Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
PLoS One. 2013 Jul 23;8(7):e68873. doi: 10.1371/journal.pone.0068873. Print 2013.
Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca(2+)-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca(2+) in the medium or when intracellular Ca(2+) was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during sperm capacitation.
射出的精子在受精前必须经历一系列称为获能的生化修饰。蛋白激酶 A（PKA）介导精子获能，但其调节机制尚不完全清楚。精子含有几种 A 激酶锚定蛋白（AKAPs），它们是锚定 PKA 的支架蛋白。在这项研究中，我们表明 AKAP3 在牛精子在获能条件下孵育时会降解。不同公牛精子的降解速率不同，与获能能力相关。AKAP3 的降解被蛋白酶体抑制剂 MG-132 显著抑制，表明 AKAP3 降解是通过蛋白酶体机制发生的。用 Ca（2+）离子载体处理诱导 AKAP3 的进一步降解；然而，当培养基中没有 Ca（2+）或细胞内 Ca（2+）螯合时，这种效应被发现增强。使用 NH4Cl 使细胞内空间碱化，或用含有 AKAPs 的 PKA 结合域的肽 Ht31 处理精子时，AKAP3 的降解速率显著增强。当使用 H89 抑制 PKA 活性或使用 8Br-cAMP 激活 PKA 活性时，AKAP3 的降解速率也会增加。这种明显的矛盾可以通过假设 PKA 与 AKAP3 的结合来保护 AKAP3 免受降解来解释。我们得出结论，AKAP3 的降解是由精子获能过程中的细胞内碱化和 PKARII 锚定调节的。
