A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. This study shows that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH₂-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN (Terada, 2003).

In animal cells, microtubules are organized from the centrosome/microtubule-organizing center (MTOC), composed of a pair of centrioles and the surrounding pericentriolar material. Individual microtubules are nucleated from an ~25-nm γ-tubulin–containing ring complex (γ-TuRC). At the onset of M phase, the centrosome becomes 'mature' and organizes more microtubules, which is accompanied with an increased level of γ-tubulin accumulation at each spindle pole. One of the molecules that has been implicated in the mechanism of centrosome maturation is Aurora-A, a mitosis-specific Ser/Thr kinase located at mitotic poles and spindle microtubules. The kinase, originally identified as a gene product important in spindle assembly and function in Drosophila, has recently been shown to be in the Ran-signaling pathway and to play an important role in efficient transmission of Ran-GTP gradient established by the condensed chromosomes for the control of spindle assembly and dynamics. Aurora-A binds to spindle components, such as TACC/XMAP215 and TPX2. Although possible functions of those molecules and their interaction with Aurora-A in bipolar spindle formation have been elucidated, mechanisms of how Aurora-A stimulates the recruitment of γ-tubulin to the centrosome at spindle poles have not yet been evaluated. To address this question, centrosomal proteins were sought that interact with Aurora-A and regulate the process of microtubule nucleation onto the centrosome (Terada, 2003).

By screening of a Drosophila two-hybrid library, two clones were isolated encoding a molecule capable of interaction with Aurora-A. The sequence corresponds to the COOH-terminal domain of centrosomin (CNN), a core component of the centrosome important for assembly of mitotic centrosomes in Drosophila (Heuer, 1995; Megraw, 1999). Although the truncated polypeptide covered by clone CNN-C1 appears to be sufficient for interaction with Aurora-A, the binding intensity was weaker than CNN-C. Endogenous Aurora-A, but not Aurora-B, immunoprecipitates with HA-tagged CNN expressed in S2 cells. Specificity of the COOH-terminal domain of CNN for interaction with Aurora-A was further confirmed by in vitro binding assays (Terada, 2003).

To investigate the role of protein interaction in the centrosome, S2 cells were prepared from which Aurora-A or CNN was depleted by RNA interference (RNAi). In cells lacking Aurora-A, not only CNN, but also γ-tubulin, were absent at each spindle pole. When CNN was depleted, neither γ-tubulin nor Aurora-A was seen at the spindle pole. In cells with partially depleted Aurora-A or CNN, comparable amounts of γ-tubulin and CNN or Aurora-A were detected at each pole. Besides γ-tubulin, other centrosome proteins, CP190 and CP60, become dislocated from the spindle poles in RNAi cells. Therefore, it is concluded that CNN and Aurora-A are mutually dependent for localization at spindle poles, which is required for proper targeting of other centrosomal proteins to the centrosome. This is consistent with previous observations (Barbosa, 2000) that the centrosomal association of CNN is not dependent on the presence of γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the role of CNN in recruiting γ-tubulin, protein interaction was analyzed in vitro. Nickel beads conjugated with His-tagged CNN were mixed with cell extracts prepared from colcemid-treated S2 cells. γ-Tubulin was specifically sedimented by the full and NH₂-terminal sequence, but not the COOH-terminal sequence of CNN. Because neither in vitro binding assays nor two-hybrid screens demonstrated direct binding between two molecules, CNN may interact with a γ-tubulin complex, rather than γ-tubulin directly. Further, HA-tagged CNN was expressed in S2 cells. Exogenous proteins caused formation of γ-tubulin–containing cytoplasmic aggregates capable of microtubule formation and association with microtubule asters. These results clearly indicate that the NH₂-terminal domain of CNN interacts with γ-tubulin/γ-TuRC and plays an important role in assembly of MTOCs (Terada, 2003).

γ-Tubulin/γ-TuRC–mediated microtubule assembly is believed to be common among species. Thus, it is highly likely that an Aurora-A–binding molecule(s) equivalent to CNN is functioning in a variety of organisms. Although Drosophila CNN was unable to associate with mammalian Aurora-A in transfected mammalian cells as well as by two-hybrid screens, the NH₂-terminal domain of CNN still interacts with γ-tubulin/γ-TuRC in mammalian cells as in S2 cells. To analyze a possible role of CNN–γ-tubulin interaction in initiation of microtubule assembly, Drosophila CNN was overexpressed in mammalian cells. HA-tagged CNN induces cytoplasmic foci in various sizes and numbers. Significantly, the pattern of microtubule distribution is profoundly affected as a result of microtubule association with virtually every dot containing CNN. These sites can initiate microtubule formation as evidently shown in cells where short microtubules are assembled during brief recovery from nocodazole treatment. All cells overexpressing CNN induced microtubule-organizing sites, which were associated with centrosome proteins, such as pericentrin and Cep135. Particularly prominent was γ-tubulin, which was probably recruited from a large cytoplasmic pool. In support of this view, GFP-tagged exogenous γ-tubulin became colocalized with HA-CNN to participate in the formation of microtubule-nucleating sites. This was in striking contrast with cells expressing γ-tubulin alone, where cytoplasmic aggregates induced by γ-tubulin expression could not contribute to microtubule formation. These results suggest that microtubules are directly nucleated from the CNN aggregates through the mechanism mediated by γ-tubulin/γ-TuRC (Terada, 2003).

To confirm the microtubule-nucleating activity of the CNN aggregates, microtubules were polymerized in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamine–conjugated brain tubulin. There was always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes. The process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center (Terada, 2003).

Microtubules are nucleated from the pericentriolar material that surrounds the centrioles of the centrosome. To compare ultrastructure of microtubule-initiating sites induced by CNN with that of the pericentriolar material/centrosome, CHO cells expressing GFP-tagged CNN were examined by EM. Two microtubule asters were seen formed in cells that were briefly extracted before fixation. Located at each focal point of microtubule asters was an electron-dense particle in various sizes and shapes. Unlike the pericentriolar material, which has been described as an ill-defined amorphous cloud, the entire structure induced by CNN was well delineated by electron-dense materials to which microtubules were attached. In favorable sections, microtubules could be seen penetrating to the interior region of the aggregates. Neither centrioles nor centrosomal substructures, such as satellites, appendages, and CHO cell–specific virus particles, were generally seen at the site induced by CNN expression. Because CNN is a coiled-coil structural protein (Heuer, 1995), the dense particles likely represent the aggregated form of overexpressed CNN proteins (Terada, 2003).

Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated. In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (8–12 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM and immunostaining with centriole-specific centrin-2 antibodies. Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting γ-tubulin/γ-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with γ-tubulin/γ-TuRC suggested CNN may function as an adaptor for connecting γ-tubulin to the centrosome (Terada, 2003).

By expressing truncated polypeptides, it was concluded that CNN's ability to interact with γ-tubulin/γ-TuRC and induce ectopic microtubule-nucleating sites resides in the NH₂-terminal sequence of CNN from which the Aurora-A–binding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells. These results lead to the conclusion that CNN consists of two functionally distinct subdomains: the Aurora-A–binding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH₂-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting γ-tubulin/γ-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells (Terada, 2003). Control of mitotic spindle assembly onto the centrosome could be achieved by several mechanisms, including nucleation of individual microtubules onto γ-tubulin–containing protein complexes, stimulation of microtubule nucleation and stabilization of polymerized microtubules by MAPs, and recruitment of minus ends of preexisting microtubules by the action of motor activity to the centrosome. Aurora-A binds not only CNN but also the D-TACC/MSPS/XMAP215 complex. These components appear to be required for microtubule assembly on mitotic centrosomes/poles controlled through the distinct mechanisms from that of γ-tubulin recruitment. Therefore, it is reasonable that Aurora-A plays a role in regulating the overall process of centrosome maturation by orchestrating multiple pathways of microtubule assembly during mitosis. It is worth mentioning that individual mechanisms of microtubule assembly may show a distinct requirement for protein phosphorylation and the Aurora-A kinase activity; although both Aurora-A and CNN are still able to locate at the centrosome, D-TACC/MSPS complex failed to be recruited to spindle poles in the absence of enzymatic activity of Aurora-A kinase (Terada, 2003).

Aurora kinases are highly expressed in cells derived from many human tumor cell types, which frequently contain multiple centrosomes. Because defects in the number, structures, and function of centrosomes are closely associated with the genetic instability in transformed cells, Aurora-A might be involved in tumorigenesis by inducing abnormal numbers of MTOCs as a result of inappropriate distribution of CNN-like molecule(s) (Terada, 2003).

GENE STRUCTURE

cDNA clone length - 4335 bp (isoform A)

Bases in 5' UTR - 127

Exons - 6

Bases in 3' UTR - 761

PROTEIN STRUCTURE

Amino Acids - 1148 (cnn-PA)

Structural Domains

The expression pattern of cnn suggested homeotic regulation, i.e., there were higher levels of expression in the thoracic region of the CNS compared to the abdominal region. The 6.8 kb SalI fragment hybridizes to two major species of polyadenylated RNA on a northern blot of sizes 4.8 and 5 kb. To characterize the gene further this fragment was used as a probe to isolate a cDNA clone from an embryonic library. A single cDNA of 4,332 bp in size was isolated that recognizes the same species of mRNA on a northern blot as the 6.8 kb genomic fragment. Assuming a poly A tail addition of 400 bases, the size of this cDNA is in close agreement to the sizes of polyadenylated RNA species observed on a northern blot (4.8 and 5 kb) and thus appears to be near full length. Both strands of the entire 4,332 bp cDNA were sequenced by a combination of nested primers and exonuclease III treatment of both strands. Within the entire 4,332 bp, a single long open reading frame (ORF) of 3,102 bp was found. The cDNA sequence therefore contains 129 bp of 5' non-translated sequence, 1,101 bp of 3' non-translated sequence and a polyadenylation signal sequence AATAAA is found at its most 3' end. The long ORF encodes a potential polypeptide of 1,034 amino acids in length. The most striking structural features of the protein are the three putative leucine zipper motifs. The first putative leucine zipper is located from amino acids 100-126 and is bounded by several proline residues, the second is located at amino acids 525-551 and the third is from amino acids 946-972. All three zippers are within predicted coiled-coil regions. Helical wheel plots of the three zipper regions show that all contain a continuous spine of hydrophobicity greater than six helical turns and the opposite faces are rich in charged amino acid residues for the formation of salt bridges. No other protein motifs were identified, although the protein does contain a number of protein kinase recognition sites. These include 2 sites for cAMP-dependent kinase, 2 sites for Ca2+- dependent kinase II, 2 sites for GSK3 kinase, 2 sites for protein kinase C and one site for tyrosine kinase. The region of CNN extending from amino acids 85-970 shows limited homology with coiled-coil domains of myosin heavy chain from several species. This region of CNN contains long stretches of alpha helices interrupted by small regions that lack heptad periodicity, often containing proline residues which are known to disrupt alpha helices. There are no other significant homologies to known proteins in the databases (Heuer, 1995)

EVOLUTIONARY HOMOLOGS

Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. From an insertional mutagenesis screen a novel gene, mod20+, was identified, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. Mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. It is concluded that Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells (Sawin, 2004).

From an insertional mutagenesis screen, a novel gene, mto2+, was isolated involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. In mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex (Samejima, 2005).

date revised: 24 September 2006

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