PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified c entrosomal P 4.1‐a ssociated p rotein (CPAP), a human homologue of SAS‐4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage‐specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho‐resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo‐like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo‐like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.     

Fidgetin-like 1 is a ciliogenesis-inhibitory centrosome protein

Fidgetin-like 1 (FIGL-1) is a homolog of fidgetin, an AAA protein that was identified as the protein encoded by the gene mutated in fidget mice. Because the phenotypes of fidget mice are reminiscent of the phenotypes of ciliopathy diseases, and because fidgetin has microtubule-severing activity, we hypothesize that these proteins participate in cilia-related processes. Indeed, overexpression of FIGL-1 interfered with ciliogenesis in cultured cells. In particular, overexpressed FIGL-1 strongly accumulated at the centrosome, and, when highly expressed, perturbed the localization of centrosomal proteins such as pericentrin, CP110, and centrin. Using a polyclonal antibody against human FIGL-1, we found that endogenous FIGL-1 localized preferentially around the mother centriole. Consistently, depletion of FIGL-1 by shRNA treatment enhanced ciliogenesis in HEK293T cells. By checking the integrity of the cytoplasmic microtubule network in FIGL-1-overexpressing cells, we found that FIGL-1 probably has microtubule-severing activity, as suggested by its sequence homology with other microtubule-severing proteins. Furthermore, we showed that overexpression of FIGL-1 in zebrafish embryo decreased the length of cilia and perturbed the heart laterality. Taken together, these results demonstrate that FIGL-1 is a new centrosomal protein and inhibits ciliogenesis. These results extend the already long list of centrosomal proteins and provide new insights into the regulation of ciliogenesis.     

Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/- neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear "cage" to the centrosome. Lis1+/- neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.     

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A–mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.     

Suppression of centrosome duplication and amplification by deacetylases

Centrosome duplication is controlled both negatively and positively by a number of proteins. The activities and stabilities of those regulatory proteins are in many cases controlled by posttranslational modifications. Although acetylation and deacetylation are highly common posttranslational modifications, their roles in the regulation of centrosome duplication had not been closely examined. Here, through focusing on the deacetylases, we investigated the role of acetylation/deacetylation in the regulation of centrosome duplication and induction of abnormal amplification of centrosomes. We found that the deacetylation event negatively controls centrosome duplication and amplification. Of the 18 total known deacetylases (HDAC1–11, SIRT1–7), ten deacetylases possess the activity to suppress centrosome amplification, and their centrosome amplification suppressing activities are strongly associated with their abilities to localize to centrosomes. Among them, HDAC1, HDAC5 and SIRT1 show the highest suppressing activities, but each of them suppresses centrosome duplication and/or amplification with its unique mechanism.     

Suppression of centrosome duplication and amplification by deacetylases

Centrosome duplication is controlled both negatively and positively by a number of proteins. The activities and stabilities of those regulatory proteins are in many cases controlled by posttranslational modifications. Although acetylation and deacetylation are highly common posttranslational modifications, their roles in the regulation of centrosome duplication had not been closely examined. Here, through focusing on the deacetylases, we investigated the role of acetylation/deacetylation in the regulation of centrosome duplication and induction of abnormal amplification of centrosomes. We found that the deacetylation event negatively controls centrosome duplication and amplification. Of the 18 total known deacetylases (HDAC1–11, SIRT1–7), ten deacetylases possess the activity to suppress centrosome amplification, and their centrosome amplification suppressing activities are strongly associated with their abilities to localize to centrosomes. Among them, HDAC1, HDAC5 and SIRT1 show the highest suppressing activities, but each of them suppresses centrosome duplication and/or amplification with its unique mechanism.     

Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability

The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum . We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatin-binding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosome-nucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.     

Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis

Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes.     

Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.     

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.     

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.     

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.     

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portions of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434–1465, and 1661–1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434–1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.     

Myomegalin regulates Hedgehog pathway by controlling PDE4D at the centrosome

Mutations in the hedgehog (Hh) signaling are implicated in birth defects and cancers, including medulloblastoma (MB), one of the most malignant pediatric brain tumors. Current Hh inhibitors face the challenge of drug resistance and tumor relapse, urging new insights in the Hh pathway regulation. Our previous study revealed how PDE4D controls global levels of cAMP in the cytoplasm to positively regulate Hh signaling; in the present study, we found that a specific isoform PDE4D3 is tethered to the centrosome by Myomegalin (Mmg), a centrosome/Golgi-associated protein. Mmg loss dislocates PDE4D3 from the centrosome, leading to local PKA overactivation and inhibition of the Hh signaling, leaving other PKA-related pathways unaffected. Mmg loss suppresses the proliferation of granule neuron precursors and blocks the growth of MB in mouse model. Our findings specify a new regulatory mechanism of the Hh pathway and highlight an exciting therapeutic avenue for Hh-related cancers with reduced side effects.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.     

Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome “maturation” requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd‐2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.     

POPX2 phosphatase regulates cell polarity and centrosome placement

Proper centrosome positioning is critical for many cellular functions, such as cell migration and maintenance of polarity. During wound healing, fibroblasts orient their centrosomes such that they face the wound edge. The centrosome orientation determines the direction of cells’ migration so that they can close the wound effectively. In this study, we investigated the regulation of centrosome polarization and have identified the phosphatase POPX2 as an important regulator of centrosome orientation. We found that POPX2 inhibits centrosome centration, but not rearward nuclear movement, by regulating multiple proteins that function in centrosome positioning. High POPX2 levels result in reduced motility of the kinesin-2 motor, which, in turn, inhibits the transport of N-cadherin to the cell periphery and cell junctions. Loss of N-cadherin localization to the cell membrane affects the localization of focal adhesions and perturbs CDC42-Par6/PKCζ signaling. In addition, overexpression of POPX2 also results in a loss of Par3 localization to the cell periphery and reduced levels of LIC2 (dynein light intermediate chain 2), leading to defects in microtubule tethering and dynamics at cell-cell contacts. Therefore, POPX2 functions as a regulator of signaling pathways to modulate the positioning of centrosome in fibroblast during wound healing.