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.

Keywords: :

• 14-3-3
• Protein Phosphatase 1
• spermatogenesis
• Tandem Affinity Purification
• YWHA

Disclosure of Potential Conflicts of Interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

Acknowledgments

This research was Supported by National Institute of Health grant HD38520 to S.V and a Kent State University Graduate Student Senate grant to P.P. We thank Dr. Mike Kinter for doing LC-MS in Lerner Research Institute Proteomics laboratory, Cleveland. We also thank Dr. Deborah A. O'Brien (Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC for her generous gifts of PGK2 antibody. We also thank Dr. William H Walker, Dr. Natalia Kostereva and Tejasvi Dudiki for their help in results and discussion.