A Testis Specific Isoform of Endophilin B1, Endophilin B1t, Interacts Specifically with Protein Phosphatase-1cγ2 in Mouse Testis and Is Abnormally Expressed in PP1cγ Null Mice

Abstract

Male mice homozygous for a null mutation in the protein phosphatase-1c gamma (PP1c gamma) gene are infertile, displaying a severe impairment in spermatogenesis that is not compensated by the presence of PP1c alpha and PP1c beta in mutant testes. A lack of the PP1c gamma2 splice variant seems the most likely cause of the mutant phenotype, as it is the most heavily expressed PP1c gamma isoform in wild type testes. Yeast two-hybrid screening using PP1c gamma2 has identified several new binding partners, including endophilin B1t, a testis enriched isoform of endophilin B1a which differs from the somatic form by virtue of a carboxy terminal deletion spanning the last 10 amino acids. The testis isoform did not show an interaction with PP1c alpha, or with a truncated PP1c gamma2 mutant lacking the unique carboxy terminus. In contrast, somatic endophilin B1a did not interact with any of the PP1c isoforms. Sedimentation and co-immunoprecipitation experiments using native testis proteins verified binding of endophilin B1t to PP1c gamma2. Immunohistochemistry on wild type testis sections revealed a stage specific expression pattern for endophilin that appeared concentrated at discrete puncta throughout the seminiferous epithelium. Punctate endophilin expression in cells adjacent to the lumen was absent in PP1c gamma null mice. Phosphatase assays indicate that chimeric endophilin B1t is able to inhibit recombinant PP1c gamma2 activity toward phosphorylase a while having little effect on the activity of PP1c alpha. A potential role for endophilin B1t in mammalian spermatogenesis is discussed within the context of the PP1c gamma knockout testis phenotype.