Explore the vast range of titles available.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers 1 – 3 . Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation 4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer 5 and active RAF dimer 5 – 8 bound to 14-3-3 9 , 10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF 11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS 11 – 13 , but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway. Cryo-electron microscopy structure, molecular dynamics and biochemical analyses of the SHOC2–PP1C–MRAS complex demonstrate the dependence of the complex formation on RAS–GTP and identify the determinants of RAS isoform preference for SHOC2–PP1C and specificity of the complex for RAF dephosphorylation.

Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets. Protein delivery with cell-penetrating peptides suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting treatment success. Here the authors present an intracellular protein delivery system and demonstrate efficient delivery in vitro and therapeutic efficacy in a liver failure model in vivo.

Receptor tyrosine kinase (RTK)–RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2–MRAS–PP1C holophosphatase complex functions as a key regulator of RTK–RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling 1 . SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes 2 – 5 . However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2–MRAS–PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure–function model comprehensively defines key binding interactions within the SHOC2–MRAS–PP1C holophosphatase complex and will inform therapeutic development .

RAS–MAPK signalling is fundamental for cell proliferation and is altered in most human cancers 1 – 3 . However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF–MEK1–14-3-3 complexes 4 , the intermediate steps that lead to RAF activation remain unclear. The MRAS–SHOC2–PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF–RAS association 3 , 5 , 6 . MRAS, SHOC2 and PP1C are mutated in rasopathies—developmental syndromes caused by aberrant MAPK pathway activation 6 – 14 —and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)–RAS-driven tumours 15 – 18 . Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS–SHOC2–PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein–protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS–MAPK signalling, therefore providing the structural basis for therapeutic interventions. A structure of the MRAS–SHOC2–PP1C complex supports a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to activate the downstream pathway.

The activation and coordination of phosphatase activity is important during mitotic exit; here, a mitotic phosphatase relay is described in fission yeast between the two major phosphatases, PP1 and PP2A, a mode of regulation that may be a feature of signalling networks across eukaryotes. This way to the exit Phosphorylation by mitotic kinases drives the initiation and progression of mitosis, the type of cell division that takes place during ordinary tissue growth. This process is facilitated by the suppression of phosphatases, but during mitotic exit phosphatase activity must be restored. Here, Iain Hagan and colleagues describe a mitotic phosphatase relay in fission yeast between the two major phosphatases, PP1 and PP2A, a mode of regulation that may be a feature of signalling networks across eukaryotes. The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes 1 , 2 , 3 , 4 , 5 , 6 . Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1–cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate 1 , 7 , 8 , 9 , 10 . PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs 11 , 12 suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.

Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling.