hNinein is required for targeting spindle-associated protein Astrin to the centrosome during the S and G2 phases

Human Ninein (hNinein) is implicated in centrosomal microtubule nucleation and microtubule anchoring in interphase cells and may act as a scaffold protein, but its direct interaction partners remain unexplored in the centrosome. In this report, we show clearly that a spindle-associated protein, Astrin, interacts and co-localizes with hNinein at the centrosome during the S and G2 phases, and this complex may dissociate in the M phase. We also demonstrate that the truncated forms of hNinein, which could interfere with gamma-tubulin and function as dominant-negative mutants, are able to affect Astrin localization to the centrosome. Moreover, siRNA-mediated knockdown of hNinein in HeLa cells causes Astrin to fail to target to the centrosome, whereas hNinein can localize at the centrosome in the absence of Astrin. In addition, reduction in hNinein protein levels causes mislocalization of Astrin with the spindle apparatus and results in the formation of an aberrant mitotic spindle. Collectively, these data suggest that hNinein is required for targeting Astrin to the centrosome during the S and G2 phases. We therefore propose a model wherein hNinein regulates the dynamic movement of Astrin throughout the cell cycle and this interaction, in turn, is required for maintenance of centrosome/spindle pole integrity.     

Characterization and Functional Aspects of Human Ninein Isoforms that Regulated by Centrosomal Targeting Signals and Evidence for Docking Sites to Direct Gamma-Tubulin

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring, and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3β. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits γ-tubulin to centrosomal or non-centrosomal hNinein-containing sites, implying that the γ-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the γ-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for γ-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full-length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit γ-tubulin at centrosomal and non-centrosomal sites.     

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.     

Assembly of centrosomal proteins and microtubule organization depends on PCM-1

The protein PCM-1 localizes to cytoplasmic granules known as "centriolar satellites" that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1-containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization.     

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.     

TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification.     

Human Ninein is a Centrosomal Autoantigen Recognized by CREST Patient Sera and Plays a Regulatory Role in Microtubule Nucleation

Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of ?-tubulin, recruiting it to ectopic (non-centrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.     

Relocalization of a microtubule-anchoring protein,ninein, from the centrosome to dendrites during differentiation of mouse neurons

Microtubules in typical cells form radial arrays with their plus-ends pointing toward the cell periphery. In contrast, microtubules in dendrites of neurons are free from centrosomes and have a unique arrangement in which about half have a polarity with a minus-end distal orientation. Mechanisms for generation and maintenance of the microtubule arrangement in dendrites are not well understood. Here, we examined dendritic localization of a centrosomal protein, ninein, which has microtubule-anchoring and stabilizing functions. Immunohistochemical analysis of developing mouse cerebral and cerebellar cortices showed that ninein is localized at the centrosome in undifferentiated neural precursors. In contrast, ninein was barely detected in migrating neurons, such as those in the intermediate layer of the cerebral cortex and the internal granular layer of the cerebellar cortex. High expression was observed in thick dendrite-bearing neurons such as pyramidal neurons of the cerebral cortex and Purkinje neurons in the cerebellar cortex. Ninein was not detected at the centrosome of these cells, but was diffusely present in cell soma and dendrites. In cultured cortical neurons, ninein formed granular structures in soma and dendrites, being not associated with γ-tubulin. About 60% of these structures showed resistance to detergent and association with microtubules. Our observations suggest that the minus-ends of microtubules may be anchored and stabilized by centrosomal proteins localized in dendrites.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.     

Microtubule release from the centrosome in migrating cells

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63-71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration.     

Regulation of REGγ cellular distribution and function by SUMO modification

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.     

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, α- and especially with γ-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways.     

Inhibition of proteasome activity impairs centrosome-dependent microtubule nucleation and organization

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.