Alternatively spliced protein variants as potential therapeutic targets for male infertility and contraception

Mammalian sperm were previously shown to express the PP1 gamma 2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1 gamma 1 and PP1 gamma 2 (the two known alternatively spliced variants of PP1 gamma). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two-hybrid method. Among the various proteins detected, the most abundant interactors with PP1 gamma 2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A-T and NIPP1-T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT-PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A-T and NIPP1-T variants appear to bind PP1 gamma 2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis-specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1 gamma 2, Nek2A-T, and NIPP1-T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.