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

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 26 (2015): 3520-3534, doi:10.1091/mbc.E15-02-0095.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.Work in F.V.’s laboratory is supported by National Institutes of Health R01 Grant GM095867. Part of this work was also supported by National Science Foundation Grant 0745129. J.R.Y. is supported by National Institutes of Health Grants P41 GM103533 and R01 MH 067880

T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest

Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites

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 orbF303, 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

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

Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests

<div><p>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 <i>in vitro</i>, 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.</p></div

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

Role of Oxysterol Binding Protein in Hepatitis C Virus infection▿ †

Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (ΔPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process

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

Phosphorylation state defines discrete roles for monopolin in chromosome attachment and spindle elongation

Background: it is unknown how oscillations in Cdk1 activity drive the dramatic changes in chromosome and spindle dynamics that occur at the metaphase/anaphase transition. Results: we show that the Schizosaccharomyces pombe monopolin complex has distinct functions in metaphase and anaphase that are determined by the phosphorylation state of its Mde4 subunit. When Cdk1 activity is high in metaphase, Mde4 is hyperphosphorylated on Cdk1 phosphorylation sites and localizes to kinetochores. A nonphosphorylatable mutant of Mde4 does not localize to kinetochores, appears prematurely on the metaphase spindle, and interferes with spindle dynamics and chromosome segregation, illustrating the importance of Cdk1 phosphorylation in regulating metaphase monopolin activity. When Cdk1 activity drops in anaphase, dephosphorylation of Mde4 triggers monopolin localization to the mitotic spindle, where it promotes spindle elongation and integrity, coupling the late mitotic loss of Cdk1 activity to anaphase spindle dynamics. Conclusions: together, these findings illustrate how the sequential phosphorylation and dephosphorylation of monopolin helps ensure the orderly execution of discrete steps in mitosis