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IDENTIFICATION OF PHOSPHOPROTEINS INVOLVED IN SPERM MATURATION AND FERTILITY.

Goswami, Suranjana
http://rave.ohiolink.edu/etdc/view?acc_num=kent1532952768427828

Abstract Details

2018, PHD, Kent State University, College of Arts and Sciences / Department of Biological Sciences.
Cyclic-AMP plays an important role in sperm motility and activation. The actions of cAMP in sperm involve Protein Kinase A mediated protein phosphorylation. The crucial roles of sperm cAMP and PKA have also been demonstrated using genetic approaches. Understanding how a cAMP act to sustain sperm motility and fertility requires identification of the protein substrates of PKA. Identification of substrates of specific protein kinases and determination of changes in phosphorylation of these substrates during sperm motility initiation and fertilization will be a major advance in understanding signaling mechanisms underlying male gamete function. In our study we are using a novel chemical-genetic approach to identify proteins which are phosphorylated by PKA during sperm motility. In this approach the structurally conserved ATP-binding pocket in Protein Kinase A is genetically modified (analog-sensitive-Protein Kinase A) to generate mutant allele, this mutant protein will in addition to ATP, can also utilize specific ATP analogs. The mutation replaces a conserved amino acid with a bulky side chain with a smaller residue (alanine or glycine) creates a “gap” or an enlarged ATP binding pocket. The engineered ``gap’’, located in the active site of the enzyme where the N6 amino group on the purine moiety of ATP is positioned, allows binding of not only ATP but also structurally modified ATP analogs with substitutions at the N6 position, such as N6-(benzyl)-ATP. Only the as-mutant kinase, but not the wild type kinase, can use N6-substituted ATP analogs as phosphate donors. Thus, only substrates of the as-mutant kinase are labeled by the ATP analogs. Another recent advance in this approach was the development of an affinity tagging technique where the ATP-analog labeled proteins can be recognized by specific antibodies. In this approach following labeling of substrates with N6-(benzyl)-ATP-¿S, the thio-phosphate group on the polypeptides is alkylated by para-nitrobenzylmesylate (PNBM) to create an epitope that can be detected by specific antibodies to the thio-phosphate ester. Mammalian sperm exiting the seminiferous tubules undergo maturation in the epididymis acquiring motility and the ability to fertilize eggs. Despite the proposed roles for calcium, pH, and cAMP the biochemical basis for epididymal sperm maturation remains unknown. We proposed that high catalytic activity of the sperm specific serine/threonine phosphatase (PP1) isoform, holds motility in check in immature spermatozoa. The PP1g2 isoform is present in all mammalian spermatozoa studied - mouse, rat, hamster, bovine, non-human primate, and human. The serine/threonine protein phosphatase 1 (PP1) inhibitors, PPP1R2, PPP1R7 and PPP1R11, are highly conserved and evolutionarily ancient proteins in a variety of organisms from yeast to mammals. Surprisingly, these three ubiquitous PP1 inhibitors were identified as regulators of sperm-specific PP1¿2. The purpose of this study was to investigate how changes in the activity of PP1g2 occur in epididymal sperm. We hypothesized that changes in sperm PP1g2 activity effected by binding to its regulatory proteins are critical for the development and regulation of sperm function. We show that the association of PPP1R2, PPP1R7, and PPP1R11 to PP1¿2, changes during epididymal sperm maturation. In immotile caput sperm, PPP1R2 and PPP1R7 are not bound to PP1¿2. In motile caudal sperm PPP1R2, PPP1R7 and PPP1R11 are bound to PP1g2 as hetero-dimers or -trimers. Binding of PPP1R11 remains unchanged in caput and caudal sperm. The spatiotemporal expression of PPP1R2, PPP1R11 and PPP1R7 matches with PP1y2 expression during spermatogenesis. The inhibitors and PP1y2 are co-localized within mature spermatozoa. Changes in association of these inhibitors are likely due to changes in their phosphorylation status and the interrelated roles of GSK3, PKA, and PP1¿2. In mature caudal sperm from mice lacking GSK3, PPP1R7 is not bound to PP1¿2, resembling immature caput sperm. Moreover, ATP and net adenine nucleotides levels also resemble immature caput sperm. Altered binding of PPP1R7 is also found in sperm lacking cAMP due to the absence of soluble adenylyl cyclase. All the three regulators of PP1g2 contain consensus sites for GSK3 and PKA phosphorylation. Our studies show that GSK3, PKA, and PP1g2 are mechanistically interrelated in regulating the development of motility and fertilizing ability of sperm during their passage through the epididymis.
Srinivasan Vijayaraghavan, PHD (Committee Chair)
Douglas Kline, PHD (Committee Member)
Gary Koski, PHD (Committee Member)
Sanjaya Abeysirigunawardena, PHD (Committee Member)
Aaron Jasnow, PHD (Committee Member)
137 p.
Biochemistry; Biology; Cellular Biology; Molecular Biology
Chemical genetics; PKA; PP1g2; GSK3; PPP1R11; PPP1R7; PPP1R2; Epididymal Sperm maturation;

Recommended Citations

Citations

  • Goswami, S. (2018). IDENTIFICATION OF PHOSPHOPROTEINS INVOLVED IN SPERM MATURATION AND FERTILITY. [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532952768427828

    APA Style (7th edition)

  • Goswami, Suranjana. IDENTIFICATION OF PHOSPHOPROTEINS INVOLVED IN SPERM MATURATION AND FERTILITY. 2018. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1532952768427828.

    MLA Style (8th edition)

  • Goswami, Suranjana. "IDENTIFICATION OF PHOSPHOPROTEINS INVOLVED IN SPERM MATURATION AND FERTILITY." Doctoral dissertation, Kent State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532952768427828

    Chicago Manual of Style (17th edition)

kent1532952768427828
543
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This open access ETD is published by Kent State University and OhioLINK.