scholarly journals A molecular mechanism for the procentriole recruitment of Ana2

2019 ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Sarah K. Speed ◽  
Daniel W. Buster ◽  
Carey J. Fagerstrom ◽  
Brian J. Galletta ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Kinase Activity ◽  
Surface Protein ◽  
Single Site ◽  
Centriole Duplication ◽  
C Terminus ◽  
N Terminus ◽  
Mother Centriole ◽  
Centriole Assembly ◽  
Daughter Centriole

AbstractCentriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STIL’s C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2’s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2’s N-terminus which increases Ana2’s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

scholarly journals A molecular mechanism for the procentriole recruitment of Ana2

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Tiffany A. McLamarrah ◽  
Sarah K. Speed ◽  
John M. Ryniawec ◽  
Daniel W. Buster ◽  
Carey J. Fagerstrom ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Kinase Activity ◽  
Outer Wall ◽  
Single Site ◽  
Structural Motif ◽  
Centriole Duplication ◽  
Short Region ◽  
N Terminus ◽  
Mother Centriole ◽  
Daughter Centriole

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2–G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.

scholarly journals Autophosphorylation of Polo-like Kinase 4 and Its Role in Centriole Duplication

2010 ◽  
Vol 21 (4) ◽  
pp. 547-561 ◽  
Author(s):  
James E. Sillibourne ◽  
Frederik Tack ◽  
Nele Vloemans ◽  
An Boeckx ◽  
Sathiesan Thambirajah ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Activity ◽  
Proteasome Inhibition ◽  
Centrosome Duplication ◽  
Active Fraction ◽  
Kinase Activation ◽  
Centriole Duplication ◽  
M Phase ◽  
Mother Centriole ◽  
Daughter Centriole

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.

scholarly journals Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state

2018 ◽  
Vol 29 (23) ◽  
pp. 2874-2886 ◽  
Author(s):  
Cody J. Boese ◽  
Jonathan Nye ◽  
Daniel W. Buster ◽  
Tiffany A. McLamarrah ◽  
Amy E. Byrnes ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Dominant Role ◽  
Kinase Domain ◽  
Feedback Mechanism ◽  
Phosphorylation State ◽  
Negative Feedback Mechanism ◽  
Centriole Duplication ◽  
C Terminus ◽  
N Terminus ◽  
Opposing Effects

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole “targeting-factor.” In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4’s kinase domain. Thus, Asl-A’s phosphorylation state determines which of Asl-A’s two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

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 CD4+ T Lymphocytes from Anaplasma marginale Major Surface Protein 2 (MSP2) Vaccinees Recognize Naturally Processed Epitopes Conserved in MSP3

2004 ◽  
Vol 72 (6) ◽  
pp. 3688-3692 ◽  
Author(s):  
Wendy C. Brown ◽  
Guy H. Palmer ◽  
Kelly A. Brayton ◽  
Patrick F. M. Meeus ◽  
Anthony F. Barbet ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
T Lymphocyte ◽  
Antigenic Variation ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Lymphocyte Response ◽  
Major Surface Protein ◽  
C Terminus ◽  
N Terminus ◽  
Major Surface

ABSTRACT Major surface protein 2 (MSP2) and MSP3 of the persistent bovine ehrlichial pathogen Anaplasma marginale are immunodominant proteins that undergo antigenic variation. The recently completed sequence of MSP3 revealed blocks of amino acids in the N and C termini that are conserved with MSP2. This study tested the hypothesis that CD4+ T cells specific for MSP2 recognize naturally processed epitopes conserved in MSP3. At least one epitope in the N terminus and two in the C terminus of MSP2 were also processed from MSP3 and presented to CD4+ T lymphocytes from MSP2-immunized cattle. This T-lymphocyte response to conserved and partially conserved epitopes may contribute to the immunodominance of MSP2 and MSP3.

scholarly journals A Reduced Risk of Infection with Plasmodium vivax and Clinical Protection against Malaria Are Associated with Antibodies against the N Terminus but Not the C Terminus of Merozoite Surface Protein 1

2006 ◽  
Vol 74 (5) ◽  
pp. 2726-2733 ◽  
Author(s):  
Paulo Afonso Nogueira ◽  
Fabiana Piovesan Alves ◽  
Carmen Fernandez-Becerra ◽  
Oliver Pein ◽  
Neida Rodrigues Santos ◽  
...  
Keyword(s):  
Immune Responses ◽  
Plasmodium Vivax ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Risk Of Infection ◽  
Merozoite Surface Protein 1 ◽  
C Terminus ◽  
N Terminus ◽  
Clinical Protection ◽  
Reduced Risk

ABSTRACT Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondônia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite.

scholarly journals Centrobin–tubulin interaction is required for centriole elongation and stability

2011 ◽  
Vol 193 (4) ◽  
pp. 711-725 ◽  
Author(s):  
Radhika Gudi ◽  
Chaozhong Zou ◽  
Jun Li ◽  
Qingshen Gao
Keyword(s):  
Molecular Mechanism ◽  
Early Stage ◽  
Centrosome Duplication ◽  
Centriole Duplication ◽  
The Stability ◽  
Daughter Centriole

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins.

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 Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly

2010 ◽  
Vol 191 (2) ◽  
pp. 331-346 ◽  
Author(s):  
Moe R. Mahjoub ◽  
Zhigang Xie ◽  
Tim Stearns
Keyword(s):  
Basal Bodies ◽  
Animal Cells ◽  
Tracheal Epithelial Cells ◽  
Centriole Duplication ◽  
Photobleaching Recovery ◽  
Mother And Daughter ◽  
Tracheal Epithelial ◽  
Centriole Assembly ◽  
And Function ◽  
Daughter Centriole

Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase–arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.

scholarly journals N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization

2008 ◽  
Vol 411 (2) ◽  
pp. 407-414 ◽  
Author(s):  
Ritu Garg ◽  
Kirsi Riento ◽  
Nicholas Keep ◽  
Jonathan D. H. Morris ◽  
Anne J. Ridley
Keyword(s):  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Actin Reorganization ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Coiled Coil Region ◽  
N Terminus ◽  
In Cells

ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I1–420 is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.

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