Interaction between salt-inducible kinase 2 and protein phosphatase 2A regulates the activity of calcium/calmodulin-dependent protein kinase I and protein phosphatase methylesterase-1.

Salt-inducible kinase 2 (SIK2) is the only AMP-activated kinase (AMPK) family member known to interact with protein phosphatase 2 (PP2A). However, the functional aspects of this complex are largely unknown. Here we report that the SIK2-PP2A complex preserves both kinase and phosphatase activities. In this capacity,SIK2 attenuates the association of the PP2A repressor, the protein phosphatase methylesterase-1 (PME-1), thus preserving the methylation status of the PP2A catalytic subunit. Furthermore, the SIK2-PP2A holoenzyme complex dephosphorylates and inactivates Ca2(+)/calmodulin-dependent protein kinase I (CaMKI), an upstream kinase for phosphorylating PME-1/Ser(15). The functionally antagonistic SIK2-PP2A and CaMKI and PME-1 networks thus constitute a negative feedback loop that modulates the phosphatase activity of PP2A. Depletion of SIK2 led to disruption of the SIK2-PP2A complex, activation of CaMKI, and downstream effects, including phosphorylation of HDAC5/Ser(259), sequestration of HDAC5 in the cytoplasm, and activation of myocyte-specific enhancer factor 2C (MEF2C)-mediated gene expression. These results suggest that the SIK2-PP2A complex functions in the regulation of MEF2C-dependent transcription. Furthermore, this study suggests that the tightly linked regulatory loop comprised of the SIK2-PP2A and CaMKI and PME-1 networks may function in fine-tuning cell proliferation and stress response.

Interaction between salt-inducible kinase 2 (SIK2) and p97/valosin-containing protein (VCP) regulates endoplasmic reticulum (ER)-associated protein degradation in mammalian cells.

Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.

Reversible acetylation regulates salt-inducible kinase (SIK2) and its function in autophagy.

Salt-inducible kinase 2 (SIK2) is a serine/threonine protein kinase belonging to the AMP-activated protein kinase (AMPK) family. SIK2 has been shown to function in the insulin-signaling pathway during adipocyte differentiation and to modulate CREB-mediated gene expression in response to hormones and nutrients. However, molecular mechanisms underlying the regulation of SIK2 kinase activity remains largely elusive. Here we report a dynamic, post-translational regulation of its kinase activity that is coordinated by an acetylation-deacetylation switch, p300/CBP-mediated Lys-53 acetylation inhibits SIK2 kinase activity, whereas HDAC6-mediated deacetylation restores the activity. Interestingly, overexpression of acetylation-mimetic mutant of SIK2 (SIK2-K53Q), but not the nonacetylatable K53R variant, resulted in accumulation of autophagosomes. Further consistent with a role in autophagy, knockdown of SIK2 abrogated autophagosome and lysosome fusion. Consequently, SIK2 and its kinase activity are indispensable for the removal of TDP-43Δ inclusion bodies. Our findings uncover SIK2 as a critical determinant in autophagy progression and further suggest a mechanism in which the interplay among kinase and deacetylase activities contributes to cellular protein pool homeostasis.

A functional genomic approach reveals the transcriptional role of EDD in the expression and function of angiogenesis regulator ACVRL1.

EDD (E3 isolated by differential display) was initially isolated as a progestin-regulated gene in breast cancer cells, and represents the human ortholog of the Drosophila melanogaster hyperplastic discs gene (hyd). It encodes a highly conserved and predominantly nuclear ubiquitin E3 ligase of the HECT family, with potential multifunctional roles in development and tumorigenesis. In this study, we further examined the largely uncharacterized role of EDD in transcriptional regulation by uncovering the spectrum of its direct target genes at a genome-wide level. Use of a systematic approach that integrates gene expression and chromatin binding profiling identified several candidate EDD-target genes, one of which is ACVRL1, a TGF-ß receptor with functional implications in blood vessel development. Further characterization revealed a negative regulation of ACVRL1 gene expression by EDD that is exerted at the promoter. Consistent with the aberrant upregulation of ACVRL1 and downstream Smad signaling, abrogation of EDD led to deregulated vessel development and endothelial cell motility. Collectively, these results extended the known cellular roles of EDD to critical functions in transcriptional regulation as well as angiogenesis, and may provide mechanistic explanations for EDD's tumorigenic and developmental roles.

Cytoskeletal protein vimentin interacts with and regulates peroxisome proliferator-activated receptor gamma via a proteasomal degradation process.

Peroxisome proliferators-activated receptor gamma (PPARγ) receptor is a transcription factor that is located in and functions primarily in the nucleus. PPARγ is exported from the nucleus upon mitogen and ligand stimulation under certain circumstances. However, a cytoplasmic PPARγ interacting protein and its function have not been previously identified. Here, we report for the first time that cytosolic PPARγ interacts directly with cytoskeletal vimentin. We performed PPARγ immunoprecipitation followed by mass spectrometry to identify the vimentin-PPARγ complex. This interaction was confirmed by reciprocal vimentin and PPARγ immunoprecipitation and co-immunofluorescence examination. We demonstrated that PPARγ colocalized with vimentin in certain organelles that is golgi, mitochondria, and endoplasmic reticulum. In cells depleted of vimentin, PPARγ was ubiquitinated and targeted to a proteasomal degradation pathway. Together, these findings indicate a direct interaction of PPARγ with vimentin in the cytosolic compartment, in which vimentin appears to play a role in regulating the turnover rate of PPARγ, which may further regulate genomic or non-genomic activities through the regulation of PPARγ protein degradation.

FACT facilitates chromatin transcription by RNA polymerases I and III.

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co-purify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Transcriptional regulation of tristetraprolin by NF-κB signaling in LPS-stimulated macrophages.

Lipopolysaccharide (LPS) treatment causes the marked changes of gene expression in macrophages. Tristetraprolin (TTP), which is an mRNA-destabilizing protein that negatively regulates the expression of pro-inflammatory mediators, is induced by LPS. To delineate the molecular mechanism of LPS-stimulated TTP expression, several inhibitors blocking different signaling pathways were used initially. We observed that inhibitors of the NF-κB signaling pathway could down-regulate the TTP expression during LPS-induction. Consistently, TTP expression was increased upon recombinant TNFα stimulation which activates NF-κB signaling. The 5' regulatory region of zfp36 gene spanning from -2 k to +50 was isolated, which contained a putative NF-κB-binding site located in -1859 to -1850. Analysis of luciferase reporter activity driven by a serial 5'-deletion of TTP promoter showed that NF-κB inhibitor-mediated suppression of LPS or TNFα induced activity was through the predicted κB-binding sites. When the NF-κB-binding site was mutated, the TTP promoter decreased its response to the ectopic expression of NF-κB. Physical interaction analysis including oligonucleotides competition, gel shift and chromatin immunoprecipitation assays demonstrated that NF-κB activated by LPS or TNFα bound to the TTP promoter specifically. These results suggested that during LPS stimulation, NF-κB signaling were activated to regulate the transcription of TTP mRNA.

WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway.

The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1α localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1's involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene.

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.

Differential regulation of CHOP translation by phosphorylated eIF4E under stress conditions.

Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORF(chop)-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2alpha/S51. Contrary to phospho-eIF2alpha/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2alpha/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation.

Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation.

BACKGROUND: Functional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood. METHODS & RESULTS: Here we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities. CONCLUSIONS: Together, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.

Phosphorylation at Ser473 regulates heterochromatin protein 1 binding and corepressor function of TIF1beta/KAP1.

BACKGROUND: As an epigenetic regulator, the transcriptional intermediary factor 1beta (TIF1beta)/KAP1/TRIM28) has been linked to gene expression and chromatin remodeling at specific loci by association with members of the heterochromatin protein 1 (HP1) family and various other chromatin factors. The interaction between TIF1beta and HP1 is crucial for heterochromatin formation and maintenance. The HP1-box, PXVXL, of TIF1beta is responsible for its interaction with HP1. However, the underlying mechanism of how the interaction is regulated remains poorly understood. RESULTS: This work demonstrates that TIF1beta is phosphorylated on Ser473, the alteration of which is dynamically associated with cell cycle progression and functionally linked to transcriptional regulation. Phosphorylation of TIF1beta/Ser473 coincides with the induction of cell cycle gene cyclin A2 at the S-phase. Interestingly, chromatin immunoprecipitation demonstrated that the promoter of cyclin A2 gene is occupied by TIF1beta and that such occupancy is inversely correlated with Ser473 phosphorylation. Additionally, when HP1beta was co-expressed with TIF1beta/S473A, but not TIF1beta/S473E, the colocalization of TIF1beta/S473A and HP1beta to the promoters of Cdc2 and Cdc25A was enhanced. Non-phosphorylated TIF1beta/Ser473 allowed greater TIF1beta association with the regulatory regions and the consequent repression of these genes. Consistent with possible inhibition of TIF1beta's corepressor function, the phosphorylation of the Ser473 residue, which is located near the HP1-interacting PXVXL motif, compromised the formation of TIF1beta-HP1 complex. Finally, we found that the phosphorylation of TIF1beta/Ser473 is mediated by the PKCdelta pathway and is closely linked to cell proliferation. CONCLUSION: The modulation of HP1beta-TIF1beta interaction through the phosphorylation/de-phosphorylation of TIF1beta/Ser473 may constitute a molecular switch that regulates the expression of particular genes. Higher levels of phosphorylated TIF1beta/Ser473 may be associated with the expression of key regulatory genes for cell cycle progression and the proliferation of cells.

Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation.

Chromatin-modifying factors play key roles in transcription, DNA replication and DNA repair. Post-translational modification of these proteins is largely responsible for regulating their activity. The FACT (facilitates chromatin transcription) complex, a heterodimer of hSpt16 and SSRP1, is a chromatin structure modulator whose involvement in transcription and DNA replication has been reported. Here we show that nucleosome binding activity of FACT complex is regulated by poly(ADP-ribosyl)ation. hSpt16, the large subunit of FACT, is poly(ADP-ribosyl)ated by poly(ADP-ribose) polymerase-1 (PARP-1) resulting from physical interaction between these two proteins. The level of hSpt16 poly(ADP-ribosyl)ation is elevated after genotoxic treatment and coincides with the activation of PARP-1. The enhanced hSpt16 poly(ADP-ribosyl)ation level correlates with the dissociation of FACT from chromatin in response to DNA damage. Our findings suggest that poly(ADP-ribosyl)ation of hSpt16 by PARP-1 play regulatory roles for FACT-mediated chromatin remodeling.

Functional analysis of the subunits of the chromatin assembly factor RSF.

The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Transcriptional activation of C/EBPbeta gene by c-Jun and ATF2.

C/EBPbeta is one of the key transcription factors responsible for the induction of a wide array of genes. Like many proto-oncogenes and transcription factors, transcription of C/EBPbeta gene can be induced by multiple extracellular signals. Using nuclear extracts from lipopolysaccharide (LPS)-stimulated mouse liver, five trans-acting factor-binding motifs, URE1 (-376 to -352), URE2 (-253 to -223), URE3 (-220 to -190), URE4 (-123 to -103), and URE5 (-72 to -45) were identified by DNAse I footprinting assays. Competition and supershift analysis of the complexes formed at the URE2 and URE4 indicated that they contain CREB/ATF and AP-1 family factors. Furthermore, recombinant ATF2 and c-Jun proteins from mammalian and bacterial cells can bind to URE2 and URE4 but not URE1. Cotransfection experiments showed that ATF2 and c-Jun activate the C/EBPbeta gene expression cooperatively through URE2 and URE4, and this activation was greatly increased under the treatment of low concentration of anisomycin. During acute phase response, the phosphorylation of c-Jun and ATF2 was found to correlate with C/EBPbeta gene expression. Taken together, our results provide the evidences that both c-Jun and ATF2 are the regulators of C/EBPbeta gene.

Phosphorylation of mRNA decapping protein Dcp1a by the ERK signaling pathway during early differentiation of 3T3-L1 preadipocytes.

BACKGROUND: Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5' cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway-mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.

The ATM substrate KAP1 controls DNA repair in heterochromatin: regulation by HP1 proteins and serine 473/824 phosphorylation.

The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor that binds to HP1 during gene silencing but is also robustly phosphorylated by Ataxia telangiectasia mutated (ATM) at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells that lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin.

Phosphorylation of NuSAP by Cdk1 regulates its interaction with microtubules in mitosis.

Cell division in eukaryotes depends on a fine control of the dynamic changes of microtubules. Nucleolar and spindle-associated protein (NuSAP) is a microtubule-binding and -bundling protein essential for the integrity of the anaphase spindle and cell division. NuSAP contains two consensus cdk phosphorylation sites in its microtubule-binding domain. Here we show NuSAP is phosphorylated by cdk1 in early mitosis. This phosphorylation inhibits the binding of NuSAP to microtubules. During metaphase-to anaphase transition, NuSAP is dephosphorylated to promote spindle midzone formation and cell cycle progression. Expression of cdk1 phosphorylation-null mutant causes extensive bundling of microtubules in the prometaphase spindle. Our results suggest that phosphorylation and dephosphorylation of NuSAP during progression of mitosis regulate spindle organization through modulation of the dynamics of microtubules.

Aurora-C kinase deficiency causes cytokinesis failure in meiosis I and production of large polyploid oocytes in mice.

We previously isolated Aurora-C/Aie1 in a screen for kinases expressed in mouse sperm and eggs. Here, we show the localization of endogenous Aurora-C and examine its roles during female mouse meiosis. Aurora-C was detected at the centromeres and along the chromosome arms in prometaphase I-metaphase I and was concentrated at centromeres at metaphase II, in which Aurora-C also was phosphorylated at Thr171. During the anaphase I-telophase I transition, Aurora-C was dephosphorylated and relocalized to the midzone and midbody. Microinjection of the kinase-deficient Aurora-C (AurC-KD) mRNA into mouse oocytes significantly inhibited Aurora-C activity and caused multiple defects, including chromosome misalignment, abnormal kinetochore-microtubule attachment, premature chromosome segregation, and cytokinesis failure in meiosis I. Furthermore, AurC-KD reduced Aurora-C and histone H3 phosphorylation and inhibited kinetochore localization of Bub1 and BubR1. Similar effects also were observed in the oocytes injected with INCNEP-delIN mRNAs, in which the Aurora-C binding motif was removed. The most dramatic effect observed in AurC-KD-injected oocytes is cytokinesis failure in meiosis I, resulting in producing large polyploid oocytes, a pattern similar to Aurora-C deficiency human spermatozoa. Surprisingly, we detected no Aurora-B protein in mouse oocytes. We propose that Aurora-C, but not Aurora-B, plays essential roles in female mouse meiosis.