title: Identification of testis 14-3-3 binding proteins by tandem affinity purification.
abstract: The 14-3-3 family of proteins interacts with various cellular phosphoproteins and regulates multiple cell signaling cascades. Identification of 14-3-3 interactors is important to define 14-3-3 functions in various biological pathways. The binding partners of protein 14-3-3 in testis are not known. The main goal of this study was to identify the 14-3-3 interactome in testis to determine the 14-3-3 regulated cellular processes in testis. We used transgenic mice expressing tandem affinity tagged 14-3-3ζ (TAP-14-3-3ζ) driven by the ubiquitin promoter to isolate 14-3-3 binding proteins. The 14-3-3 complexes in testis were isolated using a two-step tandem affinity purification (TAP) followed by identification with liquid chromatography/tandem mass spectrometry (LC-MS/MS). A total of 135 proteins were found to be associated with 14-3-3 in vivo in testis. Comparison of the testis 14-3-3 proteome with known 14-3-3 binding proteins showed that 71 of the proteins identified in this study are novel 14-3-3 interactors. Eight of these novel 14-3-3 interacting proteins are predominantly expressed in testis. The 14-3-3 interactors predominant in testis are: protein phosphatase1γ2 (PP1γ2), spermatogenesis associated 18 (SPATA18), phosphoglycerate kinase-2 (PGK2), testis specific gene A-2 (TSGA-2), dead box polypeptide 4 (DDX4), piwi homolog 1, protein kinase NYD-SP25 and EAN57. The fact that some of these proteins are indispensable for spermatogenesis suggests that their binding to 14-3-3 may be important for their function in germ cell division and maturation. These findings are discussed in context of the putative functions of 14-3-3 in spermatogenesis.
authors.affiliation: Department of Biological Sciences; Kent State University; Kent, OH.
authors.forename: Pawan
authors.initials: P
authors.lastname: Puri
authors.forename: Amparo
authors.initials: A
authors.lastname: Acker-Palmer
authors.forename: Ryan
authors.initials: R
authors.lastname: Stahler
authors.forename: Yijing
authors.initials: Y
authors.lastname: Chen
authors.forename: Douglas
authors.initials: D
authors.lastname: Kline
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Vijayaraghavan