KAUST Global Collaborative Research Program, Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
PLoS One. 2013 Jul 29;8(7):e69510. doi: 10.1371/journal.pone.0069510. Print 2013.
The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.
p38 丝裂原活化蛋白激酶（p38MAPK）在藤壶幼虫附着中起着关键作用。为了研究幼虫附着过程中与 p38MAPK 相关的信号通路，我们试图鉴定 p38MAPK 的上游激酶。在藤壶中克隆并重组表达了三种 MKKs（MKK3、MKK4 和 MKK7）和三种 MAPKs（p38MAPK、ERK 和 JNK）。通过激酶测定，我们发现 MKK3 而不是 MKK4 或 MKK7 磷酸化 p38MAPK。此外，MKK3 活性特异性地针对 p38MAPK，因为它不磷酸化 ERK 或 JNK。为了进一步研究 MKK3 和 p38MAPK 在体内的功能关系，我们通过免疫染色研究了磷酸化 MKK3（pMKK3）和 MKK3 的定位。与 p38MAPK 和磷酸化 p38MAPK（pp38MAPK）的模式一致，pMKK3 和 MKK3 主要定位于刚毛幼虫的触角上。Western blot 分析显示，与无节幼虫和幼体阶段相比，pMKK3 水平（如 pp38MAPK 水平）在刚毛幼虫阶段升高。此外，pMKK3 水平在处理成年藤壶粗提取物后增加，表明 MKK3 可能介导成年藤壶提取物对 p38MAPK 途径的刺激作用。
