Huang Zaohua, Khatra Balwant, Bollen Mathieu, Carr Daniel W, Vijayaraghavan Srinivasan
Biological Sciences Department, Kent State University, Ohio 44242, USA.
Biol Reprod. 2002 Dec;67(6):1936-42. doi: 10.1095/biolreprod.102.004093.
Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa. Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates. We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP1gamma2. A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.
丝氨酸/苏氨酸磷酸酶PP1γ2是精子中一种睾丸特异性的蛋白磷酸酶同工型。这种酶似乎在运动启动和刺激中起关键作用。与有运动能力的精子相比，PP1γ2在无运动能力的精子中的催化活性更高。抑制PP1γ2的活性会导致运动启动和运动刺激。一般来说，蛋白磷酸酶是由其结合蛋白调节的。本文的目的是通过首先鉴定其调节蛋白来了解PP1γ2的调节机制。我们之前已经表明，一部分牛精子PP1γ2存在于精子超声裂解物的胞质部分。我们通过免疫亲和色谱法从可溶性牛精子提取物中纯化了PP1γ2。纯化酶的凝胶电泳显示它与一种大小为43 M(r) x 10(-3) 的蛋白质复合。微量测序表明这种蛋白质是sds22的哺乳动物同源物，sds22是一种酵母PP1结合蛋白。磷酸酶活性测量表明，与sds22复合的PP1γ2没有催化活性。该复合物不能通过有限的蛋白水解作用被激活。该复合物无法与微囊藻毒素琼脂糖结合。这表明sds22可能会阻断PP1γ2中的微囊藻毒素结合位点。精子提取物中一部分可能未与sds22复合的PP1γ2具有催化活性。荧光免疫细胞化学被用于确定PP1γ2和sds22在精子内的定位。这两种蛋白质都存在于尾部。它们也存在于头部的不同位置。我们的数据表明，PP1γ2与sds22的结合会抑制其催化活性。调节sds22与PP1γ2结合的机制可能对于理解精子功能发育和调节的生化基础很重要。
