Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry.

Top-down mass spectrometry is a valuable tool for understanding gene expression through characterization of combinatorial histone post-translational modifications (i.e., histone code). In this protocol, we describe a top-down workflow that employs liquid chromatography (LC) coupled to mass spectrometry (MS), for fast global profiling of changes in histone proteoforms, and apply LCMS top-down approach for comparative analysis of a wild-type and a mutant fungal species. The proteoforms exhibiting differential abundances can be subjected to further targeted studies by other MS or orthogonal (e.g., biochemical) assays. This method can be generally adapted for screening of changes in histone modifications between samples such as wild type vs. mutant or healthy vs. diseased.

Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry.

As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (PTMs) along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here we used state-of-the-art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as a search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

Identification of Differential Gene Expression Patterns after Acute Exposure to High and Low Doses of Low-LET Ionizing Radiation in a Reconstituted Human Skin Tissue.

In this study we utilized a systems biology approach to identify dose- (0.1, 2.0 and 10 Gy) and time- (3 and 8 h) dependent responses to acute ionizing radiation exposure in a complex tissue, reconstituted human skin. The low dose used here (0.1 Gy) falls within the range of certain medical diagnostic procedures. Of the two higher doses used, 2.0 Gy is typically administered for radiotherapy, while 10 Gy is lethal. Because exposure to any of these doses is possible after an intentional or accidental radiation events, biomarkers are needed to rapidly and accurately triage potentially exposed individuals. Here, tissue samples were acutely exposed to X-ray-generated low-linear-energy transfer (LET) ionizing radiation, and direct RNA sequencing (RNA-seq) was used to quantify altered transcripts. The time points used for this study aid in assessing early responses to exposure, when key signaling pathways and biomarkers can be identified, which precede and regulate later phenotypic alterations that occur at high doses, including cell death. We determined that a total of 1,701 genes expressed were significantly affected by high-dose radiation, with the majority of genes affected at 10 Gy. Expression levels of a group of 29 genes, including GDF15, BBC3, PPM1D, FDXR, GADD45A, MDM2, CDKN1A, TP53INP1, CYCSP27, SESN1, SESN2, PCNA and AEN, were similarly altered at both 2 and 10 Gy, but not 0.1 Gy, at both time points. A much larger group of upregulated genes, including those involved in inflammatory responses, was significantly altered only after 10 Gy irradiation. At high doses, downregulated genes were associated with cell cycle regulation and exhibited an apparent linear response between 2 and 10 Gy. While only a few genes were significantly affected by 0.1 Gy irradiation, using stringent statistical filters, groups of related genes regulating cell cycle progression and inflammatory responses consistently exhibited opposite trends in their regulation compared to high-dose irradiated groups. Differential regulation of PLK1 signaling at low- and high-dose irradiation was confirmed using qRT-PCR. These results indicate that some alterations in gene expression are qualitatively different at low and high doses of ionizing radiation in this model system. They also highlight potential biomarkers for radiation exposure that may precede the development of overt physiological symptoms in exposed individuals.

Data integration reveals key homeostatic mechanisms following low dose radiation exposure.

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time - with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24-72h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

High-throughput proteomics.

Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

Quantitative Proteomic Profiling of Low-Dose Ionizing Radiation Effects in a Human Skin Model.

To assess responses to low-dose ionizing radiation (LD-IR) exposures potentially encountered during medical diagnostic procedures, nuclear accidents or terrorist acts, a quantitative proteomic approach was used to identify changes in protein abundance in a reconstituted human skin tissue model treated with 0.1 Gy of ionizing radiation. To improve the dynamic range of the assay, subcellular fractionation was employed to remove highly abundant structural proteins and to provide insight into radiation-induced alterations in protein localization. Relative peptide quantification across cellular fractions, control and irradiated samples was performing using 8-plex iTRAQ labeling followed by online two-dimensional nano-scale liquid chromatography and high resolution MS/MS analysis. A total of 107 proteins were detected with statistically significant radiation-induced change in abundance (>1.5 fold) and/or subcellular localization compared to controls. The top biological pathways identified using bioinformatics include organ development, anatomical structure formation and the regulation of actin cytoskeleton. From the proteomic data, a change in proteolytic processing and subcellular localization of the skin barrier protein, filaggrin, was identified, and the results were confirmed by western blotting. This data indicate post-transcriptional regulation of protein abundance, localization and proteolytic processing playing an important role in regulating radiation response in human tissues.

Annexin A2 modulates radiation-sensitive transcriptional programming and cell fate.

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Enhanced top-down characterization of histone post-translational modifications.

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.

Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.

Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low- and high-dose ionizing radiation-dependent signalling in a complex three-dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

Two-dimensional liquid chromatography system for online top-down mass spectrometry.

An online metal-free weak cation exchange-hydrophilic interaction LC/RPLC system has been developed for sensitive, high-throughput top-down MS. Here, we report results for analyzing PTMs of core histones, with a focus on histone H4, using this system. With just ∼24 µg on-column of core histones (H4, H2B, H2A, and H3) purified from human fibroblasts, 41 H4 isoforms were identified, with the type and location of PTMs unambiguously mapped for 20 of these variants. Compared to corresponding offline studies reported previously, the online weak cation exchange-hydrophilic interaction LC/RPLC platform offers significant improvement in sensitivity, with several orders of magnitude reduction in sample requirements and a reduction in the overall analysis time. To the best of our knowledge, this study represents the first online 2-D LC-MS/MS characterization of core histone mixture at the intact protein level.

Combined pulsed-Q dissociation and electron transfer dissociation for identification and quantification of iTRAQ-labeled phosphopeptides.

Here, we report a new approach that integrates pulsed Q dissociation (PQD) and electron transfer dissociation (ETD) techniques for confident and quantitative identification of iTRAQ-labeled phosphopeptides. The use of isobaric tags for relative and absolute quantification enables a high-throughput quantification of peptides via reporter ion signals in the low m/z range of tandem mass spectra. PQD, a form of ion trap collision activated dissociation, allows for detection of low mass-to-charge fragment ions, and electron transfer dissociation is especially useful for sequencing peptides that contain post-translational modifications. Analysis of the phosphoproteome of human fibroblast cells using a sensitive linear ion trap mass spectrometer demonstrated that this hybrid approach improves both identification and quantification of phosphopeptides. ETD improved phosphopeptide identification, while PQD provides improved quantification of iTRAQ-labeled phosphopeptides.

Linear discriminant analysis-based estimation of the false discovery rate for phosphopeptide identifications.

The development of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has made it possible to characterize phosphopeptides in an increasingly large-scale and high-throughput fashion. However, extracting confident phosphopeptide identifications from the resulting large data sets in a similar high-throughput fashion remains difficult, as does rigorously estimating the false discovery rate (FDR) of a set of phosphopeptide identifications. This article describes a data analysis pipeline designed to address these issues. The first step is to reanalyze phosphopeptide identifications that contain ambiguous assignments for the incorporated phosphate(s) to determine the most likely arrangement of the phosphate(s). The next step is to employ an expectation maximization algorithm to estimate the joint distribution of the peptide scores. A linear discriminant analysis is then performed to determine how to optimally combine peptide scores (in this case, from SEQUEST) into a discriminant score that possesses the maximum discriminating power. Based on this discriminant score, the p- and q-values for each phosphopeptide identification are calculated, and the phosphopeptide identification FDR is then estimated. This data analysis approach was applied to data from a study of irradiated human skin fibroblasts to provide a robust estimate of FDR for phosphopeptides. The Phosphopeptide FDR Estimator software is freely available for download at http://ncrr.pnl.gov/software/.

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus.

The chromosomal passenger complex (CPC) is a crucial regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, using liquid chromatography coupled to mass spectrometry (LC-MS), we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation-specific antibody that labels the CPC. A mitotic phosphorylation motif {PX[G/T/S][L/M]S(P) P or WGLS(P) P} was identified by MS in 11 proteins, including FZR1 (Cdh1) and RIC8A-two proteins with potential links to the CPC. Phosphoprotein complexes contained the known CPC components INCENP, Aurora-B (Aurkb) and TD-60 (Rcc2, RCC1-like), as well as SMAD2, 14-3-3 proteins, PP2A and Cdk1 (Cdc2a), a probable kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins, including SMAD2, PLK3 and INCENP. Mitotic SMAD2 and PLK3 phosphorylation was confirmed using phosphorylation-specific antibodies, and, in the case of Plk3, phosphorylation correlated with its localization to the mitotic apparatus and the midbody. A mutagenesis approach was used to show that INCENP phosphorylation is required for its localization to the midbody. These results provide evidence for a shared phosphorylation event that regulates localization of crucial proteins during mitosis.

Cellular trafficking of phospholamban and formation of functional sarcoplasmic reticulum during myocyte differentiation.

Phospholamban (PLB) associates with the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to beta-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca(2+)-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca(2+)-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca(2+)-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca(2+)-ATPase.

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation.

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.

Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility.

Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10(-12) to 10(-5)) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression.

Regulation of inducible nitric oxide synthase by aggresome formation.

Misfolding and aggregation of proteins play an important part in the pathogenesis of several genetic and degenerative diseases. Recent evidence suggests that cells have evolved a pathway that involves sequestration of aggregated proteins into specialized "holding stations" called aggresomes. Here we show that cells regulate inducible NO synthase (iNOS), an important host defense protein, through aggresome formation. iNOS aggresome formation depends on a functional dynein motor and the integrity of the microtubules. The iNOS aggresome represents a "physiologic aggresome" and thus defines a new paradigm for cellular regulation of protein processing. This study indicates that aggresome formation in response to misfolded proteins may merely represent an acceleration of an established physiologic regulatory process for specific proteins whose regulation by aggresome formation is deemed necessary by the cell.

Dynamic motion of helix A in the amino-terminal domain of calmodulin is stabilized upon calcium activation.

Calcium-dependent changes in the internal dynamics and average structures of the opposing globular domains of calmodulin (CaM), as well as their relative spatial arrangement, contribute to the productive association between CaM and a range of different target proteins, affecting their functional activation. To identify dynamic structural changes involving individual alpha-helical elements and their modulation by calcium activation, we have used site-directed mutagenesis to engineer a tetracysteine binding motif within helix A near the amino terminus of calmodulin (CaM), permitting the selective and rigid attachment of the fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) with full retention of function. The rigid tetracoordinate linkage of FlAsH to CaM, in conjunction with frequency domain fluorescence anisotropy measurements, allows assessment of dynamic changes associated with calcium activation without interference from independent probe motion. Taking advantage of the large fluorescence enhancement associated with binding of FlAsH to CaM, we determined rates of binding of FlAsH to apo-CaM and calcium-activated CaM to be 2800 +/- 80 and 310 +/- 10 M(-)(1) s(-)(1), respectively. There is no difference in the solvent accessibility of the bound FlAsH irrespective of calcium binding to CaM. Thus, given that FlAsH selectively labels disordered structures, the large difference in rates of FlAsH binding indicates that calcium binding stabilizes helix A. Frequency domain anisotropy measurements of bound FlAsH indicate that prior to calcium activation, helix A undergoes large amplitude nanosecond motions. Following calcium activation, helix A becomes immobile, and structurally coupled to the overall rotation of CaM. We discuss these results in the context of a model that suggests stabilization of helix A relative to other domain elements in the CaM structure is critical to defining high-affinity binding clefts, and in promoting specific and ordered binding of the opposing lobes of CaM to target proteins.

Aurora-C kinase is a novel chromosomal passenger protein that can complement Aurora-B kinase function in mitotic cells.

The function of Aurora-C kinase, a member of the Aurora kinase family identified in mammals, is currently unknown. We present evidence that Aurora-C, like Aurora-B kinase, is a chromosomal passenger protein localizing first to centromeres and then to the midzone of mitotic cells. Aurora-C transcript is expressed at a moderate level albeit about an order of magnitude lower than Aurora-B transcript in diploid human fibroblasts. The level of Aurora-C transcript is elevated in several human cancer cell types. Aurora-C and Aurora-B mRNA and protein expressions are maximally elevated during the G2/M phase but their expression profiles in synchronized cells reveal differential temporal regulation through the cell cycle with Aurora-C level peaking after that of Aurora-B during the later part of the M phase. Aurora-C, like Aurora-B, interacts with the inner centromere protein (INCENP) at the carboxyl terminal end spanning the conserved IN box domain. Competition binding assays and transfection experiments revealed that, compared with Aurora-C, Aurora-B has preferential binding affinity to INCENP and co-expression of the two in vivo interferes with INCENP binding, localization, and stability of these proteins. A kinase-dead mutant of Aurora-C had a dominant negative effect inducing multinucleation in a dose-dependent manner. siRNA mediated silencing of Aurora-C and Aurora-B also gave rise to multinucleated cells with the two kinases silenced at the same time displaying an additive effect. Finally, Aurora-C could rescue the Aurora-B silenced multinucleation phenotype, demonstrating that Aurora-C kinase function overlaps with and complements Aurora-B kinase function in mitosis.