title: Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.
abstract: The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.
authors.affiliation: Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Jagathpala
authors.initials: J
authors.lastname: Shetty
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota, USA.
authors.forename: Rondedrick
authors.initials: R
authors.lastname: Sinville
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Shumilin
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA.
authors.forename: Wladek
authors.initials: W
authors.lastname: Minor
authors.affiliation: Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA, USA; Shanxi Key Laboratory of Ecological Animal Science and Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, China.
authors.forename: Jianhai
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota, USA.
authors.forename: Jon E
authors.initials: JE
authors.lastname: Hawkinson
authors.affiliation: Institute for Therapeutics Discovery and Development, Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 717 Delaware Street SE, Minneapolis, Minnesota, USA.
authors.forename: Gunda I
authors.initials: GI
authors.lastname: Georg
authors.affiliation: Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA, USA.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Flickinger
authors.affiliation: Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VA, USA. Electronic address: jch7k@virginia.edu.
authors.forename: John C
authors.initials: JC
authors.lastname: Herr
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D003272:Contraceptive Agents, Male / Q000494:pharmacology*; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008297:Male; D010750:Phosphoproteins; D010766:Phosphorylation / Q000187:drug effects; D000072417:Protein Domains; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D002364:Caseins; D003272:Contraceptive Agents, Male; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C432423:TSKS protein, human; C497812:TSSK2 protein, human; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine
keywords: Male contraceptive drug targets; Mobility shift assay; Recombinant kinases; TSKS; TSSK2