Binding and inactivation of the germ cell-specific protein phosphatase PP1γ2 by sds22 during epididymal sperm maturation
Binding and inactivation of the germ cell-specific protein phosphatase PP1γ2 by sds22 during epididymal sperm maturation
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DOI：
10.1095/biolreprod.103.018739
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发表时间：
2003-11-01
影响因子：
3.6
通讯作者：
Vijayaraghavan, S
中科院分区：
文献类型：
--
作者：
Mishra, S;Somanath, PR;Vijayaraghavan, S
睾丸和精子特异性蛋白磷酸酶PP1gamma2是调节精子功能的关键酶。它的活性在精子在附睾成熟过程中降低。抑制PP1gamma2导致运动启动和刺激。我们的实验室专注于确定在附睾精子运动起始过程中PP1gamma2活性下降的机制。此前，我们利用免疫亲和层析技术发现，酵母sds22的哺乳动物同源物与运动尾端精子中的PP1gamma2结合（Huang Z, et al.）。生物再生2002；67:1936 - 1942)。本研究的目的是确定：1)PP1gamma2-sds22结合的化学计量学和2)固定附睾头精子中PP1gamma2是否与sds22结合。用柱层析法纯化了精子尾部和头部提取物中的酶。在deae -纤维素柱的流过部分中发现了尾端和头端精子的免疫反应性PP1gamma2和sds22。然而，来自尾端精子的PP1gamma2是不活跃的，而在头形精子中它是活跃的。deae -纤维素流动组分随后通过SP-sepharose柱。尾端精子sds22和PP1gamma2在梯度分数中析出。相比之下，顶精子sds22和PP1gamma2分别在流动和梯度部分分离。通过Superose 6柱进一步纯化表明，来自尾端精子的PP1gamma2-sds22复合物的大小为88 kDa。精子sds22和PP1gamma2分别以60 kDa和39 kDa洗脱。这些纯化组分的SDS-PAGE显示，在尾端精子中，88 kDa的物种由sds22 （43 kDa）和PP1gamma2 （39 kDa）组成，表明这两种蛋白质之间的复合物为1:1。PP1gamma2与sds22的结合是无活性的。以60 kda洗脱的头精子sds22被发现与17 kda蛋白相关（p17）。这表明sds22与p17的解离或sds22的一些其他翻译后修饰是其结合和失活PP1gamma2所必需的。目前正在进行研究，以确定在附睾精子成熟过程中sds22与PP1gamma2结合的机制。
Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1gamma2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936-1942). The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1gamma2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.