioem>) CaMKI未 (CaMKI未-S/L). In the present study we cloned a new CaMKI未 isoform, CaMKI未-LL, encoded by a different gene from CaMKI未-S/L. While the catalytic domain of CaMKI未-LL showed 86% identity that of CaMKI未-S/L, it had a unique C-terminal sequence. To clarify the functional role of CaMKI未-LL, we investigated the biological significance of this new isoform during zebrafish embryogenesis. Although CaMKI未-LL exhibited essentially the same catalytic properties and substrate specificities as the other CaMKI未 isoforms, it showed different temporal and spatial expression. During zebrafish embryogenesis, RT-PCR analysis detected CaMKI未-LL expression after 48 h post-fertilization. Western blotting in adult zebrafish demonstrated that CaMKI未-LL is expressed in the brain, the eye, and, abundantly, in fins. Knockdown of CaMKI未-LL expression using morpholino-based antisense oligonucleotides resulted in an increase in abnormal embryos with small fins and underdeveloped cartilage. These phenotypes were rescued by co-injection with recombinant CaMKI未-LL. These results clearly indicated that CaMKI未-LL plays an important role in the generation of cartilage and fins during zebrafish embryogenesis.