Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility.
Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility.
复制标题
cAMP 依赖性蛋白激酶对轴激肽的磷酸化足以激活精子鞭毛运动。
DOI：
10.1083/jcb.103.2.649
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Means,AR
中科院分区：
文献类型：
--
作者：
Tash,JS;Hidaka,H;Means,AR
使用cAMP依赖的蛋白激酶选择性抑制剂N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide(H-8)，研究了犬精子鞭毛运动启动过程中对cAMP依赖的磷蛋白的需求。H-8以剂量依赖的方式抑制活体精子的活力，并使精子复活。在相同的条件下，对重新激活的运动的一半最大抑制(32微米)与对cAMP依赖的蛋白激酶的纯催化亚单位的抑制(50微米)平行。H-8抑制了整个模型和分离的诺尼特P-40(NP-40)提取液中的蛋白质磷酸化。Axokinin是一种耐热的NP-40可溶性蛋白，其磷酸化是鞭毛重新激活所必需的，它占cAMP刺激后NP-40提取物中从头合成磷酸的97%。500微米H-8对Axokinin的磷酸化抑制达87%。当精子在5 mM H-8和NP-40提取物的存在下重新激活时，cAMP或cAMP依赖的蛋白激酶活性都不需要完全重新激活鞭毛。如果H-8使精子完全静止，那么在H-8继续存在的情况下，所得到的模型仍然是静止的，除非加入预磷酸化的Axokinin。这些结果表明，磷酸化的Axokinin不仅是鞭毛重新激活所必需的，而且也是足够的。
Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.