Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1(Mph1) kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1(Mph1) kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1(Mph1) kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.

Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis.

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24-Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.

The VPS-20 subunit of the endosomal sorting complex ESCRT-III exhibits an open conformation in the absence of upstream activation.

Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodelling processes during multivesicular endosome (MVE) biogenesis, cytokinesis, retroviral budding and plasma membrane repair. During luminal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit vacuolar protein sorting (VPS)-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an auto-inhibited closed conformation in solution and its activation depends on an association with ESCRT-II specifically at membranes [1]. However, we show in the present study that Caenorhabditis elegans ESCRT-II and VPS-20 interact directly in solution, both in cytosolic cell extracts and in using recombinant proteins in vitro. Moreover, we demonstrate that purified VPS-20 exhibits an open extended conformation, irrespective of ESCRT-II binding, in contrast with the closed auto-inhibited architecture of another ESCRT-III subunit, VPS-24. Our data argue that individual ESCRT-III subunits adopt distinct conformations, which are tailored for their specific functions during ESCRT-mediated membrane reorganization events.

Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice.

Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Dynactin integrity depends upon direct binding of dynamitin to Arp1.

Dynactin is a multiprotein complex that works with cytoplasmic dynein and other motors to support a wide range of cell functions. It serves as an adaptor that binds both dynein and cargoes and enhances single-motor processivity. The dynactin subunit dynamitin (also known as p50) is believed to be integral to dynactin structure because free dynamitin displaces the dynein-binding p150(Glued) subunit from the cargo-binding Arp1 filament. We show here that the intrinsically disordered dynamitin N-terminus binds to Arp1 directly. When expressed in cells, dynamitin amino acids (AA) 1-87 causes complete release of endogenous dynamitin, p150, and p24 from dynactin, leaving behind Arp1 filaments carrying the remaining dynactin subunits (CapZ, p62, Arp11, p27, and p25). Tandem-affinity purification-tagged dynamitin AA 1-87 binds the Arp filament specifically, and binding studies with purified native Arp1 reveal that this fragment binds Arp1 directly. Neither CapZ nor the p27/p25 dimer contributes to interactions between dynamitin and the Arp filament. This work demonstrates for the first time that Arp1 can directly bind any protein besides another Arp and provides important new insight into the underpinnings of dynactin structure.

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane.

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus.

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the 'partitioning complex' is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage.

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8(-/-) mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.

A novel Rac1 GAP splice variant relays poly-Ub accumulation signals to mediate Rac1 inactivation.

Spatial control of RhoGTPase-inactivating GAP components remains largely enigmatic. We describe a brain-specific RhoGAP splice variant, BARGIN (BGIN), which comprises a combination of BAR, GAP, and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42-amino acid N-terminal stretch. The partial CIN domain is a poly-ubiquitin (poly-Ub)-binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as reactive oxygen species (ROS) generation by the Nox1 complex. Given that Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/Ub interactions are observed through colocalization in tangle aggregates in the Alzheimer's disease (AD) brain. Moreover, enhanced BGIN membrane distribution correlates with reduced Rac1 activity in AD brain tissue. Finally, BGIN contributes to Rac1 inhibition and ROS generation in an amyloid precursor protein (APP) proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relay poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context.

Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex.

Chromatin boundaries subdivide eukaryotic chromosomes into functionally autonomous domains of genetic activity. This subdivision insulates genes and/or regulatory elements within a domain from promiscuous interactions with nearby domains. While it was previously assumed that the chromosomal domain landscape is fixed, there is now growing evidence that the landscape may be subject to tissue and stage specific regulation. Here we report the isolation and characterization of a novel developmentally restricted boundary factor, Elba. We show that Elba is an unusual hetero-tripartite protein complex that requires all three proteins for DNA binding and insulator activity.DOI:http://dx.doi.org/10.7554/eLife.00171.001.

Identification of novel targets for miR-29a using miRNA proteomics.

MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3' Untranslated Regions (3' UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.

PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential.

Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development.

The functioning of the Drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity.

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orb(F303), which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach we identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, we show that ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, we find that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, we show that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase.

Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor.

LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.

Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation.

Protein kinase CK2 is one of the most conserved kinases in eukaryotic cells and plays essential roles in diverse processes. While we know that CK2 plays a role(s) in cell division, our understanding of how CK2 regulates cell cycle progression is limited. In this study, we revealed a regulatory role for CK2 in kinetochore function. The kinetochore is a multi-protein complex that assembles on the centromere of a chromosome and functions to attach chromosomes to spindle microtubules. To faithfully segregate chromosomes and maintain genomic integrity, the kinetochore is tightly regulated by multiple mechanisms, including phosphorylation by Aurora B kinase. We found that a loss of CK2 kinase activity inhibits anaphase spindle elongation and results in chromosome missegregation. Moreover, a lack of CK2 activates the spindle assembly checkpoint. We demonstrate that CK2 associates with Mif2, the Saccharomyces cerevisiae homologue of human CENP-C, which serves as an important link between the inner and outer kinetochore. Furthermore, we show Mif2 and the inner kinetochore protein Ndc10 are phosphorylated by CK2, and this phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of these targets, respectively.

Accumulation of noncoding RNA due to an RNase P defect in Saccharomyces cerevisiae.

Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5' leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition, as bacterial RNase P contains a catalytic RNA core and one protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine protein subunits. The additional eukaryotic protein subunits most likely provide additional functionality to RNase P, with one possibility being additional RNA recognition capabilities. To investigate the possible range of additional RNase P substrates in vivo, a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turn over pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively nonspecific capacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.

Mitotic spindle proteomics in Chinese hamster ovary cells.

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

TFG-1 function in protein secretion and oncogenesis.

Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.

Integrin αß1, αvß, α6ß effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance.

Ligand engagement by integrins induces receptor clustering and formation of complexes at the integrin cytoplasmic face that controls cell signaling and cytoskeletal dynamics critical for adhesion-dependent processes. This study searches for a subset of integrin effectors that coordinates both tumor cell invasion and resistance to the chemotherapeutic drug cisplatin in oral carcinomas. Candidate integrin effectors were identified in a proteomics screen of proteins recruited to clustered integrin αß1, α(v)ß or α(6)ß receptors in oral carcinomas. Proteins with diverse functions including microtubule and actin binding proteins, and factors involved in trafficking, transcription and translation were identified in oral carcinoma integrin complexes. Knockdown of effectors in the oral carcinoma HN12 cells revealed that p130Cas, Dek, Src and talin were required for invasion through Matrigel. Disruption of talin or p130Cas by RNA interference increased resistance to cisplatin, whereas targeting Dek, Src or zyxin reduced HN12 resistance to cisplatin. Analysis of the spreading of HN12 cells on collagen I and laminin I revealed that a decrease in p130Cas or talin expression inhibited spreading on both matrices. Interestingly, a reduction in zyxin expression enhanced spreading on laminin I and inhibited spreading on collagen I. Reduction of Dek, Src, talin or zyxin expression reduced HN12 proliferation by 30%. Proliferation was not affected by a reduction in p130Cas expression. We conclude that p130Cas, Src and talin function in both oral carcinoma invasion and resistance to cisplatin.