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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Tissue-specific Pka Inhibition Using A Chemical Genetic Approach And Its Application To Studieson Sperm Capacitation
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Tissue-specific Pka Inhibition Using A Chemical Genetic Approach And Its Application To Studieson Sperm Capacitation

机译：化学遗传方法抑制组织特异性Pka及其在精子获能研究中的应用

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Studies on cAMP signaling and protein kinase A (PKA) function in vivo are limited by the lack of highly specific inhibitors that can be used in primary cell culture and whole animals. Previously we reported that a mutation in the ATP binding pocket of a catalytic subunit (Cα) of PKA confers sensitivity to the pyrazolo[3,4-d]pyri-midine inhibitor, 1NM-PP1. We have now engineered the mouse Pkraca gene such that after Cre-mediated recombination in vivo, the CαM120A mutant protein is expressed and the wild-type Cα is turned off. We demonstrate the utility of this approach by examining the requirement for PKA activity during capacitation of sperm from mice that express CαM120A mutant protein. For CαM120A sperm, 10 μM of 1NM-PP1 prevented PKA-dependent phosphory-lation and the activation of motility that are both rapidly ( < 90 s) evoked by the HCO_3~- anion. A continuous (90 min) inhibition with 10 μM of 1NM-PP1 prevented the protein tyrosine phosphorylation of late-stage capacitation. Delayed application of 1NM-PP1 demonstrated that PKA activity was required for at least the initial 30 min of capacitation to produce subsequent protein tyrosine phosphorylation. Acute application of 1NM-PP1 rapidly slowed the accelerated beat of activated motility but did not affect the established waveform asymmetry of hyperactivated sperm. Our results demonstrate that PKA in CαM120A mutant sperm is rapidly and reversibly inhibited by 1NM-PP1 and that this blockade has selective and time-dependent effects on multiple aspects of capacitation. The conditional CαM120A-expressing mouse lines will be valuable tools for studying PKA function in vivo.
机译：由于缺乏可用于原代细胞培养和整个动物的高特异性抑制剂，因此对体内cAMP信号传导和蛋白激酶A（PKA）功能的研究受到限制。以前我们报道过，PKA催化亚基（Cα）的ATP结合口袋中的突变赋予对吡唑并[3,4-d]吡啶-idine抑制剂1NM-PP1敏感。现在，我们已经对小鼠Pkraca基因进行了工程改造，以便在体内Cre介导的重组后，表达CαM120A突变蛋白，并关闭野生型Cα。我们通过检查表达CαM120A突变蛋白的小鼠的精子获能过程中PKA活性的要求，证明了该方法的实用性。对于CαM120A精子，10μM的1NM-PP1阻止了PKA依赖性的磷酸化作用和运动性的激活，而HCO_3〜-阴离子可迅速激活这些运动（<90 s）。用10μM的1NM-PP1进行连续（90分钟）抑制可防止后期捕获的蛋白质酪氨酸磷酸化。 1NM-PP1的延迟施用证明，至少在最初的30分钟捕获过程中需要PKA活性才能产生随后的蛋白酪氨酸磷酸化。急性应用1NM-PP1迅速减慢了活化运动的加速搏动，但并未影响已建立的过度活化精子的波形不对称性。我们的结果表明，1NM-PP1可快速和可逆地抑制CαM120A突变精子中的PKA，并且这种封锁对获能的多个方面具有选择性和时间依赖性。有条件表达CαM120A的小鼠系将是研究体内PKA功能的有价值的工具。

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