FAM122A (also known as PABIR1) regulates cell cycle progression through inhibition of the PP2A-B55 phosphoprotein phosphatase. Recent structural work has uncovered helical elements in the N-terminus of FAM122A as binding determinants for PP2A-B55 but whether FAM122A inhibition towards PP2A-B55 is regulated is presently unclear. To address this, we performed a systematic analysis of the PP2A-B55 interaction with FAM122A in cells uncovering a novel region in the C-terminus of FAM122A, spanning residues 150-170, required for binding. This C-terminal region and the N-terminal helices are both required for efficient binding to PP2A-B55 suggesting a bipartite binding mechanism. We perform amino acid resolution scans of FAM122A 150-170 uncovering several residues in this region contributing to binding including the conserved Ser158, a reported phosphorylation site. We show that Ser158 is important for PP2A-B55 inhibition in human cells as well as efficient stimulation of mitotic entry in Xenopus laevis egg extracts. In human cells and in Xenopus laevis Ser158 phosphorylation is regulated, with increased occupancy correlating with cell cycle stages requiring PP2A-B55 inhibition. Collectively our work uncovers novel aspects of the FAM122A interaction with PP2A-B55 and provides a possible mechanism for how the inhibitory activity of FAM122A can be regulated during the cell cycle.

Meiotic progression relies on maintaining a precise balance between cyclin B/CDK1 activity and the phosphatase PP2A-B55. The latter is negatively regulated by the Greatwall kinase (Gwl). In maturing Xenopus oocytes, we show that the loss of Gwl and the subsequent hyperactivation of PP2A-B55 prevents phosphorylation of key proteins involved in spindle formation, chromosome condensation and spindle migration as well as the phosphorylation of Wee1/Myt1 and the APC/C complex. As a consequence, in these oocytes bipolar spindles cannot be formed and migrate to the cortex and chromosomes are partially decondensed preventing meiotic I progression. The APC/C remains inactive impairing cyclin B3 degradation and ultimately preventing Erp1 accumulation. Finally, the c-Mos-MAPK-Rsk1/2 pathway fails to activate due to the absence of c-Mos as a consequence of its improper degradation. Overall, our findings reveal a crucial role of Gwl in the coordination and progression of meiotic divisions.

The evolutionarily conserved, intrinsically disordered protein Bora is critical for initiating the activation of mitotic kinases. Once phosphorylated at Ser112 by Cyclin A-Cdk1 kinase, phospho-Bora activates unphosphorylated Aurora A kinase (AURKA), directing it towards Polo-like kinase 1 (Plk1), thus promoting Cyclin B-Cdk1 activation and mitotic entry. Here, by combining structural modeling and in vitro assays, we provide evidence that Bora wraps around the N-terminal lobe of AURKA to position its phospho-Ser112 near AURKA’s T-loop, mimicking T-loop phosphorylation. Additionally, Bora transiently interacts with the αC helix of the Plk1 kinase domain through a conserved motif, guiding AURKA activity towards the Plk1 T-loop, which is otherwise impervious to phosphorylation by AURKA. We highlight the importance of this motif for Bora function in vitro and during mitotic entry in Xenopus laevis egg extracts. Our results reveal critical molecular details of mitotic kinase activation, which could lead to the development of pathway-specific inhibitors.

Cell adhesion tightly controls cell proliferation and homeostasis in stratified epithelia by mechanisms that remain unclear. Focal adhesion kinase (FAK) transduces cell adhesion signals, is frequently deregulated in epithelial cancer, and it has been associated with proliferation and resistance to treatments. The mechanisms by which FAK controls the epithelial cell cycle are still intriguing. We previously unraveled a mitosis-differentiation checkpoint that is the limiting factor in the keratinocyte cell cycle. To investigate whether FAK plays a role in this checkpoint, we inactivated the protein in normal human oral keratinocytes by specific shRNAs or by the specific inhibitor defactinib. Inactivation of FAK very rapidly and strikingly blocked entry into mitosis and triggered a differentiation response. This response was independent of DNA damage. Tumor suppressor P53 was induced shortly after inhibition of FAK, while mitotic Cyclin B was not translocated into the nucleus. Human epidermal N-TERT cells that were synchronized in prometaphase failed to execute mitosis. Concomitant inhibition of FAK-downstream Rho-associated kinase (Rock) rescued mitotic progression. The results unveil a rapid Rock-dependent mitosis switch upon inactivation of FAK, inducing terminal differentiation, pointing at a mitotic automatic mechanism of epithelia to suppress suprabasal proliferation of precancerous cells.

Microtubules are dynamic, conserved cytoskeletal filaments that are essential for all eukaryotic cells. Microtubule dynamic properties are primarily characterized by measuring how fast they grow and shrink (growth and shrinkage rates) and how often they switch between assembly and disassembly (catastrophe and rescue frequencies). These four parameters measured for individual filaments can inform on the behavior of the entire microtubule network at the cell level. By comparing microtubule dynamics in Caenorhabditis elegans one-cell embryos using different genetically-encoded fluorescent probes, we observed an unexpected high variability in these parameters. Microtubule dynamics parameters were consistently higher in C. elegans strains expressing a fluorescently labelled microtubule end-binding protein than in strains relying on tubulin labelling, with microtubule growth rates differing by nearly a factor of two between the two conditions. This discrepancy was not limited to C. elegans, as we observed a similar effect in embryos of the tunicate Phallusia mammillata. Despite this, spindle size and assembly timing were only mildly affected. However, embryos expressing labelled end-binding protein exhibited higher frequency of mitotic defects and perturbed embryonic development upon exposure to various stresses such as elevated temperature or a compromised spindle assembly checkpoint. Thus, our work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions. Our findings should also serve as a cautionary note when using tagged end-binding proteins to measure microtubule dynamics.

MAST-like, or Greatwall (Gwl), an atypical protein kinase related to the evolutionarily conserved MAST kinase family, is crucial for cell cycle control during mitotic entry. Mechanistically, Greatwall is activated by Cyclin B-Cdk1 phosphorylation of a 550 amino acids-long insertion in its atypical activation segment. Subsequently, Gwl phosphorylates Endosulfine and Arpp19 to convert them into inhibitors of PP2A-B55 phosphatase, thereby preventing early dephosphorylation of M-phase targets of Cyclin B-Cdk1. Here, searching for an elusive Gwl-like activity in C. elegans, we show that the single worm MAST kinase, KIN-4, fulfills this function in worms and can functionally replace Greatwall in the heterologous Xenopus system. Compared to Greatwall, the short activation segment of KIN-4 lacks a phosphorylation site, and KIN-4 is active even when produced in E. coli. We also show that a balance between Cyclin B-Cdk1 and PP2A-B55 activity, regulated by KIN-4, is essential to ensure asynchronous cell divisions in the early worm embryo. These findings resolve a long-standing puzzle related to the supposed absence of a Greatwall pathway in C. elegans, and highlight a novel aspect of PP2A-B55 regulation by MAST kinases.

Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.

p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often down-regulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P 2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P 2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P 2 -interacting proteins at endocytic sites.

Cells require major physical changes to induce a proper repartition of the DNA. Nuclear envelope breakdown, DNA condensation and spindle formation are promoted at mitotic entry by massive protein phosphorylation and reversed at mitotic exit by the timely and ordered dephosphorylation of mitotic substrates. This phosphorylation results from the balance between the activity of kinases and phosphatases. The role of kinases in the control of mitosis has been largely studied, however, the impact of phosphatases has long been underestimated. Recent data have now established that the regulation of phosphatases is crucial to confer timely and ordered cellular events required for cell division. One major phosphatase involved in this process is the phosphatase holoenzyme PP2A-B55. This review will be focused in the latest structural, biochemical and enzymatic insights provided for PP2A-B55 phosphatase as well as its regulators and mechanisms of action.

Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) plays an instrumental role in driving CME initiation. The F-BAR domain only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remains elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining bottom-up synthetic approaches on in vitro and cellular membranes. Our results indicate that PI(4,5)P 2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like shape protein patches. We show that binding of FCHo2 on cellular membranes promotes PI(4,5)P 2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P 2 -interacting proteins at endocytic sites.

PP2A is a major serine/threonine phosphatase class involved in the regulation of cell signaling through the removal of protein phosphorylation. This class of phosphatases is comprised of different heterotrimeric complexes displaying distinct substrate specificities. The present review will focus on one specific heterocomplex, the phosphatase PP2A-B55. Herein, we will report the direct substrates of this phosphatase identified to date, and its impact on different cell signaling cascades. We will additionally describe its negative regulation by its inhibitors Arpp19 and ENSA and their upstream kinase Greatwall. Finally, we will describe the essential molecular features defining PP2A-B55 substrate specificity that confer the correct temporal pattern of substrate dephosphorylation. The main objective of this review is to provide the reader with a unique source compiling all the knowledge of this particular holoenzyme that has evolved as a key enzyme for cell homeostasis and cancer development.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.