KIBRA protein phosphorylation is regulated by mitotic kinase aurora and protein phosphatase 1

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.     

Phosphorylation of KIBRA by the extracellular signal-regulated kinase (ERK)–ribosomal S6 kinase (RSK) cascade modulates cell proliferation and migration

In mammals, KIBRA is defined as a memory performance-associated protein. The physiological function and regulation of KIBRA in non-neuronal cells are much less understood. Recent studies have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in tumorigenesis by inhibiting cell proliferation and promoting apoptosis. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora and cyclin-dependent kinase 1 during mitosis. In this current study, we show that KIBRA is also phosphorylated by the ERK (extracellular signal-regulated kinases)–RSK (p90 ribosomal S6 kinases) cascade. We demonstrated that ERK1/2 phosphorylate KIBRA at Ser⁵⁴⁸ in cells as well as in vitro. Moreover, we found that RSK1/2 specifically phosphorylates KIBRA at two highly conserved sites (Thr⁹²⁹ and Ser⁹⁴⁷) in vitro and in cells. RSK-mediated phosphorylation is required for KIBRA binding to RSK1, but not RSK2. Surprisingly, KIBRA knockdown impaired cell migration and proliferation in breast cancer cells. By using inducible-expression cell lines, we further show that phospho-regulation of KIBRA by ERK1/2 and RSK1/2 is required for proper cell proliferation and RSK-mediated phosphorylation also modulates KIBRA's migratory activity in MDA-MB-231 breast cancer cells. Our findings uncover unexpected results and a new mechanism through which KIBRA regulates cell migration and proliferation.     

Phospho-regulation of KIBRA by CDK1 and CDC14 phosphatase controls cell-cycle progression

KIBRA (kidney- and brain-expressed protein) is a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by regulating both cell proliferation and apoptosis. In mammals, KIBRA is associated with memory performance. The physiological function and regulation of KIBRA in non-neuronal cells remain largely unclear. We reported recently that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. In the present study, we have expanded our analysis of KIBRA's role in cell-cycle progression. We show that KIBRA is also phosphorylated by CDK1 (cyclin-dependent kinase 1) in response to spindle damage stress. We have identified KIBRA Ser542 and Ser931 as main phosphorylation sites for CDK1 both in vitro and in vivo. Moreover, we found that the CDC (cell division cycle) 14A/B phosphatases associate with KIBRA, and CDK1-non-phosphorylatable KIBRA has greatly reduced interaction with CDC14B. CDC14A/B dephosphorylate CDK1-phosphorylated KIBRA in vitro and in cells. By using inducible-expression cell lines, we show further that phospho-regulation of KIBRA by CDK1 and CDC14 is involved in mitotic exit under spindle stress. Our results reveal a new mechanism through which KIBRA regulates cell-cycle progression.     

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Aurora-A and an interacting activator,the LIM protein Ajuba,are required for mitotic commitment in human cells

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.     

Urochordate Ascidians Possess a Single Isoform of Aurora Kinase That Localizes to the Midbody via TPX2 in Eggs and Cleavage Stage Embryos

Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner) and INCENP (a vertebrate AURKB partner) and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody in eggs and cleavage stage embryos.     

What's so Bor(a)ing about Aurora-A activation?

Aurora-A kinases are highly conserved mitotic kinases required for cell division. The regulation of Aurora-A activity is less highly conserved and currently poorly understood. Work by Knoblich and coworkers in this issue of Developmental Cell identifies the conserved protein, Aurora Borealis (Bora), as a key regulator of Aurora-A activity during mitosis.     

Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis

BACKGROUND: During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. RESULTS: We show here that the mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA(37), Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA(37) mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, we show that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. CONCLUSIONS: Our results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells.     

Roles of Aurora kinases in mitosis and tumorigenesis

Aurora kinases, which have been implicated in several vital events in mitosis, represent a protein kinase family highly conserved during evolution. The activity of Aurora kinases is delicately regulated, mainly by phosphorylation and degradation. Deregulation of Aurora kinase activity can result in mitotic abnormality and genetic instability, leading to defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis. Both the expression level and the kinase activity of Aurora kinases are found to be up-regulated in many human cancers, indicating that these kinases might serve as useful targets for the development of anticancer drugs. This review focuses on recent progress on the roles of Aurora kinases in mitosis and tumorigenesis.     

Aurora B kinases restrict chromosome decondensation to telophase of mitosis

The Aurora kinases comprise a family of evolutionary conserved serine/threonine kinases that have important functions in centrosome duplication, mitotic spindle assembly, chromosome condensation, chromosome biorientation on the spindle and chromosome segregation. Vertebrates have three Aurora kinases, Aurora-A, -B and -C, while invertebrates have only Aurora-A and -B and yeasts have a single Aurora kinase, Ipl1 in S. cerevisiae and Ark1 in S. pombe. Recently, the role of Aurora kinases in chromosome condensation has been defined; Aurora B plays a crucial role in the axial shortening of chromosomes during anaphase, presumably in order to prevent chromosome arms from becoming trapped within the cytokinetic plate.     

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.     

Bora在细胞功能调控中的作用和机制研究进展

Aurora蛋白激酶A及Polo样蛋白激酶1（PLK在）作为重要的细胞周期调节蛋白可参与调控纺锤体组装、有丝分裂等细胞进程,但其激活机制及在有丝分裂中的作用机制仍然不是很清楚。Bora作为Aurora蛋白激酶A的结合蛋白,在果蝇和脊椎动物中功能高度保守,其主要通过结合Aurora蛋白激酶A从而调节Aurora蛋白激酶A的活性、促进PLK1的磷酸化、调节纺锤体的组装以及调控细胞周期进程等。随着对Bora研究的深入,人们对AuroraA和PLK1的激活机制以及Bora、Aurora蛋白激酶A、PLK1三者对细胞的调控也有了进一步的认识。主要综述Bora在细胞功能调控中的作用和研究机制。     

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.     

Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach

Aurora-A, -B, and -C are members of a small family of mitotic serine/threonine kinases that regulate centrosome maturation, chromosome segregation, and cytokinesis. They are often overexpressed in different human tumor types and have been identified as attractive targets for anticancer drug development. As specific inhibitors of the Aurora kinases are entering phase I clinical trials, there is a high need for appropriate Aurora-A biomarkers to follow mechanism of action or response. To identify novel Aurora-A substrates potentially useful as specific biomarkers we applied several modifications to the original KESTREL (Kinase Substrate Tracking and Elucidation) method in conjunction with gel electrophoresis and MALDI-MS and LC-MS/MS. The major modifications to the method included the introduction of a heating step to inactivate endogenous kinases after cell lysis and the execution of the in vitro kinase reaction in the presence of 5 mM Mg(2+) and at high (1 mM) ATP concentration. Total and fractionated extracts from nocodazole-treated HeLa cells were used as a source of Aurora-A substrates. Using this approach, we were able to detect a number of Aurora-A specific phospholabeled signals and to identify vimentin as a putative Aurora-A substrate. Vimentin was then confirmed as an in vitro substrate of Aurora-A by the phosphorylation of the recombinant protein followed by MS and antibody detection.