Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo
Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo
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DOI：
10.1126/scisignal.aao7232
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发表时间：
2019-03-26
影响因子：
7.3
通讯作者：
Fujita, Jun
中科院分区：
文献类型：
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作者：
Itoh, Katsuhiko;Kondoh, Gen;Fujita, Jun
与其他组织一样，组蛋白的翻译后修饰在精子发生中至关重要;然而，在精子发生过程中，组蛋白被鱼精蛋白取代，鱼精蛋白对精子细胞中DNA的紧密包装至关重要。鱼精蛋白也通过磷酸化和去磷酸化进行后修饰，这促使我们研究其调节的潜在机制和生物学后果。在筛选的基础上，牵连热休克蛋白Hspa 4l在精子发生，我们产生的小鼠缺乏Hspa 4l（Hspa 4l-null小鼠），这表明男性不育和畸形的精子头部。这些表型与Ppp 1cc缺陷小鼠的表型相似，我们发现，在Hspa 4l缺失的精子中，睾丸和精子特异性Ppp 1cc磷酸酶（Ppp 1cc 2）同工型的染色质结合部分的量显著低于野生型小鼠。我们进一步表明，Ppp 1cc 2是伴侣Hsc 70和Hsp 70的底物，并且Hspa 4l增强了Ppp 1cc 2从这些复合物中的释放，使得释放的Ppp 1cc 2能够定位于染色质。下拉和体外磷酸酶测定表明Ppp 1cc 2可使鱼精蛋白2在丝氨酸56（Prm 2 Ser（56））处脱磷酸化。为了证实Prm 2 Ser（56）去磷酸化的生物学重要性，我们将Prm 2中的Ser（56）突变为丙氨酸（Prm 2 S56 A）。将该突变引入Hspa 4l-敲除小鼠（Hspa 4l（-/-）; Prm（2S 56 A/S56 A））中恢复了精子头部的畸形和Hspa 4l（-/-）小鼠的不育。这些结果揭示了Prm 2去磷酸化对精子成熟的作用机制和生物学意义。
The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis-and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser(56)) by Ppp1cc2. To confirm the biological importance of Prm2 Ser(56) dephosphorylation, we mutated Ser(56) to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l(-/-); Prm(2S56A/S56A)) restored the malformation of sperm heads and the infertility of Hspa4l(-/-) mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.