title: Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation.
abstract: 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.
authors.affiliation: Biological Sciences Department, Kent State University, Kent, Ohio 44242, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Mishra
authors.forename: Payaningal R
authors.initials: PR
authors.lastname: Somanath
authors.forename: Zaohua
authors.initials: Z
authors.lastname: Huang
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Vijayaraghavan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018797:Cell Cycle Proteins / Q000494:pharmacology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005854:Germ Cells / Q000201:enzymology*; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013079:Sperm Maturation / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases