scholarly journals Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

2015 ◽  
Vol 208 (4) ◽  
pp. 401-414 ◽  
Author(s):  
Joseph E. Klebba ◽  
Brian J. Galletta ◽  
Jonathan Nye ◽  
Karen M. Plevock ◽  
Daniel W. Buster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Binding Domain ◽  
Terminal Region ◽  
Scaffolding Protein ◽  
Binding Partner ◽  
Centriole Duplication ◽  
Binding Domains ◽  
C Terminus ◽  
N Terminus

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

scholarly journals Regulation of Cell Cycle Transcription Factor Swi4 through Auto-Inhibition of DNA Binding

1999 ◽  
Vol 19 (10) ◽  
pp. 6729-6741 ◽  
Author(s):  
Kristin Baetz ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Domain ◽  
Terminal Region ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Dna Binding Domain ◽  
C Terminus ◽  
Binding Factor

ABSTRACTInSaccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G1/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G1phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA intrans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA.

scholarly journals Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

2015 ◽  
Vol 112 (22) ◽  
pp. 6991-6996 ◽  
Author(s):  
Takashi Suzuki ◽  
Miho Suzuki ◽  
Shinji Ogino ◽  
Ryo Umemoto ◽  
Noritaka Nishida ◽  
...  
Keyword(s):  
Shear Stress ◽  
Conformational Change ◽  
Mechanical Force ◽  
Binding Domain ◽  
Terminal Region ◽  
Hematopoietic Progenitor Cell ◽  
High Shear ◽  
Cell Rolling ◽  
High Affinity ◽  
C Terminus

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD–HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

Mimicking cotranslational folding of prosubtilisin E in vitro

2020 ◽  
Vol 167 (5) ◽  
pp. 473-482 ◽  
Author(s):  
Sung-Gun Kim ◽  
Yu-Jen Chen ◽  
Liliana Falzon ◽  
Jean Baum ◽  
Masayori Inouye
Keyword(s):  
Crucial Role ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Secondary Structures ◽  
Terminal Region ◽  
Folding Intermediates ◽  
C Terminus ◽  
Cotranslational Folding ◽  
N Terminus

Abstract Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

scholarly journals Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

1991 ◽  
Vol 279 (3) ◽  
pp. 787-792 ◽  
Author(s):  
D M Poole ◽  
A J Durrant ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Catalytic Domain ◽  
Binding Capacity ◽  
Binding Domains ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Fusion Enzymes ◽  
Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

scholarly journals Binding of STIL to Plk4 activates kinase activity to promote centriole assembly

2015 ◽  
Vol 209 (6) ◽  
pp. 863-878 ◽  
Author(s):  
Tyler C. Moyer ◽  
Kevin M. Clutario ◽  
Bramwell G. Lambrus ◽  
Vikas Daggubati ◽  
Andrew J. Holland
Keyword(s):  
Cell Cycle ◽  
Gene Editing ◽  
Genetic System ◽  
Genome Integrity ◽  
Chemical Genetic ◽  
Centriole Duplication ◽  
C Terminus ◽  
A Cell ◽  
Direct Binding ◽  
Centriole Assembly

Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

scholarly journals Dissociating the Centrosomal Matrix Protein AKAP450 from Centrioles Impairs Centriole Duplication and Cell Cycle Progression

2003 ◽  
Vol 14 (6) ◽  
pp. 2436-2446 ◽  
Author(s):  
Guy Keryer ◽  
Oliwia Witczak ◽  
Annie Delouvée ◽  
Wolfram A. Kemmner ◽  
Danielle Rouillard ◽  
...  
Keyword(s):  
Cell Division ◽  
Hela Cells ◽  
Cellular Localization ◽  
Cell Cycle Progression ◽  
Matrix Protein ◽  
Coiled Coil ◽  
Cell Division Cycle ◽  
Scaffolding Protein ◽  
Centriole Duplication ◽  
C Terminus

Centrosomes provide docking sites for regulatory molecules involved in the control of the cell division cycle. The centrosomal matrix contains several proteins, which anchor kinases and phosphatases. The large A-Kinase Anchoring Protein AKAP450 is acting as a scaffolding protein for other components of the cell signaling machinery. We selectively perturbed the centrosome by modifying the cellular localization of AKAP450. We report that the expression in HeLa cells of the C terminus of AKAP450, which contains the centrosome-targeting domain of AKAP450 but not its coiled-coil domains or binding sites for signaling molecules, leads to the displacement of the endogenous centrosomal AKAP450 without removing centriolar or pericentrosomal components such as centrin, γ-tubulin, or pericentrin. The centrosomal protein kinase A type II α was delocalized. We further show that this expression impairs cytokinesis and increases ploidy in HeLa cells, whereas it arrests diploid RPE1 fibroblasts in G1, thus further establishing a role of the centrosome in the regulation of the cell division cycle. Moreover, centriole duplication is interrupted. Our data show that the association between centrioles and the centrosomal matrix protein AKAP450 is critical for the integrity of the centrosome and for its reproduction.

scholarly journals Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

2021 ◽  
Author(s):  
Seiji Kojima ◽  
Hiroki Kajino ◽  
Keiichi Hirano ◽  
Yuna Inoue ◽  
Hiroyuki Terashima ◽  
...  
Keyword(s):  
Basal Body ◽  
Ring Formation ◽  
Terminal Region ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Bacterial Flagellum ◽  
Transmembrane Segments ◽  
Ring Assembly ◽  
C Terminus ◽  
N Terminus

AbstractThe MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

scholarly journals Repression of a herpes simplex virus immediate-early promoter by the Oct-2 transcription factor is dependent on an inhibitory region at the N terminus of the protein

1994 ◽  
Vol 14 (11) ◽  
pp. 7633-7642
Author(s):  
K A Lillycrop ◽  
S J Dawson ◽  
J K Estridge ◽  
T Gerster ◽  
P Matthias ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Critical Role ◽  
Terminal Region ◽  
Early Gene ◽  
Immediate Early ◽  
C Terminus ◽  
N Terminus ◽  
Simplex Virus

The B-cell form of the Oct-2 transcription factor Oct 2.1 can activate the herpes simplex virus immediate-early gene 3 (IE3) promoter, whereas the neuronally expressed Oct 2.4 and 2.5 forms of the protein, which contain a different C terminus, can repress this promoter. Here we show that partial or full deletion of the C terminus of Oct 2.1 in the presence of an intact N terminus results in a protein which can strongly repress the IE3 promoter. In contrast, deletion of the entire N terminus or a short region within it leaving the C terminus intact results in a very strong activator. Deletion of both N and C termini leaving only the isolated POU domain generates only a very weak repressor. The N-terminal region defined in this way can repress a heterologous promoter when linked to the DNA-binding domain of the GAL4 factor, indicating that it can function as an independent inhibitory domain. These results indicate that a specific region within the N terminus common to Oct 2.1, 2.4, and 2.5 plays a critical role in the ability of neuronally expressed forms of Oct-2 to repress the IE3 promoter but can do so only when the C-terminal region of Oct 2.1 is altered or deleted.

scholarly journals Purification and Properties of the Klebsiella aerogenes UreE Metal-Binding Domain, a Functional Metallochaperone of Urease

2005 ◽  
Vol 187 (10) ◽  
pp. 3581-3585 ◽  
Author(s):  
Scott B. Mulrooney ◽  
Sarah K. Ward ◽  
Robert P. Hausinger
Keyword(s):  
Metal Binding ◽  
Binding Domain ◽  
Klebsiella Aerogenes ◽  
Nickel Ions ◽  
Binding Domains ◽  
C Terminus ◽  
Purification And Properties ◽  
Metal Binding Domain ◽  
Urease Activation ◽  
Distinct Peptide

ABSTRACT Klebsiella aerogenes UreE, a metallochaperone that delivers nickel ions during urease activation, consists of distinct “peptide-binding” and “metal-binding” domains and a His-rich C terminus. Deletion analyses revealed that the metal-binding domain alone is sufficient to facilitate urease activation. This domain was purified and shown to exhibit metal-binding properties similar to those of UreE lacking only the His-rich tail.

scholarly journals Mdm2 Directs the Ubiquitination of β-Arrestin-sequestered cAMP Phosphodiesterase-4D5

2009 ◽  
Vol 284 (24) ◽  
pp. 16170-16182 ◽  
Author(s):  
Xiang Li ◽  
George S. Baillie ◽  
Miles D. Houslay
Keyword(s):  
Terminal Region ◽  
Scaffolding Protein ◽  
Double Knockout ◽  
Camp Phosphodiesterase ◽  
Small Interference Rna ◽  
Mouse Embryo Fibroblasts ◽  
C Terminus ◽  
Rna Knockdown ◽  
Degrading System

β-Arrestin plays a key role in regulating β2-adrenoreceptor signaling by interdicting activation of adenylyl cyclase and selectively sequestering cAMP phosphodiesterase-4D5 (PDE4D5) for delivery of an active cAMP degrading system to the site of cAMP synthesis. Here we show that the β-agonist, isoprenaline, triggers the rapid and transient ubiquitination of PDE4D5 in primary cardiomyocytes, mouse embryo fibroblasts, and HEK293B2 cells constitutively expressing β2-adrenoceptors. Reconstitution analyses in β-arrestin1/2 double knockout cells plus small interference RNA knockdown studies indicate that a β-arrestin-scaffolded pool of the E3-ubiquitin ligase, Mdm2, mediates PDE4D5 ubiquitination. Critical for this is the ubiquitin-interacting motif located in the extreme C terminus of PDE4D5, which is specific to the PDE4D sub-family. In vitro SUMOylation of a PDE4D5 spot-immobilized peptide array, followed by a mutagenesis strategy, showed that PDE4D5 ubiquitination occurs at Lys-48, Lys-53, and Lys-78, which are located within its isoform-specific N-terminal region, as well as at Lys-140 located within its regulatory UCR1 module. We suggest that mono-ubiquitination at Lys-140 primes PDE4D5 for a subsequent cascade of polyubiquitination occurring within its isoform-specific N-terminal region at Lys-48, Lys-53, and Lys-78. PDE4D5 interacts with a non-ubiquitinated β-arrestin sub-population that is likely to be protected from Mdm2-mediated ubiquitination due to steric hindrance caused by sequestered PDE4D5. Ubiquitination of PDE4D5 elicits an increase in the fraction of PDE4D5 sequestered by β-arrestin in cells, thereby contributing to the fidelity of PDE4D5-β-arrestin interaction, as well as decreasing the fraction of PDE4D5 sequestered by the scaffolding protein, RACK1.

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