Abstract Glioblastoma (GBM) is the deadliest brain cancer with a five-year survival rate of 7% and a lack of targeted therapies for its treatment. One of the main factors limiting better treatment outcomes is the invasiveness of GBM cells, a property which reduces the efficacy of surgical resection and makes recurrence almost inevitable. The development of therapeutics for restraining cell invasion is therefore a crucial goal in the biomedical community. The invasive capacity of GBM cells is largely driven by a rewiring of signal transduction networks which results in abnormal activity of the motility-regulating RHO family GTPases, and activation of pro-invasive gene expression programs. However, the molecular mechanisms controlling this signalling switch are not fully understood, and their elucidation represents a significant challenge in biomedical research. Our goal is to improve the understanding of rewired signalling in GBM cells by studying the functions of the CNK scaffold protein family. Through genetic knockdown/knockout and in vitro cell migration and invasion assays with GBM cell lines we identified the CNK3 member as a new pro-invasive factor. Crucially, this is also the case in vivo, as loss of CNK3 expression greatly reduced tumour volume in an orthotopic mouse xenograft model. Using GTPase pulldown assays, we then determined that CNK3 supports cell motility by promoting the activation of the RHO GTPases RAC1 and CDC42, while inhibiting the activity of RHOA. Consistent with these findings, CNK3 is required for normal actin-based protrusion formation at the leading-edge of migrating cells, and limits cell contractility and cell-substrate adhesion. To determine the molecular mechanism of action by which CNK3 functions as a scaffold protein, we then identified its proximal interactome by TurboID and validated its functional binding partners through co-immunoprecipitation and functional rescue experiments. These include the RAC1/CDC42 GTPase-activating protein ARHGAP39, and the MAP4K kinases MAP4K4, TNIK, and MINK1. Finally, we identified upstream growth factors and receptor tyrosine kinases (RTKs) that regulate CNK3-dependent motility and RHO GTPase activity. Thus, CNK3 functions by linking signalling by upstream RTKs to downstream regulation of RHO GTPases. Current efforts focus on the identification of pro-motility transcription programs regulated by this signalling axis. Our research identifies the CNK3 scaffold protein as a new driver of motility in GBM cells and advances our understanding of the molecular mechanisms underlying their invasive behaviour. Ultimately, CNK3 and its binding partners could be new therapeutic targets for limiting the spread of GBM and improving patient survival. Citation Format: Guillaume Serwe, David Kachaner, Thomas Perreault, Marc K. Saba-El-Leil, Driss Lajoie, Geneviève Arseneault, Vincent Quoc-Huy Trinh, Marc Therrien. The scaffold protein CNK3 mediates pro-invasive signalling in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3462.

Cell motility is a critical feature of invasive tumour cells that is governed by complex signal transduction events. Particularly, the underlying mechanisms that bridge extracellular stimuli to the molecular machinery driving motility remain partially understood. Here, we show that the scaffold protein CNK2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to downstream activation of ARF6 GTPase. Mechanistically, AXL signalling induces PI3K-dependent recruitment of CNK2 to the plasma membrane. In turn, CNK2 stimulates ARF6 by associating with cytohesin ARF GEFs and with a novel adaptor protein called SAMD12. ARF6-GTP then controls motile forces by coordinating the respective activation and inhibition of RAC1 and RHOA GTPases. Significantly, genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model. Together, this work identifies CNK2 and its partner SAMD12 as key components of a novel pro-motility pathway in cancer cells, which could be targeted in metastasis.

Mitotic entry involves inhibition of protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-B55/Tws), which dephosphorylates substrates of mitotic kinases. This inhibition is induced when Greatwall phosphorylates Endos, turning it into an inhibitor of PP2A-Tws. How this mechanism operates spatiotemporally in the cell is incompletely understood. We previously reported that the nuclear export of Greatwall in prophase promotes mitotic progression. Here, we examine the importance of the localized activities of PP2A-Tws and Endos for mitotic regulation. We find that Tws shuttles through the nucleus via a conserved nuclear localization signal (NLS), but expression of Tws in the cytoplasm and not in the nucleus rescues the development of tws mutants. Moreover, we show that Endos must be in the cytoplasm before nuclear envelope breakdown (NEBD) to be efficiently phosphorylated by Greatwall and to bind and inhibit PP2A-Tws. Disrupting the cytoplasmic function of Endos before NEBD results in subsequent mitotic defects. Evidence suggests that this spatiotemporal regulation is conserved in humans.

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term polo-box domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop. How Polo activation and localization are coupled during mitotic entry is unknown. Here we report that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila . These results uncover spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.

Entry into mitosis requires the phosphorylation of multiple substrates by cyclin B-Cdk1, while exit from mitosis requires their dephosphorylation, which depends largely on the phosphatase PP2A in complex with its B55 regulatory subunit (Tws in Drosophila). At mitotic entry, cyclin B-Cdk1 activates the Greatwall kinase, which phosphorylates Endosulfine proteins, thereby activating their ability to inhibit PP2A-B55 competitively. The inhibition of PP2A-B55 at mitotic entry facilitates the accumulation of phosphorylated Cdk1 substrates. The coordination of these enzymes involves major changes in their localization. In interphase, Gwl is nuclear while PP2A-B55 is cytoplasmic. We recently showed that Gwl suddenly relocalizes from the nucleus to the cytoplasm in prophase, before nuclear envelope breakdown and that this controlled localization of Gwl is required for its function. We and others have shown that phosphorylation of Gwl by cyclin B-Cdk1 at multiple sites is required for its nuclear exclusion, but the precise mechanisms remained unclear. In addition, how Gwl returns to its nuclear localization was not explored. Here we show that cyclin B-Cdk1 directly inactivates a Nuclear Localization Signal in the central region of Gwl. This phosphorylation facilitates the cytoplasmic retention of Gwl, which is exported to the cytoplasm in a Crm1-dependent manner. In addition, we show that PP2A-Tws promotes the return of Gwl to its nuclear localization during cytokinesis. Our results indicate that the cyclic changes in Gwl localization at mitotic entry and exit are directly regulated by the antagonistic cyclin B-Cdk1 and PP2A-Tws enzymes.

Mitosis and cytokinesis allow faithful distribution of the duplicated genetic material from one mother to two daughter cells. These cell cycle events are regulated precisely by several kinase families including Cyclin-dependent kinases (CDKs), Aurora kinases and Polo-like kinases (Plks). Interestingly, these proteins are able to collaborate with each other during cell division and defects in their regulations could lead to aneuploidy. Discovered in Drosophila and conserved from yeast to humans, the Polo kinase has emerged as a potent regulator of cell division [1]. The Polo kinase is the founding member of the Plk family [2]. While budding yeast and fission yeast each have a single Plk (Cdc5 and Plo1 respectively), mammals have five Plk members (Plk1-5). Apart from Polo, the fly has only one additional member named Sak, which is orthologous to Plk4 and appears dedicated to the regulation of centriole duplication. The fly Polo kinase, the single yeast Plks and human Plk1 all promote similar functions during cell division, including mitotic entry, centrosome maturation, spindle assembly, kinetochore function and cytokinesis. Plks share a conserved amino-terminal Ser/Thr kinase domain (KD) and a carboxy-terminal Polo-Box Domain (PBD) [3]. The PBD acts as a phosphopeptide-binding domain and allows Polo to bind substrates and adaptor proteins that recruit Polo to its subcellular locations including centrosomes, kinetochores and the central spindle during mitosis and the midbody during cytokinesis. The multiple functions of the Polo kinase are possible because this protein is submitted to a complex regulation. Amongst other modes of regulation, Plks are activated by phosphorylation of their activation loop in the KD [2]. In humans, this activation of Plk1 begins in G2 and is mediated by the Aurora A kinase, whereas in flies the activation of Polo in early mitosis on kinetochores requires its phosphorylation by Aurora B, a subunit of the Chromosomal Passenger Complex. The activity of Polo has long been known to be essential for cytokinesis [4]. However, how Polo is regulated in this process was unknown. In a recent paper from our laboratory, we have investigated this question in Drosophila cells in culture. We found that Polo phosphorylation by Aurora B is required for successful cytokinesis [5]. This phosphorylation occurs in the activation loop of the KD at Thr182. Our results indicate that upon phosphorylation, Polo dissociates from (or is prevented from binding to) the PBD-bound Map205, a microtubule-associated protein which sequesters unphosphorylated Polo on microtubules and inhibits its kinase activity [6] (Figure ​(Figure1).1). Then, freed from this inhibitor, activated Polo is recruited to the midbody. To determine the physiological importance of Polo phosphorylation during cytokinesis, we performed time-lapse microscopy in cells where Aurora B is inhibited or when endogenous Polo was replaced with a nonphosphorylatable form (Polo T182A) fused to the GFP. In both cases, Polo failed to localize properly to the midbody and more than 80% of the cells attempting division failed cytokinesis and became binucleated. Figure 1 Model for the regulation of Drosophila Polo during cytokinesis Our findings can be interpreted in structural terms in the light of the recent publication by Xu et al. which solved the cocrystal structure of a fragment of Drosophila Map205 in complex with the separate PBD and KD of zebrafish Plk1 [7]. This work reveals that Map205 binds and stabilizes the closed conformation of the PBD, which in turn binds and stabilizes the inactive conformation of the KD. Our findings now suggest that during cytokinesis, phosphorylation on the Polo kinase at its activation-loop negatively regulates the interaction between Polo and its PBD-bound inhibitor Map205 by an interdomain allosteric mechanism and induces a conformational change that is required for full activation of the kinase. Drosophila Map205 is poorly conserved in vertebrates, but the observation that human Plk1 is competent for an inhibiting interaction with Map205 suggests that there may exist other physiological interactors of Plk1 that regulate it by a similar mechanism. Such proteins remain to be identified.

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

This review highlights recent advances in our understanding of the mechanisms of Optineurin (Optn) action and its implication in diseases. Optn has emerged as a key player regulating various physiological processes, including membrane trafficking, protein secretion, cell division and host defense against pathogens. Furthermore, there is growing evidence for an association of Optn mutations with human diseases such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone. Optn functions depend on its precise subcellular localization and its interaction with other proteins. Here, we review the mechanisms that allow Optn to ensure a timely and spatially coordinated integration of different physiological processes and discuss how their deregulation may lead to different pathologies.

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.