Mammalian sperm were previously shown to express the PP1gamma2 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 PP1gamma1 and PP1gamma2 (the two known alternatively spliced variants of PP1gamma). 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 PP1gamma2 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 PP1gamma2 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. PP1gamma2, Nek2A-T, and NIPP1-T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.

BackgroundThe BRCA1/2 mutation profile varies in Spain according to the geographical area studied. The mutational profile of BRCA1/2 in families at risk for hereditary breast and ovarian cancer has not so far been reported in Andalusia (southern Spain).MethodsWe analysed BRCA1/2 germline mutations in 562 high-risk cases with breast and/or ovarian cancer from Andalusian families from 2010 to 2015.ResultsAmong the 562 cases, 120 (21.4%) carried a germline pathogenic mutation in BRCA1/2; 50 in BRCA1 (41.7%) and 70 in BRCA2 (58.3%). We detected 67 distinct mutations (29 in BRCA1 and 38 in BRCA2), of which 3 in BRCA1 (c.845C > A, c.1222_1223delAC, c.2527delA) and 5 in BRCA2 (c.293 T > G, c.5558_5559delGT, c.6034delT, c.6650_6654delAAGAT, c.6652delG) had not been previously described. The most frequent mutations in BRCA1 were c.5078_5080delCTG (10%) and c.5123C > A (10%), and in BRCA2 they were c.9018C > A (14%) and c.5720_5723delCTCT (8%). We identified 5 variants of unknown significance (VUS), all in BRCA2 (c.5836 T > C, c.6323G > T, c.9501 + 3A > T, c.8022_8030delGATAATGGA, c.10186A > C). We detected 76 polymorphisms (31 in BRCA1, 45 in BRCA2) not associated with breast cancer risk.ConclusionsThis is the first study reporting the mutational profile of BRCA1/2 in Andalusia. We identified 21.4% of patients harbouring BRCA1/2 mutations, 58.3% of them in BRCA2. We also characterized the clinical data, mutational profile, VUS and haplotype profile.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4537-9) contains supplementary material, which is available to authorized users.

In testis we find mainly PPP1gamma2 isoform. We hypothesize that in different cell types we can find different regulatory subunits that may constitute targets for therapeutics of diseases such as male infertility, cancer and Alzheimer's disease. We identified a novel alternative splicing isoform of IIIG9 in testis, a known regulator of PPP1, IIIG9sT, and the aim of this study was its further characterization. We used a specific antibody for IIIG9sT in order to characterize its localization in bovine sperm cells. We also transfected IIIG9sT-GFP construct in mouse spermatogonia cells (GC-1 cells) and we used specific antibodies for each PPP1 isoform for the colocalization studies. We observed them under a fluorescent microscope and a LSM and quantified a high co-localization with PPP1gamma1 and 2 isoforms. INTRODUCTIONA common translation modification is protein phosphorylation/dephosphorylation; that is a reversible process that enables cells to respond to several different signals in a dynamic way. In humans we can find at least 13 PPPs (protein serine/threonine phosphatases). However the diversity of members is increased by the existence of different isoforms, e.g. there are two gamma isoforms of PPP1 in humans, the most ubiquitous PPP, PPP1gamma1 and PPP1gamma2, codified by a single gene (PPP1CC). That is, the splicing machinery recognizes the exons with precision, removes the introns from the pre-mRNA molecule and ligates exons, caused by exon skipping or intron maintenance, will constitute a different protein [1]. PPP1gamma1 is ubiquity distributed in different human tissues; however gamma2 isoform is exclusive of testis and sperm. PPP1 is regulated, at the subcellular level, by protein subunits, so we hypothesize that in different cell types we can find different regulators, and the ones exclusive of a tissue/cell type can be used for therapy in several diseases such as cancer, male infertility and Alzheimer's disease. In order to identify the regulatory subunits involved in the regulation of PPP1gamma2 we performed a yeast two-hybrid screen in a testis cDNA library [2]. We found a new splice variant, that seems to be exclusive of testis [3] of a known PPP1 regulator, IIIG9 [4], that we named IIIG9sT.

Protein phosphatase 1 (PPP1) is the PPP most ubiquitous and each isoform interact with regulatory subunits that may be responsible for their subcellular localization. We identified PPP1R15B, C1ORF71 as novel regulators and the aim of this study was their further characterization in carcinoma cells. We analysed localization of each regulator in MDA-MB-468 cells and we transfected with constructs that we made with each as a GFP-fusion protein. For PPP1 cellular localization we used specific antibodies for each isoform. We observed the cells under a fluorescent microscope and LSM and we quantified co-localization. We found a high overlap coefficient of both the novel proteins with PPP1alpha and PPP1gamma1.We propose a model in which PPP1 regulator interacts with one or two regulatory subunits that may be used as target for therapeutic strategies.KEY-WORDS Fluorescent microscopy, laser scanning microscopy, overlap coefficient, PPP1, novel regulators, breast carcinoma ACKNOWLEDGMENTS This work was supported by FCT: CS received a PhD fellowship, and A.P.V. and M.F. received pos-doctoral fellowships.

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