Mónica Ferreira

Protein Ser/Thr phosphatase PP1 is always associated with one or two regulatory subunits or RIPPOs. One of the earliest evolved RIPPOs is PPP1R2, also known as Inhibitor-2. Since its discovery nearly 5 decades ago, PPP1R2 has been variously described as an inhibitor, activator or (metal) chaperone of PP1, but it is still unknown how PPP1R2 affects the function of PP1 in intact cells. Here, using specific research tools, we demonstrate that PPP1R2 stabilises a subgroup of PP1 holoenzymes, exemplified by PP1:RepoMan, thereby promoting the dephosphorylation of their substrates. Mechanistically, the recruitment of PPP1R2 disrupts an inhibitory, fuzzy interaction between the C-terminal tail and catalytic domain of PP1, and generates an additional C-terminal RepoMan-interaction site. The resulting holoenzyme is further stabilized by a direct PPP1R2:RepoMan interaction, which renders it refractory to competitive disruption by RIPPOs that do not interact with PPP1R2. Our data demonstrate that PPP1R2 modulates the function of PP1 by altering the balance between holoenzymes through stabilisation of specific subunit interactions.

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

Nuclear Inhibitor of PP1 (NIPP1) is a conserved regulatory subunit of protein phosphatase PP1. The selective deletion of NIPP1 in mouse liver parenchymal cells or skin epidermal cells culminates in a late-onset hyperproliferation of a subset of resident progenitor cells. Although a hyperplastic phenotype is usually tumor promoting, we show here that the absence of NIPP1 conferred a strong resistance to chemically induced hepatocellular or skin carcinoma. The ablation of NIPP1 did not affect the metabolism of the administered mutagens (diethylnitrosamine or 7,12-dimethylbenz[a]anthracene), but reduced the conversion of mutagen-induced covalent DNA modifications into cancer-initiating mutations. This reduced sensitivity to mutagens correlated with an enhanced DNA-damage response and an augmented expression of rate-limiting DNA-repair proteins (MGMT in liver, XPD and XPG in skin), hinting at an increased DNA-repair capacity. Our data identify NIPP1 as a repressor of DNA repair and as a promising target for novel cancer prevention and treatment therapies.

NIPP1 is one of the major nuclear interactors of protein phosphatase PP1. The deletion of NIPP1 in mice is early embryonic lethal, which has precluded functional studies in adult tissues. Hence, we have generated an inducible NIPP1 knockout model using a tamoxifen-inducible Cre recombinase transgene. The inactivation of the NIPP1 encoding alleles (Ppp1r8) in adult mice occurred very efficiently in testis and resulted in a gradual loss of germ cells, culminating in a Sertoli-cell only phenotype. Before the overt development of this phenotype Ppp1r8 ^−/− testis showed a decreased proliferation and survival capacity of cells of the spermatogenic lineage. A reduced proliferation was also detected after the tamoxifen-induced removal of NIPP1 from cultured testis slices and isolated germ cells enriched for undifferentiated spermatogonia, hinting at a testis-intrinsic defect. Consistent with the observed phenotype, RNA sequencing identified changes in the transcript levels of cell-cycle and apoptosis regulating genes in NIPP1-depleted testis. We conclude that NIPP1 is essential for mammalian spermatogenesis because it is indispensable for the proliferation and survival of progenitor germ cells, including (un)differentiated spermatogonia.

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting proteins (PIPs) that guide the phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation.