Binding of STIL to Plk4 activates kinase activity to promote 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.

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Fission Yeast F-box Protein Pof3 Is Required for Genome Integrity and Telomere Function

Molecular Biology of the Cell ◽

10.1091/mbc.01-07-0333 ◽

2002 ◽

Vol 13 (1) ◽

pp. 211-224 ◽

Cited By ~ 35

Author(s):

Satoshi Katayama ◽

Kenji Kitamura ◽

Anna Lehmann ◽

Osamu Nikaido ◽

Takashi Toda

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Ubiquitin Ligase ◽

Repeat Motif ◽

High Rate ◽

Genome Integrity ◽

Gene Encoding ◽

Cell Cycle Delay ◽

C Terminus ◽

F Box Protein

The Skp1-Cullin-1/Cdc53-F-box protein (SCF) ubiquitin ligase plays an important role in various biological processes. In this enzyme complex, a variety of F-box proteins act as receptors that recruit substrates. We have identified a fission yeast gene encoding a novel F-box protein Pof3, which contains, in addition to the F-box, a tetratricopeptide repeat motif in its N terminus and a leucine-rich-repeat motif in the C terminus, two ubiquitous protein–protein interaction domains. Pof3 forms a complex with Skp1 and Pcu1 (fission yeast cullin-1), suggesting that Pof3 functions as an adaptor for specific substrates. In the absence of Pof3, cells exhibit a number of phenotypes reminiscent of genome integrity defects. These include G2 cell cycle delay, hypersensitivity to UV, appearance of lagging chromosomes, and a high rate of chromosome loss.pof3 deletion strains are viable because the DNA damage checkpoint is continuously activated in the mutant, and this leads to G2 cell cycle delay, thereby preventing the mutant from committing lethal mitosis. Pof3 localizes to the nucleus during the cell cycle. Molecular analysis reveals that in this mutant the telomere is substantially shortened and furthermore transcriptional silencing at the telomere is alleviated. The results highlight a role of the SCFPof3 ubiquitin ligase in genome integrity via maintaining chromatin structures.

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The γ-tubulin meshwork assists in the recruitment of PCNA to chromatin in mammalian cells

Communications Biology ◽

10.1038/s42003-021-02280-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Matthieu Corvaisier ◽

Jingkai Zhou ◽

Darina Malycheva ◽

Nicola Cornella ◽

Dimitrios Chioureas ◽

...

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Mammalian Cells ◽

Nuclear Accumulation ◽

Genome Integrity ◽

Nuclear Antigen ◽

Free System ◽

Nuclear Compartment ◽

C Terminus ◽

A Cell ◽

Proliferating Cell

AbstractChanges in the location of γ-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of γ-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the γ-tubulin meshwork assists in the recruitment of PCNA to chromatin. Also, decreased levels of γ-tubulin reduce the nuclear pool of PCNA. In addition, the γ-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a γ-tubulin–PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and γ-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the γ-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.

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

The Journal of Cell Biology ◽

10.1083/jcb.201410105 ◽

2015 ◽

Vol 208 (4) ◽

pp. 401-414 ◽

Cited By ~ 26

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.

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CEP120 interacts with CPAP and positively regulates centriole elongation

The Journal of Cell Biology ◽

10.1083/jcb.201212060 ◽

2013 ◽

Vol 202 (2) ◽

pp. 211-219 ◽

Cited By ~ 61

Author(s):

Yi-Nan Lin ◽

Chien-Ting Wu ◽

Yu-Chih Lin ◽

Wen-Bin Hsu ◽

Chieh-Ju C. Tang ◽

...

Keyword(s):

Cell Cycle ◽

Dimerization Domain ◽

Interacting Protein ◽

Centrosomal Protein ◽

Microtubule Binding ◽

Protein 4.1 ◽

Centriole Duplication ◽

Localization Domain ◽

Microtubule Binding Domain ◽

A Cell

Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1–associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle–regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding–defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.

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APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer

The EMBO Journal ◽

10.1038/emboj.2011.490 ◽

2012 ◽

Vol 31 (5) ◽

pp. 1160-1176 ◽

Cited By ~ 38

Author(s):

Rachel Brough ◽

Ilirjana Bajrami ◽

Radost Vatcheva ◽

Rachael Natrajan ◽

Jorge S Reis-Filho ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Genome Integrity ◽

Interacting Protein ◽

A Cell

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E1A Blocks Hyperphosphorylation of p130 and p107 without Affecting the Phosphorylation Status of the Retinoblastoma Protein

Journal of Virology ◽

10.1128/jvi.74.7.3166-3176.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3166-3176 ◽

Cited By ~ 11

Author(s):

Matilde Parreño ◽

Judit Garriga ◽

Ana Limón ◽

Xavier Mayol ◽

George R. Beck ◽

...

Keyword(s):

Cell Cycle ◽

Cdk Inhibitor ◽

Dependent Manner ◽

Pocket Proteins ◽

Cell Cycle Entry ◽

293 Cells ◽

A Cell ◽

Direct Binding ◽

Abnormal Pattern ◽

Cell Growth And Differentiation

ABSTRACT The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G1/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of “free” hyperphosphorylated p130, which could act as a CDK inhibitor.

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A molecular mechanism for the procentriole recruitment of Ana2

10.1101/647149 ◽

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.

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