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 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 PLK4-phosphorylated NEDD1 facilitates cartwheel assembly and centriole biogenesis initiations

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Wangfei Chi ◽  
Gang Wang ◽  
Guangwei Xin ◽  
Qing Jiang ◽  
Chuanmao Zhang
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Centrosome Duplication ◽  
Spatiotemporal Regulation ◽  
Mother Centriole ◽  
Pericentriolar Material ◽  
Daughter Centriole

Centrosome duplication occurs under strict spatiotemporal regulation once per cell cycle, and it begins with cartwheel assembly and daughter centriole biogenesis at the lateral sites of the mother centrioles. However, although much of this process is understood, how centrosome duplication is initiated remains unclear. Here, we show that cartwheel assembly followed by daughter centriole biogenesis is initiated on the NEDD1-containing layer of the pericentriolar material (PCM) by the recruitment of SAS-6 to the mother centriole under the regulation of PLK4. We found that PLK4-mediated phosphorylation of NEDD1 at its S325 amino acid residue directly promotes both NEDD1 binding to SAS-6 and recruiting SAS-6 to the centrosome. Overexpression of phosphomimicking NEDD1 mutant S325E promoted cartwheel assembly and daughter centriole biogenesis initiations, whereas overexpression of nonphosphorylatable NEDD1 mutant S325A abolished the initiations. Collectively, our results demonstrate that PLK4-regulated NEDD1 facilitates initiation of the cartwheel assembly and of daughter centriole biogenesis in mammals.

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 Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

scholarly journals Casein Kinase II is Required for Proper Cell Division and Acts as a Negative Regulator of Centrosome Duplication in C. elegans Embryos

2016 ◽  
Author(s):  
Jeffrey C. Medley ◽  
Megan M. Kabara ◽  
Michael D. Stubenvoll ◽  
Lauren E. DeMeyer ◽  
Mi Hye Song
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Cycle Progression ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Negative Regulator ◽  
Centrosome Duplication ◽  
Dependent Manner ◽  
C Elegans ◽  
Mother Centriole

Summary statementThe conserved protein kinase CK2 negatively regulates centrosome assembly and is required for proper cell cycle progression and cytokinesis in early C. elegans embryos.AbstractCentrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein Kinase II (CK2) in early C. elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 1017-1025 ◽  
Author(s):  
M.H. Verlhac ◽  
J.Z. Kubiak ◽  
H.J. Clarke ◽  
B. Maro
Keyword(s):  
Cell Cycle ◽  
Myelin Basic Protein ◽  
Map Kinase ◽  
Kinase Activity ◽  
Basic Protein ◽  
Chromatin Condensation ◽  
Meiotic Maturation ◽  
Protein Kinase Activity ◽  
Microtubule Organization ◽  
Kinase Activation

Oocyte meiotic maturation is triggered by different stimuli (hormones, unknown signals through cell interactions) in different species. These stimuli indirectly lead to the activation of a major cell cycle regulating activity, the maturation promoting factor (MPF). Other factors, such as the product of the proto-oncogene c-mos or enzymes of the MAP kinase family, are also involved in the process of maturation. MAP kinase activation occurs during meiotic maturation in oocytes from different species with different kinetics. The relationships between MPF activation and MAP kinase activation have been well studied in species such as clam and Xenopus. In this paper, we study the precise timing of MAP kinase activation (as measured by phosphorylation of exogenous myelin basic protein and shifts in mobility of ERK 1 and ERK 2) versus MPF activation (as measured by phosphorylation of exogenous histone H1) during mouse oocyte maturation and, in parallel, morphological events such as changes in microtubule organization and chromatin condensation. We observed that MAP kinase activation was delayed after MPF activation and that this activity persisted throughout maturation whereas MPF activity dropped between the two meiotic metaphases. After parthenogenetic activation of ovulated eggs, MAP kinase inactivation was very slow compared to MPF inactivation. During the first mitotic cell cycle, a rise in myelin basic protein kinase activity at M-phase was observed but it was not related to MAP kinase activation. Furthermore, microtubules and chromatin remained in a metaphase-like state during the complete period of maturation (including the period between the two meiotic metaphases) and a few hours after activation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cell Cycle Regulation of the Saccharomyces cerevisiae Polo-Like Kinase Cdc5p

1998 ◽  
Vol 18 (12) ◽  
pp. 7360-7370 ◽  
Author(s):  
Liang Cheng ◽  
Linda Hunke ◽  
Christopher F. J. Hardy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Kinase Activity ◽  
Anaphase Promoting Complex ◽  
Mitotic Kinases ◽  
Polo Kinase ◽  
Evolutionarily Conserved ◽  
M Phase ◽  
Cycle Regulation

ABSTRACT Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G2/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.

scholarly journals Regulation of Cdc2/Cyclin B Activation in Xenopus Egg Extracts via Inhibitory Phosphorylation of Cdc25C Phosphatase by Ca2+/Calmodium-dependent Kinase II

2003 ◽  
Vol 14 (10) ◽  
pp. 4003-4014 ◽  
Author(s):  
James R. A. Hutchins ◽  
Dina Dikovskaya ◽  
Paul R. Clarke
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Checkpoint Kinases ◽  
M Phase ◽  
Egg Extracts ◽  
Inhibitory Site

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.

scholarly journals TheSac3Homologueshd1Is Involved in Mitotic Progression in Mammalian Cells

2004 ◽  
Vol 279 (44) ◽  
pp. 46182-46190 ◽  
Author(s):  
Sefat-e- Khuda ◽  
Mikoto Yoshida ◽  
Yan Xing ◽  
Tatsuya Shimasaki ◽  
Motohiro Takeya ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Spindle Assembly Checkpoint ◽  
Fluorescent Protein ◽  
Spindle Assembly ◽  
Centrosome Duplication ◽  
Cycle Progression ◽  
M Phase ◽  
Green Fluorescent

SaccharomycesSac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to α-tubulin, at M phase. RNA interference suppression of endogenousshd1caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection withshd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated thatshd1is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function.

scholarly journals Phosphorylation of p21 in G2/M Promotes Cyclin B-Cdc2 Kinase Activity

2005 ◽  
Vol 25 (8) ◽  
pp. 3364-3387 ◽  
Author(s):  
Bipin C. Dash ◽  
Wafik S. El-Deiry
Keyword(s):  
Kinase Activity ◽  
Cyclin B1 ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
M Phase ◽  
Dependent Protein

ABSTRACT Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21−/− cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.

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