Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.     

FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis

Apical lumen formation is a key step during epithelial morphogenesis. The establishment of the apical lumen is a complex process that involves coordinated changes in plasma membrane composition, endocytic transport, and cytoskeleton organization. These changes are accomplished, at least in part, by the targeting and fusion of Rab11/FIP5‐containing apical endosomes with the apical membrane initiation site (AMIS). Although AMIS formation and polarized transport of Rab11/FIP5‐containing endosomes are crucial for the formation of a single apical lumen, the spatiotemporal regulation of this process remains poorly understood. Here, we demonstrate that the formation of the midbody during cytokinesis is a symmetry‐breaking event that establishes the location of the AMIS. The interaction of FIP5 with SNX18, which is required for the formation of apical endocytic carriers, is inhibited by GSK‐3 phosphorylation at FIP5‐T276. Importantly, we show that FIP5‐T276 phosphorylation occurs specifically during metaphase and anaphase, to ensure the fidelity and timing of FIP5‐endosome targeting to the AMIS during apical lumen formation.     

Ran localizes around the microtubule spindle in vivo during mitosis in Drosophila embryos

The GTPase Ran regulates multiple cellular functions throughout the cell cycle, including nucleocytoplasmic transport, nuclear membrane assembly, and spindle assembly. Ran mediates spindle assembly by affecting multiple spindle assembly pathways: microtubule dynamics, microtubule motor activity, and spindle pole assembly. Ran is predicted to facilitate spindle assembly by remaining in the GTP-bound state around the chromatin in mitosis. Here, we directly test the central tenet of this hypothesis in vivo by determining the cellular localization of Ran pathway components in Drosophila embryos. We find that, during mitosis, RCC1, the nucleotide exchange factor for Ran, is associated with chromatin, while Ran and RanL43E, an allele locked in the GTP-bound state, localize around the spindle. In contrast, nuclear proteins redistribute throughout the embryo upon nuclear envelope breakdown (NEB). Thus, in vivo RanGTP has the correct spatial localization within the cell to modulate spindle assembly.     

Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.     

Altered phosphorylation and activation of pp60c-src during fibroblast mitosis

At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts.     

Abnormal Tau phosphorylation of the Alzheimer-type also occurs during mitosis

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M-phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3-10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclin-dependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation-dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho-Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis-like phase, we investigated if our observation was also relevant to a neurone-like model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100-immunoreactivity and phospho-Ser422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer-type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimer-type Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process.     

Chk1 phosphorylation during mitosis: A new role for a master regulator

The human DNA damage responses are modulated by both nonessential and essential pathways. The extensively studied ATM kinase and p53 are examples of the former. While loss-of-function mutations in genes that encode ATM and p53 cause marked predispositions to cancer, the loss of these proteins does not appear to impact basic cell growth and proliferation. In contrast, the checkpoint kinase Chk1 and its upstream activator ATR are essential.1-4 What do these proteins do in undamaged cells?     

Aurora A contributes to p150glued phosphorylation and function during mitosis

Aurora A is a spindle pole–associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150^glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A''s function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150^glued. Although wild-type p150^glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150^glued. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150^glued MBD to limit the microtubule binding of the dynein–dynactin complex and thus regulates spindle assembly.     

A temperature-sensitive mutant defective in mitosis and cytokinesis

A temperature-sensitive mutant, ts2, of murine leukemic cells (L5178Y) loses its viability gradually at the non-permissive temperature (39 °C) but resumes normal growth when shifted to the permissive temperature (33 °C). At 39 °C the incorporation rate of thymidine is reduced on a per-cell-basis, whereas that of uridine and leucine is unchanged.Autoradiographic study indicates that the fraction of cells which can synthesize DNA decreases steadily with time of incubation at 39 °C. Accumulation of mitotic and multinucleate cells suggests that ts2 cells are defective in both mitosis and cytokinesis. Experiments using synchronized culture demonstrate that the cells shifted up atthe G2, but not at the G1 phase pass through the first mitotic phase normally.     

PinX1 localizes to telomeres and stabilizes TRF1 at mitosis

Human telomeres are DNA-protein complexes that cap and protect the ends of chromosomes. The protein PinX1 associates with telomeres through an interaction with the resident double-stranded telomere-binding protein TRF1. PinX1 also binds to and inhibits telomerase, the enzyme responsible for complete replication of telomeric DNA. We now report that endogenous PinX1 associates with telomeres primarily at mitosis. Moreover, knockdown of PinX1 caused delayed mitotic entry and reduced the accumulation of TRF1 on telomeres during this stage of the cell cycle. Taking these findings together, we suggest that one function of PinX1 is to stabilize TRF1 during mitosis, perhaps to promote transition into M phase of the cell cycle.     

Aurora-C and Aurora-B share phosphorylation and regulation of cenp-A and borealin during mitosis

Aurora-B and –C kinases are members of the Aurora serine/threonine kinase family of mitotic regulators. Aurora-B kinase is evolutionarily conserved from yeast to humans and has multiple functions in chromosome condensation, cohesion, biorientation, and in cytokinesis. In contrast, Aurora-C kinase has only been found in mammals, is upregulated in some tumor cell lines and tissues, and has a unique physiological role in spermiogenesis. Despite these known functions, little is known about the function of Aurora-C in mitosis. We have found that Aurora-C interacts with Borealin in addition to the other known members of the Aurora-B chromosomal passenger complex (CPC). We have also found that Aurora-C, like Aurora-B, phosphorylates the centromeric histone Centromere Protein-A (CENP-A) and Borealin in vitro. These molecular mechanisms are consistent with our observation that in the absence of Aurora-B, Aurora-C is sufficient for proper mitotic phosphorylation of CENP-A and centromeric localization of the CPC proteins. Thus, Aurora-C shares Aurora-B substrates and is capable of performing mitotic functions previously attributed only to Aurora-B.     

BIMA, a TPR-containing protein required for mitosis, localizes to the spindle pole body in Aspergillus nidulans

The Aspergillus nidulans bimA gene is required for mitosis. Loss of function mutations in bimA cause cells to arrest growth with condensed chromatin and a short, metaphaselike mitotic spindle. bimA is a member of a gene family defined by a repeated motif called the Tetratrico Peptide Repeat (TPR), which is found in genes from bacteria, yeast and insects. Several yeast TPR genes are also required for mitosis, including Saccharomyces cerevisiae CDC27 and Schizosaccharomyces pombe nuc2+, which appear to be functional homologs of bimA. We have developed antisera specific to the bimA protein (BIMA) and have characterized BIMA by western blot and immunocytochemical analyses. BIMA is heterogeneous in apparent molecular weight, consisting of a major 90-kD species and at least two minor species of approximately 105 kD. The results of BIMA localization by immunofluorescence microscopy depend on the level of BIMA expression. Overexpression of BIMA, which had no deleterious affect on growth or mitosis, resulted in localization of BIMA on or throughout most nuclei. Nuclear staining was granular, and overlapped but was not completely coincident with DNA staining by DAPI. In contrast, when expressed at normal levels, BIMA colocalized with the spindle pole body (SPB). BIMA localized to the SPB in a cell cycle independent manner. These results show that BIMA is either associated with or is a component of the SPB, and they suggest that BIMA functions at the spindle poles to promote the onset of anaphase.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 (⁶²⁵RRSRRL⁶³⁰) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.