Signaling and Signal Transduction in Endocrine Tissues
(W774) CHARACTERIZATION OF THE HUMAN TESTIS PROTEIN PHOSPHATASE 1 INTERACTOME AND ITS RELEVANCE TO SPERM MOTILITY.
da Cruz e Silva, Edgar1, Fardilha, Margarida1, da Cruz e Silva, Odete1, 1 Universidade de Aveiro, Aveiro, Portugal
ABSTRACT- Of the three mammalian protein phosphatase 1 genes, PP1 yields the ubiquitous PP11 and the testis-specific PP12 alternatively spliced isoforms. Previous work showed that not only is PP12 highly expressed in sperm, but that the development of sperm motility correlates with changes in PP1 activity, consistent with a major role for PP1 in the control of this process. These observations stimulated the study of the function of sperm/testis PP1. Since the activity of PP1 towards different substrates is mediated via binding to specific regulatory proteins that play critical regulatory and targeting roles, particular attention was devoted to the identification and characterization of testis-specific PP1 interacting proteins. An in-depth screen using the yeast two-hybrid approach identified proteins expressed in human testis capable of interacting specifically with either or both isoforms of PP1. Around 120, 160 and 85 positive cDNA clones were identified for PP11, PP12 and the PP12-specific C-terminal, respectively. Comparison to the Genbank database identified four classes of interacting proteins: (i) known PP1 interactors, (ii) known protein not previously related to PP1, (iii) unknown proteins identified as ORFs as part of the sequencing of the human genome, and (iv) previously unidentified new proteins/genes. The results obtained were validated by a variety of criteria, including the isolation of 'bona fide' PP1 binding proteins and the presence of a consensus PP1 binding motif in the identified proteins. Of particular note was the identification of the protein kinase Nek2A as the most common PP1 binding protein in human testis. Although the role of such a phosphatase/kinase signaling complex remains to be elucidated, it provides an interesting physiological control mechanism. Further investigation led to the discovery of Nek2A-T, a novel splice variant of Nek2A missing 8 amino acids near the PP1 binding consensus motif. In parallel studies an isoform-specific antibody was used to immunoprecipitate PP12 binding proteins from human sperm. A large number of spots were identified by 2D gel analysis whose identification by mass spectrometry is currently being undertaken. Our results can potentially identify new targets for the treatment of male infertility and for the development of male contraceptives.
KEY WORDS: protein phosphatase 1, proteomics, interactome, signal transduction therapeutics