scholarly journals Kinetochore recruitment of CENP-F illustrates how paralog divergence shapes kinetochore composition and function

2018 ◽  
Author(s):  
Giuseppe Ciossani ◽  
Katharina Overlack ◽  
Arsen Petrovic ◽  
Pim Huis in ‘t Veld ◽  
Carolin Körner ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Kinase Domain ◽  
Mitotic Checkpoint ◽  
Checkpoint Control ◽  
Directed Motion ◽  
Coil Structure ◽  
Evolutionary Relatedness ◽  
Chromosome Alignment ◽  
And Function ◽  
Paralogous Proteins

The metazoan proteins CENP-E and CENP-F are components of a fibrous layer of mitotic kinetochores named the corona. Several features suggest that CENP-E and CENP-F are paralogs: they are very large (approximately 2700 and 3200 residues, respectively), rich in predicted coiled-coil structure, C-terminally prenylated, and endowed with microtubule-binding sites at their termini. In addition, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus-end directed motion. Here, we show that CENP-E and CENP- F are recruited to mitotic kinetochores independently of the Rod-Zwilch-ZW10 (RZZ) complex, the main corona constituent. We identify selective interactions of CENP-E and CENP-F respectively with BubR1 and Bub1, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. While BubR1 is dispensable for kinetochore localization of CENP-E, Bub1 is stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrate that the CENP-E:BubR1 and CENP-F:Bub1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BubR1 or Bub1. Our findings are consistent with the existence of ‘pseudo-symmetric’, paralogous Bub1:CENP-F and BubR1:CENP-E axes, supporting evolutionary relatedness of CENP-E and CENP-F.

Molecular characterisation of ninein, a new coiled-coil protein of the centrosome

1996 ◽  
Vol 109 (1) ◽  
pp. 179-190 ◽  
Author(s):  
V. Bouckson-Castaing ◽  
M. Moudjou ◽  
D.J. Ferguson ◽  
S. Mucklow ◽  
Y. Belkaid ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Coiled Coil ◽  
Immunocytochemical Staining ◽  
Mouse Tissues ◽  
Coil Structure ◽  
Ef Hand ◽  
Alternatively Spliced ◽  
Polyclonal Antisera ◽  
And Function ◽  
Recombinant Fragments

We describe the cDNA cloning of ninein, a novel component of centrosomes. In the mouse, ninein is predicted to be an acidic protein (calculated pI of 4.8) with alternatively spliced forms of 245 kDa and 249 kDa that contain extensive regions of coiled-coil structure flanked by non-coiled ends. Other interesting features of this protein include an EF-hand-like domain, a potential GTP binding site and four leucine zipper domains. Specific polyclonal antisera were raised to two non-overlapping recombinant fragments of the protein and used to characterise the cellular distribution of ninein. Immunofluorescence and immunoelectron microscopy experiments with macrophage-like cells, Mm1, showed that ninein is localised specifically in the pericentriolar matrix of the centrosome. Studies with NIH3T3 fibroblasts demonstrated that ninein is associated with the centrosome throughout the cell cycle and can also be detected within nuclei at interphase. At mitosis ninein was also observed in association with the mitotic spindle. Immunocytochemical staining of mouse tissues showed that ninein was expressed in a heterogeneous fashion. Staining, if present, was always consistent with a centrosomal localisation and was never associated with nuclei. Ninein provides a new molecular tool for analysing the structure and function of the centrosome.

scholarly journals Activity of the Ste20-like kinase, SLK, is enhanced by homodimerization

2011 ◽  
Vol 301 (3) ◽  
pp. F554-F564 ◽  
Author(s):  
Sierra Delarosa ◽  
Julie Guillemette ◽  
Joan Papillon ◽  
Ying-Shan Han ◽  
Arnold S. Kristof ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Gel Filtration ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
Fk506 Binding Protein ◽  
Induced Apoptosis ◽  
Injury And Repair

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1–373) and a modified FK506 binding protein, Fv (Fv-SLK 1–373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1–373. In an in vitro kinase assay, the dimeric Fv-SLK 1–373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1–373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1–373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1–373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.

scholarly journals Roles of the novel coiled-coil protein Rng10 in septum formation during fission yeast cytokinesis

2016 ◽  
Vol 27 (16) ◽  
pp. 2528-2541 ◽  
Author(s):  
Yajun Liu ◽  
I-Ju Lee ◽  
Mingzhai Sun ◽  
Casey A. Lower ◽  
Kurt W. Runge ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Cellular Localization ◽  
Affinity Purification ◽  
Coiled Coil ◽  
The Novel ◽  
Septum Formation ◽  
Division Site ◽  
And Function

Rho GAPs are important regulators of Rho GTPases, which are involved in various steps of cytokinesis and other processes. However, regulation of Rho-GAP cellular localization and function is not fully understood. Here we report the characterization of a novel coiled-coil protein Rng10 and its relationship with the Rho-GAP Rga7 in fission yeast. Both rng10Δ and rga7Δ result in defective septum and cell lysis during cytokinesis. Rng10 and Rga7 colocalize on the plasma membrane at the cell tips during interphase and at the division site during cell division. Rng10 physically interacts with Rga7 in affinity purification and coimmunoprecipitation. Of interest, Rga7 localization is nearly abolished without Rng10. Moreover, Rng10 and Rga7 work together to regulate the accumulation and dynamics of glucan synthases for successful septum formation in cytokinesis. Our results show that cellular localization and function of the Rho-GAP Rga7 are regulated by a novel protein, Rng10, during cytokinesis in fission yeast.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

scholarly journals Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

Open Biology ◽  
2014 ◽  
Vol 4 (3) ◽  
pp. 130172 ◽  
Author(s):  
Barbara Franke ◽  
Alexander Gasch ◽  
Dayté Rodriguez ◽  
Mohamed Chami ◽  
Muzamil M. Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Quaternary Structure ◽  
E3 Ubiquitin Ligase ◽  
Coiled Coil ◽  
Trim Protein ◽  
Muscle Catabolism ◽  
And Function ◽  
Helical Region ◽  
Structural Significance

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

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