Neurofibromin is a large and multifunctional protein encoded by the tumor suppressor gene NF1, mutations of which cause the tumor predisposition syndrome neurofibromatosis type 1 (NF1). Over the last three decades, studies of neurofibromin structure, interacting partners, and functions have shown that it is involved in several cell signaling pathways, including the Ras/MAPK, Akt/mTOR, ROCK/LIMK/cofilin, and cAMP/PKA pathways, and regulates many fundamental cellular processes, such as proliferation and migration, cytoskeletal dynamics, neurite outgrowth, dendritic-spine density, and dopamine levels. The crystallographic structure has been resolved for two of its functional domains, GRD (GAP-related (GTPase-activating protein) domain) and SecPH, and its post-translational modifications studied, showing it to be localized to several cell compartments. These findings have been of particular interest in the identification of many therapeutic targets and in the proposal of various therapeutic strategies to treat the symptoms of NF1. In this review, we provide an overview of the literature on neurofibromin structure, function, interactions, and regulation and highlight the relationships between them.

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell.

The eukaryotic THO complex coordinates the assembly of so-called messenger RNA–ribonucleoprotein particles (mRNPs), a process that involves cotranscriptional coating of nascent mRNAs with proteins. Once formed, mRNPs undergo a quality control step that marks them either for active transport to the cytoplasm, or Rrp6/RNA exosome-mediated degradation in the nucleus. However, the mechanism behind the quality control of nascent mRNPs is still unclear. We investigated the cotranscriptional quality control of mRNPs in budding yeast by expressing the bacterial Rho helicase, which globally perturbs yeast mRNP formation. We examined the genome-wide binding profiles of the THO complex subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation of the mRNP biogenesis, and found that Tho2 plays two roles. In addition to its function as a subunit of the THO complex, upon perturbation of mRNP biogenesis Tho2 targets Rrp6 to chromatin via its carboxy-terminal domain. Interestingly, other THO subunits are not enriched on chromatin upon perturbation of mRNP biogenesis and are not necessary for localizing Rrp6 at its target loci. Our study highlights the potential role of Tho2 in cotranscriptional mRNP quality control, which is independent of other THO subunits. Considering that both the THO complex and the RNA exosome are evolutionarily highly conserved, our findings are likely relevant for mRNP surveillance in mammals.

Neurofibromin (Nf1) is a large multidomain protein encoded by the tumour-suppressor gene NF1 . NF1 is mutated in a frequently occurring genetic disease, neurofibromatosis type I, and in various cancers. The best described function of Nf1 is its Ras-GTPase activity, carried out by its GAP-related domain (GRD). SecPH, another structurally well-characterized domain of Nf1, is immediately adjacent to the GRD and interacts with lipids and proteins, thus connecting Nf1 to diverse signalling pathways. Here, we demonstrate, for the first time, that Nf1 and SecPH are substrates of the SUMO pathway. We identified a well-defined SUMOylation profile of SecPH and a main SUMOylation event on Lys1731 that appears to play a role in Ras-GAP activity. Our data allowed us to characterize a new set of pathogenic Nf1 missense mutants that exhibits a disrupted SUMOylation profile that may correlate with their unfolding. Accordingly, Lys1731 SUMOylation is mediated by a noncanonical structural motif, therefore allowing a read-out of SecPH conformation and folding status.

While copper is an essential micronutrient and a technologically indispensable heavy metal, it is toxic at high concentrations, harming the environment and human health. Currently, copper is monitored with costly and low-throughput analytical techniques that do not evaluate bioavailability, a crucial parameter which can be measured only with living cells. We overcame these limitations by building upon yeast S. cerevisiae ’s native copper response and constructed a promising next-generation eukaryotic whole-cell copper biosensor. We combined a dual-reporter fluorescent system with an engineered CUP1 promoter and overexpressed Cup2 transactivator, constructing through four iterations a total of 16 variants of the biosensor, with the best one exhibiting a linear range of 10 ^-8 to 10 ^-3 M of bioavailable copper. Moreover, this variant distinguishes itself by superior specificity, detection limit, and linear range, compared to other currently reported eukaryotic and prokaryotic whole-cell copper biosensors. By re-engineering the transactivator, we altered the system’s sensitivity and growth rate, while assessing the performance of Cup2 with heterologous activation domains. Thus, in addition to presenting the next-generation whole-cell copper biosensor, this work urges for an iterative design of eukaryotic biosensors and paves the way toward higher sensitivity through transactivator engineering. Graphical

Summary: LIM kinases, LIMK1 and LIMK2, play a crucial role in cytoskeleton dynamics. They are involved in many physiological processes but also in several pathologies such as cancer, neuronal diseases and neurofibromatosis. Although LIM kinases appear as promising therapeutic targets, they remain undruggable. A better understanding of their activity and regulation is thus required to better design efficient targeted therapies. Here, we have shown the impact of a single amino acid on LIMK activity on cofilin, their main substrate in actin filament remodelling. We demonstrated that Y632 and Y630, for LIMK1 and LIMK2 respectively, mediate LIMK dimerization, resulting in their transphosphorylation. This process seems to be a prerequisite for their canonical phosphorylation on their respective T508 and T505 residues within the activation loop. These Tyrosine are not phosphorylated, their aromatic nature is rather critical to ensure proper LIMK activity on cofilin. These results bring new insights into LIMK molecular features.