Identification of the spermatogenic zip protein Spz1 as a putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the PP1cgamma2 splice variant in mouse testis.
The spermatogenic zip protein (Spz1) was originally isolated from a mouse testis library and identified as a novel member of the basic helix-loop-helix family of transcription factors. Here we identify Spz1 as a specific binding partner of the gamma2 catalytic subunit of protein phosphatase-1. Male mice homozygous for a null mutation in the protein phosphatase-1cgamma (PP1cgamma) gene are infertile and display a distinct impairment in spermiogenesis despite the continued presence of closely related PP1c isoforms. Yeast two-hybrid screening using the PP1cgamma2 splice variant has identified Spz1 as an interacting protein and possible mediator of the sterile PP1cgamma mutant phenotype. Spz1 was shown to interact specifically with PP1cgamma2 but did not show an interaction with PP1calpha or with a truncated version of PP1cgamma2 lacking 18 amino acids from the C terminus. Interaction between full-length Spz1 and PP1cgamma2 was verified by co-immunoprecipitation and co-localization experiments in COS-1 cells as well as gel-shift and sedimentation assays using whole testis lysates. Immunohistochemistry on wild type testis sections reveals a stage-specific expression pattern for Spz1 during spermatogenesis that appeared grossly abnormal in the testes of PP1cgamma mutant mice. Phosphatase assays using recombinant PP1c indicate that increasing concentrations of Spz1 are able to inhibit PP1cgamma2 activity while having little effect on the activity of PP1calpha. Furthermore, an interaction between PP1cgamma2 and Spz1 was shown to prevent binding of the latter to the consensus E-box promoter sequence. We propose that the interaction between Spz1 and PP1cgamma2 may be required for proper regulation of spermatogenesis and fertility in males.
Hrabchak
Christopher
C
Department of Zoology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada.
Varmuza
Susannah
S
D017398:Alternative Splicing; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006720:Homozygote; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013091:Spermatogenesis; D013737:Testis / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D020033:Protein Isoforms; D011994:Recombinant Proteins; C426848:Spz1 protein, mouse; D014157:Transcription Factors; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1